The high proliferation rate of tumor cells demands high energy and metabolites that are sustained by a high glycolytic flux known as the ‘Warburg effect’. The activation and further metabolism of glucose is initiated by hexokinase, a focal point of metabolic regulation. The human hexokinase 2 (HK2) is overexpressed in all aggressive tumors and predominantly found on the outer mitochondrial membrane, where interactions through its N-terminus initiates and maintains tumorigenesis. Here, we report the structure of HK2 in complex with glucose and glucose-6-phosphate (G6P). Structural and biochemical characterization of the mitochondrial conformation reveals higher conformational stability and slow protein unfolding rate (ku) compared with the cytosolic conformation. Despite the active site similarity of all human hexokinases, the N-domain of HK2 is catalytically active but not in hexokinase 1 and 3. Helix-α13 that protrudes out of the N-domain to link it to the C-domain of HK2 is found to be important in maintaining the catalytic activity of the N-half. In addition, the N-domain of HK2 regulates the stability of the whole enzyme in contrast with the C-domain. Glucose binding enhanced the stability of the wild-type (WT) enzyme and the single mutant D657A of the C-domain, but it did not increase the stability of the D209A mutant of the N-domain. The interaction of HK2 with the mitochondria through its N-half is proposed to facilitate higher stability on the mitochondria. The identification of structural and biochemical differences between HK2 and other human hexokinase isozymes could potentially be used in the development of new anticancer therapies.
A high rate of glucose metabolism is essential to support the rapid growth of different types of tumors that utilize aerobic glycolysis rather than oxidative phosphorylation, a phenomenon known as the Warburg effect [1–3]. The high demand for glucose is necessary for energy production and the synthesis of building blocks required for the rapidly proliferating cancer cells . Hexokinase, the first enzyme of the glycolytic pathway, catalyzes the irreversible rate-limiting phosphorylation of glucose to glucose-6-phosphate (G6P). The role of hexokinase is essential not only for the intracellular retention of glucose, but also for its activation and further metabolism . The hexokinase reaction is also a focal point for the regulation of glucose metabolism and other biosynthetic pathways that require glycolytic intermediates for their function, making hexokinase an important target for the regulation of tumor growth and metastasis.
In mammals, four hexokinase isozymes have been characterized, including HK1, HK2, and HK3 with two domains (~100 kDa) that are structurally similar to the single domain HK4 (52 kDa), also known as glucokinase . The elevated rates of glucose metabolism, tumor progression, and the high patient mortality has been predominantly associated with the overexpression of HK2 in different cancers . This phenotype has been utilized clinically for the diagnosis of cancer by Positron Emission Tomography (PET) scan . In the liver, for example glucokinase is the predominantly expressed enzyme in normal adult hepatocytes. Tumorigenesis is initiated in the liver after a switch in gene expression from glucokinase (HK4) to high levels of HK2 [8,9]. Furthermore, the up-regulation of HK2 has been shown to facilitate the chemotherapy resistance and metastasis in different cancer cells .
HK2 is predominantly co-localized on the outer mitochondrial membrane (OMM), where its translocation is facilitated by an increase in the cellular glucose concentration [11–13]. The mitochondrial translocation of HK2 not only gives it preferential access to ATP required for its function and relieves feedback inhibition by G6P, but most importantly, suppresses apoptosis [14,15]. HK2 localization on the OMM prevents the formation of the mitochondrial permeability transition pore, which halts apoptosis and increases tumor cell viability and resistance to chemotherapeutic agents . Conversely, HK2 dissociation promotes mitochondrial injury and decreases tumor growth, which makes it an attractive target for the development of anticancer therapies .
Here, we determined the crystal structure of human HK2 in complex with glucose and G6P with structural fold similar to other mammalian hexokinases [16–23]. Mitochondrial interaction is facilitated by the mitochondrial-binding peptide (MBP), the first helix on the N-terminus of HK2. The mitochondrial conformation was generated after deleting the MBP (Δ16-HK2) and was found to possess higher conformational stability in comparison with the full-length (FL) HK2 variant.
A total loss of the catalytic activity in the N-half of HK2 was observed after altering the size of the linker helix-α13 that connects the N- to C-domain. The catalytic activity of the N-domain was proportional to the length of the linker helix-α13, where an N-domain variant with a short helix-α13 was catalytically inactivate. Computational modeling showed the linker helix-α13 to remotely modulate glucose binding by controlling the grove width of the N-domain active site. These findings can explain the inactivation of the N-half of HK1 with similar active site residues to HK2 but different conformation of its linker helix.
The catalytic residue of the N-half, but not the C-half, was found to reduce the extent of glucose stabilization on the whole enzyme. The addition of glucose to the single mutant D209A in the N-domain did not stabilize the whole enzyme, but increased the stability of the wild-type (WT) and D657A mutant enzymes. These results indicate the importance of the N-domain in controlling the stability of the whole enzyme in the addition to its role in binding HK2 to the mitochondria.
All chemicals obtained were from Sigma–Aldrich (Germany). The bacterial culture media, buffers, and supplements were purchased from Invitrogen. The synthesis of oligonucleotide DNA primers and DNA sequencing were done by Genscript Inc (Piscataway, NJ). Restriction enzymes were purchased from NEB (Beverly, MA). Ni Sepharose 6 Fast Flow was purchased from GE Healthcare (Sweden). The PCR and restriction digest products were purified using the QIAquick PCR purification kit (Qiagen). KOD DNA polymerase and dNTP mix were from EMD Millipore (Danvers, MA).
Different constructs of HK2 were expressed in E. coli using Champion pET SUMO expression system (Thermo Fisher Scientific). Point mutations were introduced using the QuickChange™ site-directed mutagenesis kit (Agilent). HK2 expression was induced by 0.2 mM IPTG and the cells were harvested by centrifugation at 8000 rpm for 10 min. The cell pellet was resuspended in 100 ml lysis buffer (100 mM Tris, pH 7.5, 150 mM NaCl, 5% glycerol, 5 mM imidazole) containing protease inhibitor cocktail (Sigma). Cells were lysed by sonication at 20% amplitude for 5 min, then centrifuged at 16000 rpm for 30 min at 4°C. The supernatant was loaded on to ProBond™ Nickel-Chelating Resin (Life Technologies) that was pre-equilibrated with lysis buffer. The column was washed, then eluted with lysis buffer containing 30 and 250 mM imidazole, respectively. The His6 tag was cleaved with thrombin protease overnight on ice then loaded on to a HiLoad Superdex 200 column using AKTA FPLC system (GE Life Sciences). Fractions containing HK2 protein were combined and concentrated to ~5 mg/ml in buffer containing 150 mM NaCl, 0.5 mM TCEP, 5% glycerol, and 10 mM HEPES, pH 7.8. The protein samples were analyzed using SDS/PAGE, and the purity of the different HK2 variants was determined from the bands on the Coomassie stained gels using densitometry on ImageJ software .
Crystals of Δ16-HK2 were grown by sitting-drop vapor diffusion in a mixture of 16% PEG 3350, 0.2 M sodium malonate, 0.1 M Bis-Tris propane (BTP) pH 8.5, 10% ethylene glycol, and 1 mM DTT at 18°C. Crystals were cryoprotected in a 50:50 mixture of Paratone-N and mineral oil before flash cooling in liquid nitrogen. Diffraction data were collected on Rigaku FR-E with Rigaku RAXIS image plate at a wavelength of 1.5418 Å and data were processed with the HKL2000 suite (Supplementary Table S1) . The crystal structure was solved by the molecular replacement method using Phaser in the CCP4 program suite with human hexokinase 1 (PDB code: 1HKB) as the structural model . The structure was refined with REFMAC5. The treatment for B-factors is isotropic and the dimer is in the asymmetric unit. The model was deposited with PDB code: 2NZT. Figures were prepared with the PyMOL Molecular Graphics System, version 1.8, Schrödinger LLC.
Since HK2 did not crystallize in the absence of substrates, the open state was modeled using the closed state of HK2 (PDB code: 2NZT). Computational modeling approach was used to generate the open state using HK4 (PDB code: 1V4T) as a template using MODELLER [19,37]. Keeping the open state model as the target state, a targetted molecular dynamics (TMD) simulation protocol implemented in Gromacs 4.05 was used with Structure-based Model for biomolecules (SMOG) potential [38–40] to obtain the open state. SMOG potential allowed rapid sampling of the conformational space while keeping the secondary structure stoichiometry intact during the closed-to-open transition. Structure-based potentials have been utilized heavily to study biomolecular processes including the mechanism of conformational transitions of large biomolecular complexes . Here, we used it for the first time to obtain the open state of HK2 based on the initial guess from homology modeling. A homodimer was constructed and energy minimized for both end states. A TMD simulation of 250000 steps in temperature 40K that is kept constant using Velocity Verlet scheme . Stochastic dynamics integrator with a time step of 0.001 ps and a friction coefficient of 0.1 amu/ps was used to integrate the equations of motion. For details on the conversion to real values, see . Non-bonded interactions were treated with 15 Å cutoff. The trajectory created by TMD was later used as the initial guess for minimum energy pathway of open-to-close transition.
is optimized. Here, R(l) is the coordinate vector as a function of arc-length l, and ∇U(R(l)) is the gradient of the potential energy. The minimum energy path from closed to open states is shown in Supplementary Movie S1.
MD was conducted in explicit water and ions using Gromacs 4.05 suite of programs  including only N-half. As in the previous section, OPLSAA force field was used for glucose and protein , and ATP parameters were adopted from . Water was modeled using SPCE . Smith and Dang  parameters were used to model K+ and Cl−, and for Mg2+ ions, method developed by  was used. The protein was solvated with a simulation box of 7.7 × 11.8 × 8.2 nm3. To neutralize the system and to mimic experimental conditions, 21 Cl− and 22 K+ were added by randomly replacing water molecules. In addition, one crystallographic Mg2+ ion was added to the catalytic site. To equilibrate water and ions, we restrained the position of the heavy atoms in the enzyme and conduct MD for 10 ns using constant pressure (NPT) simulation. The pressure was kept at 1 bar using Parrinello–Rahman scheme . NPT simulation was followed by constant volume (NVT) simulation for another 10 ns with the position restrains. Unrestrained NVT simulations were later carried out for sampling the conformational states. In all simulations, Leapfrog integration scheme was used with a time step of 2 fs. The temperature was kept ~300 K using the Velocity-Scaling method implemented in Gromacs . van der Waals interactions were calculated with 7–10 Å switching scheme. Electrostatic interactions were treated by Particle Mesh Ewald summation method with cubic interpolation order of 4 and grid spacing of 1.6 Å with a real-space cutoff of 12 Å. All bonds in the enzyme and water O–H bonds were constrained by LINCS algorithm. The dynamics of the enzyme were simulated for ~300 ns for the WT and R458A mutant. Gromacs suit of programs later used to perform trajectory analysis.
The HK2 reaction rate was measured spectroscopically using G6P dehydrogenase (G6PDH) couple reaction by monitoring NADH produced from the G6PDH reaction at 340 nm (ε340 =6220 M−1 cm−1) using a PerkinElmer Lambda 25 spectrophotometer equipped with a PTP-6 Peltier Temperature Programmer. The enzymatic reaction contained 20 mM HEPES (pH 7.5), 20 mM MgCl2, 3 mM NAD+, and 0.1 U/µl G6PDH (Sigma- G2921) at different concentrations of glucose and ATP from 0.5- to 10-times of their Km values. All the data were plotted in a double reciprocal form to determine the quality of the data then fitted using (eqn 1) for a sequential mechanism.
(1)where v and Vmax are initial and maximum velocities, respectively, A and B are substrate concentrations, and Ka and Kb are Michaelis constants for substrates A and B, respectively. Data were fitted using the Global fitting analysis in the kinetics module of SigmaPlot (Systat Software, Inc. San Jose, California, U.S.A., www.sigmaplot.com).
The TOpt was determined from the enzymatic activity of the different HK2 variants over a temperature range from 15 to 60°C. Reaction mixtures contained 10 mM HEPES pH 7.5, 1 mM ATP, 0.3 mM glucose, 1 mM NAD+, 20 mM MgCl2 were incubated at the specific temperatures for 3 min prior the addition of 0.1 unit/µl G6PD and 150 nM HK2 enzymes. The HK2 activity was monitored for 3 min at 340 nm. Error bars were calculated from triplicates of each reaction.
Thermal inactivation kinetics of HK2 at 37°C in the presence or absence of 5 mM glucose or 5 mM ATP were analyzed using two-state mechanism (N↔U). Enzyme aliquots at different time intervals were assayed as described above for residual activity and were plotted as a function of HK2 incubation time. Thermal inactivation was analyzed considering first order activity decay, and a semi-log plot of % residual activity was plotted as a function of HK2 incubation time. The enzymatic activity decay was fitted to a first order reaction, (eqn 2) or to a signal phase exponential decay using Prism 6, GraphPad Software.
(2)where Ao and A are initial and remaining enzyme activity at different time intervals, respectively. k is the rate constant for enzyme inactivation and t is the incubation time. The half-life (t1/2) is the time required to decrease the enzyme activity by half, was calculated as ln(2)/k.
Calorimetric thermal unfolding measurements were performed on a Nano-DSC (TA instruments) with HK2 concentration between 0.3 and 0.8 mg/ml in a 20 mM HEPES pH 7.5 and 20 mM MgCl2 buffer solution. All samples were heated at a scanning rate of 1°C/min from 10 to 80°C under 3 atm pressure in the presence or absence of 5 mM glucose or 5 mM ATP. Background scans were obtained using buffer (with or without substrates) and collected same as above. DSC thermograms were corrected by subtracting the corresponding buffer baseline and converted into plots of excess heat capacity (Cp) as a function of temperature. The ΔHcal was estimated from the area under the thermal transition and fitted using Nano Analyzer™ Software package from TA instruments.
CD spectra were collected from 190 to 260 nm at 120 nm/s scanning speed and 1 nm bandwidth on a Chirascan™ CD spectrometer (Applied Photophysics) at 25°C using 1 mm quartz cuvette. Enzyme concentration was 0.2 mg/ml in a 50 mM phosphate buffer pH 7.5 in the presence or absence of 5 mM glucose or 1 mM ATP. CD measurements in the presence of ATP was carried out using 0.5 mm quartz cuvette.
The urea unfolding experiments were carried out using a CS/SF attachment on the Chirascan instrument at 222 nm with a path length of 2 mm and dead time of 3 ms. A circulating water bath maintained the temperature at 25°C and 10:1 mixing ratio was used to mix HK2 to a final concentration of 0.1 mg/ml at different urea concentrations. A minimum of four shots for each experimental condition was averaged to improve the signal-to-noise ratio. Unfolding data were fitted to a single exponential growth using the Pro-Data software from Applied Photophysics to acquire the ku constants at different urea concentrations.
The rate of thermal unfolding was determined from ITD measurements by incubating HK2 in 50 mM phosphate buffer pH 7.5 at 37°C in the presence or absence of 5 mM glucose or 1 mM ATP. CD scans were collected every minute from 210 to 230nm for 50 min as described above. The protein (0.3 mg/ml) stability at 37°C was monitored at 222 nm and normalized to fraction unfolded using (eqn 3).
(3)where θ is the ellipticity of protein at specific time, and θN and θD are the ellipticities of native and unfolded states, respectively. The ellipticity of the native state (θN) was obtained prior to incubating the enzyme at 37°C, and the fully denatured state (θD) was obtained at the end of the measurement and after incubating the protein at 80°C for 1 h. The fraction of unfolded protein (FUnf) was fitted to straight line, where the slope was equal to the rate of thermal unfolding (k).
DSF was used to determine the HK2 thermal Tm using SYPRO Orange protein dye to monitor the protein unfolding on a Stratagene Mx3005P QPCR system (Agilent Technologies, La Jolla, CA) at λex =492 nm and at λem =555 nm. To each well of a 96-well thin-walled unskirted PCR microplate (Bio–Rad, catalog number : 223 94444), samples consisting of 5 µl buffer (20 mM HEPES, pH 7.5 and 20 mM MgCl2), with or without 5 mM glucose or 5 mM ATP, 0.3 mg/ml protein sample, and 3× SYPRO Orange dye in a total volume of 10 µl. Fluorescent reads were collected from 25 to 80°C at 1°C/min temperature ramp rate. The data were fitted to a Boltzmann sigmoidal function to calculate the Tm at the middle of the transition using the Excel add-on package XLfit (IDBS limited, Bridgewater, NJ, U.S.A.) as described . Final graphs were created using the GraphPad Prism 6 software (GraphPad Software, Inc. La Jolla, CA, U.S.A.).
The aggregation propensity of the HK2 variants was assessed by DSF using ProteoStat Protein Aggregation Assay (Enzo Life Sciences, PA, U.S.A.). The fluorescence intensity was recorded similar to DSF analysis in the presence of 3 mM Enzo dye in 96-well plates at temperature ramp rate of 1°C/min. Tagg was calculated at half the thermal transition.
Only HK1 and HK2 of the four human hexokinase isozymes bind to the OMM via their MBP at the N-terminus . However, the structural fold, thermodynamic, and kinetic stabilities of the mitochondrial and cytosolic conformations of HK2 are yet to be determined. When bound to the mitochondria, the MBP points into the mitochondria away from the HK2 enzyme. For in vitro biochemical characterization of the mitochondrial conformation, a variant lacking the MBP, the first 16 amino acids of the N-terminus (Δ16), will mimic the HK2 conformation on the mitochondria (Supplementary Table S1). On the other hand, the FL variant of HK2 will resemble the cytosolic conformation, where the MBP is not interacting with the mitochondria and can bind to the enzyme.
All HK2 variants were expressed in Escherichia coli and purified to >90% purity based on Coomassie staining SDS/PAGE analysis, where gel band densitometry of HK2 variants was calculated using ImageJ software (Supplementary Figure S1A) . The structural integrity of the HK2 variants was verified using far-UV CD analysis of protein conformation with spectra similar to predominantly α-helical proteins with two ellipticity minima at 208 and 222 nm (Supplementary Figure S1B).
Multiple crystal screens were used for the crystallization of the different HK2 variants including FL, Δ16, and N- and C-domains for WT, D209A, and D657A mutants in the absence or presence of substrates, products, or their analogs. Only the Δ16 variant of the WT-HK2 crystallized in complex with glucose and G6P at 2.45 Å (Supplementary Table S2). The crystal structure of human HK2 is a homodimer with two structurally identical N- and C-halves of equal size linked by a long eight-turn linker helix-α13 (Figure 1 and Supplementary Figure S2). Similar to the overall fold of mammalian hexokinases, the N- and C-domains have an identical α/β fold that is conserved amongst the hexokinase family [16–23]. Each domain is distinctly folded into large and small subdomains that each contains a five stranded mixed β-sheet. The active site is formed by a cleft between the two subdomains, where their β-sheets enclose the active site and the helices α5 and α13 (Supplementary Figure S2A inset). The N-half of HK2 is linked to the C-half by helix-α13 that protrudes out of the active site of the N-domain. Helix-α5 that is perpendicular to the linker helix-α13, carries the catalytic residue D209 of the N-half. As we will show later, this network of interactions plays an important role in maintaining the catalytic activity of the N-domain of HK2.
(A) Cartoon of the HK2 homodimer with monomers colored in white and gold. The MBP (blue) at beginning of the N-terminus and the linker helix-α13 (red) is between the N- and C-halves. (B) The monomer is divided into two structurally identical N- (gold) and C- (green) halves. The linker helix-α13 (red) is a long eight-turn helix that protrudes out of the active site of the N-half. Insets: the glucose-binding pockets in the N- and C-halves. (C) The open state of the N-terminus in complex with ATP and glucose obtained from molecular dynamics simulations (MD). The enzyme dynamics is projected on two order parameters where (θ) is the angle between helix-α13 and the large subdomain, and distance between T88 and T232 situated opposite sides of the active site. A salt bridge between R468-D202 dictates the position of helix-α13. (D) Correlation between the conformational parameters of WT (blue) and R468A (black). (E) The RMSD of glucose as a function of time for WT (blue) and R468A (black). This figure was prepared using PyMol (Schrodinger LLC) and VMD.
Substrates binding to the apo-HK2 enzyme induced conformational transition to close the active site in preparation for catalysis, which was observed by a decrease in ellipticity after the addition of glucose in the CD spectra of HK2 (Supplementary Figure S1B). Atomically detailed molecular dynamics simulations (MD) were used to study the transition mechanism of HK2 during catalysis. The minimum energy path obtained using Steepest Descent Path (SDP, ) methodology suggests a collective motion of the two halves upon transition from open to closed states (Supplementary Movie S1, supplementary results). The opening and closing of the active site during catalysis is facilitated by movement of the small subdomains of the four halves of the homodimeric HK2 enzyme. During catalysis, the large subdomain does not move and its high conformational rigidity is achieved by the long ten-turn helix-α11 that spans the large subdomain (Supplementary Figures S2A,B).
The glucose-binding pockets in the N- and C-halves of HK2 are identical to other members of the hexokinase family, with the requirement of an aspartic residue for catalysis (Supplementary Figure S2C). Here, the double mutant, D209A and D657A, of the catalytic residues in the N- and C-halves of HK2, respectively, diminished the enzymatic activity of HK2. Since co-crystallization with ATP was not successful, the ATP-binding pocket of HK2 was modeled based on the crystal structure of HK4−ATP complex (Supplementary Figure S2C) [27,28]. The catalytic residue D209 or D657 of the N- and C-domains, respectively, forms H-bonding interactions with the 4-OH and 6-OH of glucose, and a lysine residue, K173 or K621, align the γ-phosphate of ATP with the 6-OH of glucose for its phosphorylation.
The initial velocity patterns of the different HK2 variants were obtained in the forward direction by varying the concentrations of glucose at different fixed concentrations of ATP. The kinetic parameters obtained were similar to those reported previously (Supplementary Figure S1C−G and Table S3) [29,30], where the catalytic efficiency of the mitochondrial conformation Δ16-HK2 is higher than the FL variant, where the V/KGluEt and V/KATPEt of Δ16-HK2 was 0.6- and 2.0-fold higher than the FL-HK2, respectively.
The N- and C-domains of HK2 are split into large and small subdomains enclosing the active site and helices α5 and α13 (Supplementary Figure S2A,B). To kinetically characterize each domain separately, the N- (residues: 1–462) and C- (residues: 465–916) domains were expressed separately from the whole enzyme. Our initial design of the N-domain did not include the last four turns of the eight-turn linker helix-α13 at the end of the N-domain to avoid any structural misfolding of the domain. The last four turns of helix-α13 protrude outside the N-domain and are exposed to solution; therefore, it was not included in the initial construct design as not to disturb the structural fold of the N-domain.
The C-half was catalytically active, but to our surprise, the N-half with short four-turns of helix-α13 was completely inactive. The inactivation of the separate N-domain was not due to misfolded domain as CD spectral analysis (data not shown) confirmed its structural fold integrity. For this reason, a detailed kinetic study of different HK2 variants was necessary to validate our findings in comparison with what have been reported previously that the N-domain of HK2 is active [29,30]. The kinetic activity of N- and C-domains was also assessed in the FL and Δ16 enzyme variant by the introduction of a single mutation D209A or D657A one at a time, respectively (Supplementary Figure S1C−G and Table S3). The catalytic rate (V/Et) of the N-half (FL-D657A mutant) was 0.5-fold higher than the C-half (FL-D209A mutant) of HK2. However, the deletion of the MBP increased the V/Et for the C-half (Δ16-D209A mutant) by two-fold in comparison with the N-half (Δ16-D657A mutant) (Supplementary Figure S1C−G and Table S3). These findings confirm that the N-half of HK2 is catalytically active and the inactivation of the separate N-domain observed here might be related to the initial design of the construct.
In the short variant of the N-domain (residues 1–462), part of the linker helix-α13 was not included in the construct design (Figure 1B and Supplementary Figure S2A). To investigate its role in the activity of the N-domain, different variants of the N-domain were constructed with increasing the length of helix-α13 to 6- (residues 1–469), and full eight-turns (residues 1–479). We found the activity of the N-domain to be dependent on the length of the helix, where longer helix-α13 promoted higher activity of the N-domain. The V/Et of the separate N-domain was similar to the FL-D657A mutant and the C-domain was >three-fold slower than the FL-D209A mutants (Supplementary Figure S1E). These results confirm that the activity of the N-domain is dependent on the conformation of helix-α13 that is perpendicular to helix-α5 carrying the catalytic residue D209 of the N-domain (Supplementary Figure S2A inset). This network of interactions is important in determining the activity of the N-domain by the linker helix-α13.
All atom MD performed on the open state of HK2 confirmed the contribution of helix-α13 on the activity of the N-half, where it was stable with an average RMSD of 1.8 Å in 0.3 μs simulation time. One of the key interactions was the formation of a salt bridge between R468 on helix-α13 and D202 on the smaller subdomain of the N-half (Figure 1C). Local dynamics of the open state showed strong correlation between θ-angle and the active site width quantitated by the distance between T88 and T336 of the small and large subdomains, respectively (Figure 1C,D). While θ-angle was measured between helix-α13 and the first β-strand of the large subdomain, wide values of the θ-angle correlate with narrow groove width of the active site, and hence tighter H-bonding network between HK2 and the substrates, glucose, and ATP. Computationally, R468A mutant abolished the salt bridge with D202 and produced broader values for θ-angle with >10 Å wider active site and hence weaker H-bonding network with the substrates for weaker binding interactions. As a result, a rapid dissociation of glucose was observed in R468A mutant compared with the WT enzyme (Figure 1E). Similar results were obtained with truncated linker helix-α13 (residues: 1–462), where the salt bridge remotely modulates the substrates affinity (data not shown).
Multiple thermoanalytical techniques were utilized to characterize the cytosolic (FL) and mitochondrial (Δ16) conformations of HK2. The Δ16 variant lacking the MBP will mimic the enzyme’s conformation on the mitochondria. While, the FL variant will be similar to the cytosolic state, as the MBP is not interacting with the mitochondria and available to interact with the enzyme. The human HK2 enzyme has been shown previously to possess high catalytic activity when bound to the mitochondria . Similarly, the Δ16 variant has been shown previously [29,30] and here to possess higher catalytic efficiency compared with the FL variants, which indicate that the Δ16 variant has a conformation similar to the enzyme on the mitochondria, and the FL variants will be similar to the cytosolic conformation.
Differential scanning calorimetry (DSC) was used for detailed thermodynamic characterization of HK2 in the absence or presence of substrates (Figure 2). The overall shapes of the thermograms were similar for all HK2 variants with deconvolution patterns indicating a two-state transition with an early large and a late small melting transitions yielding two melting temperatures (Tm1 and Tm2). The calorimetric enthalpy (ΔHcal) was determined from the area under the thermographic peak, which corresponds to the total heat absorbed by the protein during the heat denaturation transition. Thermal unfolding of all variants was calorimetrically irreversible, and the addition of either substrate increased the Tm1 and ΔHcal by up to 6°C and 600 kJ/mol, respectively (Supplementary Figure S3A,B).
). Data are mean ± S.D., n=3.
The mitochondrial conformation (Δ16) of HK2 possessed higher ΔHcal value than the cytosolic conformation (FL) with minimal change on Tm1 (Figure 2F). In the absence of substrates, deleting the MBP of HK2 increased ΔHcal >500 kJ/mol with less than 1°C increase in Tm1. In the presence of glucose, similar effect was observed with increase in ΔHcal >350 kJ/mol and again negligible change in Tm1. These findings are not commonly observed in proteins’ DSC thermograms, as an increase in ΔHcal is usually accompanied by similar increase in Tm.
The ΔHcal value, calculated from the area under the thermographic peak, is related to the type of bonding interactions. An endothermic profile with a positive thermogram is a characteristic of H-bonding and polar interactions in the protein structure, but an exothermic pattern with negative thermogram is a result of breaking hydrophobic interactions in the protein structure as exposed to the aqueous environment [32,33]. The increase in the ΔHcal value upon the deletion of the MBP (Δ16) suggests that the hydrophobicity of HK2 decreased. This suggests that upon binding to the mitochondria, conformational changes alter the intermolecular interactions of HK2 to be more optimum for a cytosolic environment. The low total protein yield from E. coli expression for all the FL variants was at least one-fold lower than the Δ16 variants of HK2 (Supplementary Figure S1H). The increased aggregation on the gel filtration profiles (data not shown) explain the low protein expression yield for the FL variants and support its low ΔHcal values as a result of increased hydrophobicity. Furthermore, the high protein yield of Δ16-HK2 and its low hydrophobicity indicate conformational changes upon translocation to the mitochondria, where the enzyme possesses more hydrophobic character. The thermodynamically most stable enzyme was the Δ16 variant of the WT in the presence of glucose, which indicates the translocation and high occupancy of HK2 on the mitochondria can be driven by higher conformational stability.
To determine the thermodynamic contribution of each domain on the stability of the entire enzyme, glucose binding was assessed on each domain after mutating its catalytic residue one at a time in the whole enzyme. For example, the thermodynamic contribution of the C-domain was characterized from the FL and Δ16 variants of the D657A mutant, where the glucose-binding pocket of the C-domain was altered. The overall shapes of the D657A thermograms of the FL and Δ16 variants in the absence or presence of substrates were similar to the WT enzyme with similar Tm1 but lower ΔHcal values (Figure 2A,C). Similar to the WT enzyme, the addition of glucose increased Tm1 and ΔHcal of D657A variants from 2.7 to 6.4°C and from 270 to 590 kJ/mol, respectively (Supplementary Figure S3A,B). On the other hand, mutating the catalytic residue D209A of the N-domain produced thermograms that are different from the WT enzyme (Figure 2B). The addition of glucose moderately increased the thermodynamic stability of the D209A mutant with a subtle increase in Tm1 and ΔHcal of less than 2°C and 104 kJ/mol, respectively.
The addition of glucose increased the thermodynamic stability for all HK2 variants except the D209A mutant as well as the N-domain (Figure 2B,D). The Tm1 of the N-domain did not change in the presence of glucose for the FL and Δ16 variants of the N-domain. However, the deletion of the MBP of the N-domain increased its Tm1 from 45 to 48.5°C, respectively (Supplementary Figure S3A,B). These findings highlights the role of the N-half in controlling the stability of the whole enzyme, where mutating the catalytic residue of the N-domain was sufficient to eliminate the glucose stabilization effect on the entire enzyme in addition to the higher thermodynamic stability of the N-domain after truncating the MBP.
In the addition to DSC analysis, differential scanning fluorimetry (DSF) was implemented to examine the global thermal unfolding of HK2 variants in the presence of SYPRO Orange reporter dye (Supplementary Figure S3C). Similar to DSC analysis, the addition of glucose increased the melting temperature (Tm) of FL and Δ16-HK2 from 34 to 42°C for WT and D657A variants, but it did not enhance the stability of the D209A mutant (Supplementary Figure S3C). Interestingly, the deletion of the MBP substantially increased the stability of the N-domain in the presence or absence of glucose. A direct correlation was observed between Tm and Tm1 obtained from DSF and DSC analyses, respectively (Supplementary Figure S3E). However, less correlation was observed between Tm and ΔHcal values from DSF and DSC analyses, respectively (Supplementary Figure S3F). To confirm whether the DSF data were direct measure of protein unfolding and not aggregation, an Enzo ProteoStat detection dye was used to determine the aggregation temperature (Tagg) of HK2 variants at half thermal transition (Supplementary Figure S3D). The thermal unfolding preceded domain aggregation since Tagg was >5°C higher than Tm for all HK2 variants except the N-domain that was more aggregate prone.
The thermal kinetic stability of HK2 variants was determined using isothermal denaturation (ITD) by monitoring the unfolding of HK2 at 37°C in the absence or presence of substrates (Figure 3). The unfolding rate (ku) was calculated from monitoring the enzyme denaturation at 222 nm in a CD spectrophotometer. The unfolded fraction was calculated from comparison of the fully unfolded and folded states. In the absence or presence of ATP, the Δ16 variant of the WT enzyme was more stable than the FL-HK2. The deletion of the MBP decreased the ku of the WT enzyme and in the presence of glucose, both had similar kinetic stabilities (Supplementary Figure S4A).
(A–H) Time dependence of CD scans on the thermal kinetic stability of HK2 variants in the absence and presence of 5 mM glucose or 1 mM ATP at 37°C. Ellipticity readings at 222 nM were normalized to fraction unfolded protein. Thermal unfolding rates (ku) were calculated from the slope of the lines. (K) Correlation between ku measured here and Tm1 from the DSC analysis. Data are mean ± S.D., n=3. Symbols and colors are as in Figure 2.
In the absence of substrates, the separate N- and C-domains were the most stable with low ku values (Supplementary Figure S4A). As observed with the DSC and DSF analyses, the addition of glucose increased the kinetic stabilities of the WT and D657A variants from 2.0- to 4.6-fold as a result of decrease in their ku. However, the addition of glucose increased the kinetic stability of the D209A mutant of Δ16- and FL-HK2 by less than one-fold. This again underlines the role of the N-domain in stabilizing the whole enzyme, where a direct correlation was observed between ku obtained here and Tm1 from DSC analysis (Figure 3K). The kinetic and thermodynamic stabilities of all HK2 variants increased upon the addition of glucose, except the D209A mutant.
The chemical kinetic stability of the WT HK2 was also measured as a function of urea concentration using stopped-flow CD spectroscopy (Figure 4A). The urea unfolding kinetics exhibited a single exponential pattern with the ku in water (kuH2O) determined from extrapolating the rate to zero urea concentration. Similar to the ITD results, the chemical kinetic stabilities of the WT variants increased upon the addition of glucose with more than three-fold decrease in ku H2O (Figure 4B). The deletion of the MBP did not affect the kinetic stability of the Δ16 variants in comparison with the FL-HK2.
(A) The rate of unfolding (ku) of the FL- and Δ16-HK2 variants of the WT were determined as a function of urea concentration. ku was monitored at 222 nm using stopped-flow CD spectroscopy at 25°C in the absence (blue) and presence of 5 mM glucose (red) or 1 mM ATP (green). (B) The ku in water (kuH2O) of the FL- (solid bars) and Δ16- (checkered bars) HK2 variants was calculated after extrapolating the rate to zero urea concentration for experiments in (B). (C) The temperature dependence of the specific enzymatic activity of HK2 variants. (D) The TOpt calculated at the middle of the peak for variants in (C). Data are mean ± S.D., n=3. Abbreviation: TOpt, optimum temperature of catalysis .
The thermal stability of HK2 can also be assessed as a function of its catalytic rate (V/Et). The optimum temperature of catalysis (TOpt) was determined at the middle of the bell-shaped temperature profile with temperature correlated to the highest V/Et rate for each variant tested. The temperature dependence on V/Et for all HK2 variants was similar with TOpt at physiological temperature of 37°C except for the N-domain and D209A mutant with TOpt values of 30 and 33°C, respectively (Figure 4C,D). Since HK2 activity was measured in the presence of glucose, a direct correlation was observed between TOpt and Tm1 values determined from DSC analysis of HK2 in the presence of glucose (Supplementary Figure S4B). The TOpt value of the D657A mutant was similar to the WT enzyme, where mutating the catalytic residue of the C-half did not alter the TOpt of D657A mutant. However, the D209A mutant of the N-domain lowered the TOpt consistent with its low thermodynamic stability (Tm1) in the presence of glucose, which supports the regulatory role of the N-half in the function and stability of the whole HK2 enzyme.
As for the separate domains that possessed high thermodynamic stabilities based on multiple thermoanalytical analyses, the TOpt of the C-domain was similar to the WT enzyme (Figure 4C,D). However, the N-domain possessed the lowest TOpt value even though its thermodynamic stability was similar to the C-domain and the FL-enzyme (Supplementary Figure S4B). The high stability of the N-domain confirms that the loss of activity at lower TOpt is not due to structural integrity of the enzyme rather due to loss of conformation of the partially exposed helix-α13 of the N-domain. From initial velocity studies, we showed the importance of helix-α13 in maintaining the catalytic activity of the N-domain. More than half of helix-α13 is exposed to solvent and protrudes out of the active site of the N-domain. The low TOpt value of the N-domain is likely due to destabilization of the linker helix-α13 at a lower temperature that resulted in an early loss of the catalytic activity.
Furthermore, to establish the relation between the stability of HK2 and its catalytic function, the time dependence on the residual catalytic activity of HK2 was determined at 37°C in the absence or presence of substrates (Figure 5). The enzyme was incubated at physiological temperature in the presence or absence of substrate and its catalytic rate (V/Et) was measured at different time intervals. The kinetic half-life (t1/2) of the HK2 variants was determined from the time-dependent decrease for half the catalytic activity. A fast catalytic inactivation was observed for all HK2 variants in the absence of substrates with a t1/2 of less than 10 min (Supplementary Figure S4C). The addition of glucose increased the t1/2 by 10- to 24-fold for the FL and Δ16 variants of WT and D657A mutant, but it did not increase the t1/2 for the D209A mutant. Again, mutating the catalytic residue of the N-half compromised the glucose stabilization effect on HK2. This was not the case for the D657A mutant, where an intact glucose binding pocket in the N-half was sufficient to stabilize and maintain the catalytic activity for >40 min compared with <6 min for the D209A mutant.
(A–H) The residual enzymatic activity of HK2 variants at different time intervals after incubating the enzyme in the absence or presence of substrates at 37°C. (I) Correlation between t1/2 and Tm1 from DSC analysis of the different HK2 variants. Data are mean ± S.D., n 3. Symbols and colors are as in Figure 2.
Correlating the t1/2 with Tm1 obtained from DSC analysis revealed enhanced catalytic stability only for HK2 variants with Tm1 values above the physiological temperature (Figure 5I). The C-term lost only 30% of its catalytic activity after 40-min incubation at 37°C (Figure 5H). Even though the separate domains were thermodynamically most stable variants, the N-term was completely inactive in less than 5 min (Figure 5G). Similar to its low TOpt, the N-domain short t1/2 is due to early destabilization of the partially solvent exposed linker helix-α13 at the end of the N-domain that is essential for its catalytic activity.
The uncontrolled and rapid proliferation of cancer cells is initiated after a switch in glucose metabolism from mitochondrial oxidative phosphorylation to aerobic glycolysis known as the ‘Warburg effect’ [1,2]. Glucose metabolism is elevated during the transformation of normal to malignant cells, an important hallmark of tumors that up-regulate HK2 expression . The dominant presence of HK2 on the mitochondria and the regulatory role of its N-domain in the inhibition of apoptosis, highlight the molecular and structural importance for the characterization of HK2 roles in the initiation and maintenance of cancer [14,15]. In this study, biochemical and structural characterization of the cytosolic (FL) and mitochondrial (Δ16) conformation of HK2, proposed a mechanism for the translocation of HK2 to the mitochondria. In addition, the conformation of helix-α13 that links the N- with C-half of HK2 was found to be important for maintaining the catalytic activity of the N-domain as observed experimentally and from computational modeling. Further characterization of the single mutants, D209A or D657A, in HK2 reveals the regulatory role of the N- but not the C-domain in the thermodynamic and kinetic stabilities as well as the half-life of the whole enzyme.
The mitochondria, the site for energy metabolism and regulation of cell death, plays an important role in tumorigenesis and metastasis. Of all the enzymes in the glycolytic pathway, and of the four human hexokinase isozymes, only HK1 and HK2 bind to the mitochondria for enhanced catalytic activity and relief of feedback inhibition by its product, G6P [29,30]. In tumor cells, HK2 is predominantly localized on the OMM, where its translocation is facilitated by an increase in the cellular glucose concentration [11–13].
The FL and Δ16 variants that mimic the cytoplasmic and mitochondrial conformations of HK2, respectively, are used to characterize the molecular machinery and forces required for HK2 translocation to the mitochondria. Since HK2 attaches to the OMM via its MBP, the conformation of Δ16 variant lacking the MBP at the beginning of its N-terminus will mimic the mitochondrial conformation of HK2. Enhanced conformational stabilities of Δ16-HK2 variants in comparison with the FL-HK2 with >two-fold increase in ΔHcal was observed from DSC analysis with limited change in Tm1 values. The increase in ΔHcal values after the deletion of the MBP is due to induced conformational changes that decrease the hydrophobicity of the FL- compared with the Δ16 variants of HK2. In general, proteins with polar character yield positive endothermic peaks on the DSC thermograms, but negative exothermic peaks are observed for hydrophobic proteins [32,33]. These findings were also confirmed by the higher protein yield after E. coli expression for all Δ16 variants with less aggregated samples in comparison with the FL-HK2. In addition and in the absence or presence of ATP, the rate of thermal unfolding measured from ITD analysis was lower for the Δ16-HK2 variants compared with FL enzyme. As a result, the mitochondrial conformation of HK2 is kinetically and thermodynamically more stable, which supports the notion that the high occupancy of HK2 on the mitochondria and its translocation from the cytoplasm is due to higher stability.
The addition of glucose increased the thermodynamic stability of all HK2 variants with the highest thermodynamic and conformational stabilities observed for the Δ16 variant of the WT enzyme. Similarly, glucose decreased the chemical and thermal ku of HK2 making it kinetically a more stable enzyme. These results support previous reports of high occupancy of HK2 on the mitochondria [11,12], where the translocation is facilitated by a more stable conformation on the mitochondria.
The human hexokinase 1, 2, and 3 contain two domains equal in size and structurally similar, where each domain contains a conserved glucose-binding pocket with the requirement of an aspartic residue for catalysis [18,28]. Regardless of their high structural similarity and active site identity, the N-domains of HK1 and HK3 are catalytically inactive with previous reports attributing the inactivation of the N-domain to regulatory roles they play in the overall function of the enzymes [16,34]. Currently, there are no mechanistic evaluation for the molecular inactivation of the N-domain of HK1 and HK3, and from a structural and biochemical point of view, the N-domains of the human hexokinase isozymes should all be catalytically active.
Here and for the first time, we identified an inactive variant of the N-domain of HK2 in an attempt to describe the mechanism by which other hexokinase isozymes lost activity in their N-domains. We found the size of the linker helix-α13 to be essential in maintaining the catalytic activity of the N-half of HK2. In hexokinase 1 and 2, helix-α13 is a long eight-turn helix that extends out of the active site of the N-half to link it to the C-half (Figure 1B and Supplementary Figure S2A) [16,17]. To express the N-half separately from the enzyme, multiple variants have been constructed with different sizes of the linker helix-α13 including four-, six-, and eight-helical turns. To our surprise, the N-half with short four-helical turns of helix-α13 was completely inactive. However, the N-half of HK2 is catalytically active based on initial velocity studies of the D657A mutant that contain inactive C-domain. Furthermore, the catalytic activity of the N-domain was restored upon increasing the size of helix-α13 with the highest activity recorded for N-domain with the full eight-turn helix. These results show that the activity of the N-domain of HK2 is dependent on the conformation of its linker helix-α13.
MD of the HK2 reaction mechanism revealed distinct conformational changes along the linker helix-α13 (Supplementary Movie S1), where opening the N-half was possible with the bending of helix-α13. Even though the structural folds of their domains are identical, overlay of the entire homodimers of HK1 (PDB code: 1HKB)  with HK2 in complex with glucose and G6P were not possible. However, alignment was possible only through one domain but not both, where alignment on the N-domains reveals a 16° bent on helix-α13 of HK1 compared with HK2 (Supplementary Figure S2D). From the HK2 simulation (Supplementary Movie S1), the linker helix-α13 underwent conformational changes for the opening and closing of the active site of the N-domain. The bent conformation of helix-α13 in HK1 and the stiffness of its backbone may hinder the opening of its active site in the N-domain.
The catalytic role of HK2 in glucose intracellular retention and metabolism is critical for cancer cells survival and proliferation, where its binding to the OMM provides a preferential access to mitochondrial ATP needed for catalysis and evades feedback inhibition by its products, G6P [14,35]. The mitochondria binding of HK2 is also important to suppress apoptosis and cancer cell death [14,15]. It has been reported previously and confirmed here that an increase in the catalytic efficiency of HK2 was accomplished upon translocation to the mitochondria, where the catalytic efficiency of Δ16 was higher than the FL variants [29,30]. However, these findings do not explain the high occupancy of HK2 on the mitochondria.
To investigate the regulatory role of the N-half on the stability of HK2, an alanine mutation was introduced at the catalytic residue of the N- (D209) or C- (D657) domains to inactivate one domain at a time. The FL variants of the WT, D209A, and D657A of HK2 had similar kinetic and thermodynamic stabilities in the absence of substrates (Figures 2F and 5I). However, the deletion of the MBP (Δ16-HK2) or the addition of glucose increased the stability of all HK2 variants, except the D209A mutant of the N-domain. As a result, an intact glucose-binding pocket in the N-domain is required for HK2 to adopt a stable conformation in the presence of glucose. Since the mitochondrial interaction is initiated by the N-domain of HK2, its role in maintaining the stability of HK2 enzyme explain mechanistically how the enzyme shifts its conformational stability upon binding to the mitochondria.
The evasion of cell death is one of the major causes of the failure of anticancer therapy. HK2 promotes tumor survival and resilience of cell death, where its mitochondrial dissociation was found to trigger apoptosis in cancer cells . Therefore, the development of therapeutics that target HK2 specifically and facilitate its mitochondrial dissociation or inhibition of its catalytic activity will make an effective anticancer therapy. Therapeutics with high specificity for HK2 and low interactions with other human hexokinase isozymes will reduce the side effects of the new anticancer treatment. Detailed structural and biochemical analyses of the human hexokinase isozymes are necessary for the identification of unique characteristics of HK2 that can be used in the development of new anticancer drugs.
One of the major differences between HK2 and other human hexokinase isozymes is the loss of catalytic activity at the N-domain in HK1 and HK3. Here, we identified the role of helix-α13 that links the N- and C-halves of HK2 in maintaining the catalytic activity of the N-domain. Reducing the size of helix-α13 decreased or completely abolished the catalytic activity of the N-domain of HK2. These findings highlight the molecular mechanism for the metabolic adaptation of tumor cells in reprogramming their metabolism to meet the high demands of cancer cells.
This work was supported by the Research Enhancement Fund from New York University Abu Dhabi ; the Terry Fox Foundation in the U.A.E.; Al Jalila Foundation for Medical Research, Dubai, U.A.E. [grant number AFJ201436]; and the Faculty Research grant [grant number AD181 (to S.K.)].
L.N., J.C.F., W.M.R, and M.H.N. cloned and expressed the HK2 variants. W.M.R and C.C.-L. screened and mounted crystals of HK2. H.Z. solved the HK2 crystal structure. J.C.F. and M.H.N. conducted the biochemical and structural analyses. S.K. performed computational modeling. W.M.R. designed the experiments, supervised the project, and wrote the paper.
We thank Mazin Magzoub, Adrienne Chang, and Andrew Stopford for editing the manuscript. This research was carried out using Core Technology Platform resources at New York University Abu Dhabi. Computational research was carried out on the High Performance Computing resources at New York University Abu Dhabi.


Socketed? Processor was socketed (so that it could be replaced with a Z800 when/if it came out, which it never did) and Z800 support chips were present.
Arrow Keys: Mostly Clustered, some versions may have non clustered arrow keys. Why clustered? Lore has it that Tandy was trying to copy Apple’s keyboard layout. Difficulty in playing games with clustered arrows, however, resulted in later models being changed back to non-clustered.
Is the “D” for Double Sided Drives? NO! It’s for DeskMate.
RS-232 was moved to face out rather than down.
P=’Portable’ (luggable). It has no Model III ROMS. You would know one if you saw one.
TRS-80 Model 4. Compare the features of the Model 4 personal desktop computer to any other computer in its class. For power, versatility and convenience, you’ll find Model 4 is a true price breakthrough!
Software Flexibility. Model 4 can run all Model III TRSDOS and LDOS programs (in Model III mode). That means a large software library is already available, and Model III owners can upgrade to a Model 4 without making their present software obsolete. In addition, Model 4 is compatible with the CP/M Plus operating system giving you literally thousands of application programs from which to coohse.
Easy to Use. Simply add ready-to-run programs for word processing, spreadsheet analysis, data base management and much more and Model 4 is ready to go to work. The 2-disk model provides 368K of disk storage (184K each) and has a built-in RS-232C Serial Interface to access information networks by phone (requires modem and cable). Three programmable function keys let you take advantage of program-designated operations.
Advanced Features. The TRSDOS 6 operating system allows you to turn extra memory into a superfast disk drive. This “MemDisk” feature allows for keystroke-quick responses instead of time-consuming disk access. A swoftware print spooler lets you print one job while working on another. You can custom-tailor print-outs with the forms command. A Job Control Language makes it possible to set up a while series of operations that Model 4 can perform unattended. A host/terminal package makes computer communications easy. There are more features for serious programmers, too, such as routing, linking and filtering.
Complete Documentation. Model 4 comes with Microsoft Disk BASIC that includes 40-character variable names, CHAIN, COMMAND and much more. Also comes with TRSDOS 6 operating system, an owner’s manual, reference card, programming maual, and an introduction to start you computing immediately. U.L. listed.
Who is the Model 4 for? The Model 4 is the perfect computer for busy managers, professionals, educators, and home computer users. Because of a huge ready-to-run software library is currently available, Model 4 can be used for forecasting, word processing, data base management, computer assisted instruction, program development and for thousands of other applications. The TRS-80 Model 4 expands easily to meet your growing computing needs, too.
Memory: 14K ROM, 64K RAM expandable to 128K (provides for disk drive emulation in RAM).
Keyboard: 70-key typewriter-style with datapad, plus control, caps and 3 programmable function keys (F1, F2, F3).
Video Display: 80 x 24 (Model 4 mode), 64 x 16 (Model III mode) or double-width 40 or 32 characters per line. Upper and lower case and reverse video (Model 4 mode). 96 text. 64 graphics and 96 “special” characters.
Disk Drives: One or two built-in double-density 184K 5-1/4″ drives.
External Connections: Cassette operation at 500/1500 baud. Parallel print port. RS-232C serial port (2-Disk version only). Buffered input/output bus.
Model 4D with DeskMate. The perfect desktop computer for busy managers, professionals, educators and home users. The Model 4D even comes with valuable DeskMate software on diskette, featuring the applications today’s computer user wants most. Plus. because a huge software library is already available, the Model 4D can be used for forecasting, word processing, data base management, computer-assisted instruction, program development and for thousands of other applications.
Self-contained and Expandable. The Model 4D is a stylish, self-contained desktop unit with a 12″ built-in 80 x 24 display. A parallel printer interface is included. And the Model 4D expands easily to meet your growing computing needs. Add more memory, one or two more floppy drives and a hard disk.
Software Flexibility. The Model 4D runs most TRSDOS and LDOS software desinged for the Model 4 and 4P as well as for Model I/III (in the Model III mode). That means a large softwre library is already available.
Easy to Use. Simply add ready-to-run programs for wrod processing, spreadsheet analysis, data base management and much more and Model 4D is ready to go to work. The two built-in, double-sided disk drives provide 736K of storage (368K each) and has a built-in RS-232C Serial Interface to access information networks by phone (requires modem and cable). Three programmable function keys (F1, F2, and D3) let you take advantage of program-designated operations.
Advanced Features. The TRSDOS 6 operating system allows you to turn extra memory into a superfast disk drive. This “MemDisk” feature allows for keystroke quick responses instead of time-consuming disk access. A software print spooler lets you print one job while working on another. You can custom-tailor print-outs with the forms command. A Job Control Language makes it possible to set up a whole series of operations that Model 4D can perform unattended. A host/terminal package makes computer communications easy. There are more features for serious programmers too such as routing, linking and filtering. Add our Double Duty utility (26-2231) and you can load two different application programs at once and move back and forth between them.
CPU: 4MHz Z-80A (2 MHz in Model III mode).
Memory: 64K RAM expandable to 128K (provides for disk drive emulation in RAM).
Video Display: 80 x 24 (Model 4 mode), 64 x 16 (Model III mode), or double-width 40 or 32 characters per line. Upper and lower case and reverse video (Model 4 mode). 96 text, 64 graphics and 96 “special” characters.
Keyboard: 71-key typerwriter-style with datapad, plus control, caps and 3 programmable function keys (F1, F2, F3).
Sound: Obtainable from BASIC. Toggled “keyclick”.
Disk Drives: Two built-in double-sided 368K 5-1/4″ drives.
External Connections: Cassette operation at 500/1500 baud, Parallel printer port. RS-232C serial port (2-Disk version only). Buffered input/output bus.
TRS-80 Model 4P Get the versatility of our popular TRS-80 Model 4 desktop computer in a compact system you can take anywhere! The Model 4P is easy to carry – weighs just 26 pounds – and it’s small enough to tstow in overhead luggage racks on planes, trains or busses. It’s the perfect computer for our mobile society.
Use Thousands of Programs. Model 4P can use all Model 4 disk software. It can also run all Model III TRSDOS and LDOS disk programs (in Model III mode) without change. So a huge software library is already available. And Model 4P is compatible with the CP/M Plus operating system which opens the door to literally thousands of applications..
Big on Features. Unlike some transportables, Model 4P is without compromise. You can depend on it as your desktop computer because of true desktop features. Rather than a small display screen, Model 4P features a full 80-character by 24-line 9″ green-screen display that can be upgraded to provide optional 640×240 high-resolution graphics. The full-size keyboard features CONTROL, CAPS and three function keys. And you can add the internal direct-connect modem board to communicate with other computers and to access information services.
Superior Operating System. The powerful TRSDOS 6 operating system lets you use extra memory as a superfast disk drive. The result? Keystroke-quick responses instead of time-consuming disk access. The software print spooler allows you to print out a job at the same time you’re using Model 4P for other tasks. A Job Control Language lets you set up a while series of operations for your Model 4P to perform without supervision.
Thorough Documentation. Model 4P comes with Microsoft Disk BASIC and TRSDOS 6 operating system, owner and programming manuals, reference card, and an introduction to your copmuter that gets you started immediately.
Who is the Model 4P for? This transportable computer is designed for managers with work that just can’t wait until they return to the office. The compact size of the Model 4P makes it usable anyplace where there’s an AC outlet. Its software flexibility allows almost any task at hand to be performed – word processing, accounting, forecasting, programming, graphic presentations and much more.
Microprocessor: 4 MHz Z-80A (2 MHz in Model III mode).
Memory: 64K RAM, expandable to 128K. Provides for disk drive emulation in RAM, only 64K addressable from BASIC.
Video Display: 80 x 24 (Model 4 mode), 64 x 16 (Model III mode), or double-width 40 or 32 characters per line. Upper and lower case. Reverse video (Model 4 mode). 96 text, 64 graphics and 96 “special” characters.
Disk Drives: Two built-in double-density 184K 5-1/4″ drives.
External Connections: Parallel printer port. RS-232C serial port. Selectable 300-9600 baud. Buffered input/output bus.
Dimensions: 16-1/2 x 13-1/4 x 9-3/4″.
[RSC-12] Now setting up a Network 3 system is easier than ever. This fully-configured student station comes complete and ready to use. Students can load the program they want from the host disk with just a few keystrokes. Student programs can access disk files on the host for storage or retrieval. U.L. listed.
Includes 64K Memory, Built-In RS-232C Interface and Cable.
[RSC-12] An affordable personal computer that’s ideal for beginners and pros alike – easy to use and hard to outgrow. Add a cassette recorder and put the Model 4 to work or play with our library of ready-to-run software. Set up a budget, compose music, create graphics, teach your kids, play games and much more. Model 4 can be used to analyze statistics or turned into an incredibly low-cost word processor. Other languages and utility programs ar also available. An easy-to-follow beginner’s manual makes it easy to learn BASIC programming. Add a printer, plotter, RS-232, telephone interface and more as your needs grow, plus you can expand into a powerful disk-based system. U.L. listed.
From B-19: The 16K TRS-80 Model4 is the ideal choice for anyone who wants a computer that can later be expanded for advanced programming, business or professional use. It stores programs and data on inexpensive audio casselle tapes (recorder not Included) instead of disks. Learn BASIC programming with the excellent Model 4 beginner’s manual. Choose from our wide selection of ready-to-run software. Set up a household budget. track your stock Investments, teach your kids, and much more! The self-contained white cabinet includes a high resolution 30.5cm monitor that displays 16 lines of 64 characters, a 70-key typewriter-style keyboard with 12-key keypad for numeric entry, parallel printer interface and Model III BASIC in ROM. And as your computing needs grow, upgrade your cassette system to disk-based Model 4 features with a 64K RAM kit and Model 4 drive upgrade kit (intallation required – notincluded).
From B-19: Our 64K two-disk Model 4 is everything you want in a personal computer. There’s a high-resolution 80 x 24 display, keyboard with numeric keypad and CONTROL, CAPS and 3 function keys, new high clock speed (4MHz), parallel interface, and much more. It comes ready to use with thousands of programs, because it’s compatible with Model 4 and Model III TRSDOS, CP/M-Plus, and LDOS operating system. All TRS-SO programs written for the Model III will run without any changes. The 64K (64,000-characters) internal memory is expandable to 128K. To speed operations, there’s “MemDisk”, which lets you create a super-fast disk drive in memory for keystroke-quick responses instead of timeconsuming disk access. TRSDOS 6.0 features a print spooler and communications program. The new Microsoft BASIC includes CHAIN and COMMON for large programs, and more.
$1,299.00 [RSC-12] Completely self contained in a high-impact ABS case with a built-in carry handle. U.L. Listed.
$399.95 Includes a 184K 5-1/4″ double-density disk drive with TRSDOS 6 operating sstem, Disk BASIC and manual.
$199.95 [RSC-19] Each drive adds 184K of data storage to your Model 4. Uses 40-track 5-1/4″ diskettes. Internal Drive #0 required. U.L. Listed.
$279.95 Each drive adds 184K of data storage to your Model 4. Uses 40-track 5-1/4″ diskettes. Internal Drive #0 required. U.L. Listed.
$59.95 [RSC-19] Expands 64K Model 4 to 128K. Required for Model 4 cassette-to-disk upgrade. Extra memory not accessible by BASIC.
$199.95 [RSC-19] Converts Model III disk computer to a Model 4 disk system (except for cabinet). Includes new keyboard, 64K RAM, TRSDOS 6 diskette with Disk BASIC.
Works on AC or four “C” batteries (not included). With battery/record LED. 10-1/2 x 5-3/4 x 1-13/16″. U.L. Listed.
Our most compact, lowest-priced recorder. Features volume control with repset and monitor switch. 7-1/8 x 4-11/16 x 15/16″. Requires four “C” batteries or AC adapter (not included).
[RSC-19] An easy way to create business graphics, tables, charts, maps, animation and more. 640 x 240 resolution. Requires a minimum 64K 1-Disk Model 4. Installation required (not included).
B-19: Create sophisticated business graphs, tables, charts … even animation. Provides 153,600 picture elements (640 x 240) for amazing detail. The Graphics BASIC command structure is incredibly powerful (draws lines and circles, fills spaces). yet easy to master. Also lets you use assembly language subroutines. Includes user’s manual and diskette with Graphics BASIC and subroutine library. Available January 1984.
Model 4P Modem Board 26‑1084 $149.95 Direct-connect auto-dial/auto-answer 300 bps modem features full/half-duplex and originate/answer modes. Completely software compatible with our Direct-Connect Modem II (26-1173). User installable.
$49.95 [RSC-19] Allows you to communicate with information networks and other computers by telephone. Send/receive electronic mail, access builletin boards, and much more. Requires modem and software. Installation required (not included).
Model 4 5MB Hard Disk Kit (Primary) 26‑1130 $3,999.00 Get high-speed access to five million characters of programs and data. Fully compatible with most Model 4/III software. Store over 1,000 different files or create files as large as available space. Backup to multiple floppies. Primary drive includes Hard Disk Operating System with BASIC.
Model 4 5MB Hard Disk Kit (Secondary) 26‑1131 $3,499.00 Add up to three for a total of 20 megabytes storage.
Model 4 15MB Hard Disk Kit 26‑1134 $79.95 Includes cable, software and manuals for use with either our 15 or 10-Megabyte Hard Disk System. Requires 64K 1-Disk Model 4.
Orchestra-90 26‑1922 $79.95 Create highly sophisticated electronic music and sound effects. Both musicians and non-musicians can compose in six octave ranges in up to five voices. Transcribe sheet music, too. Includes cassette software and an interface that connects your home stereo to the Model III/4 computer. Plays programmed sounds only.
Model 4P Travel Case 26‑1085 $24.95 Heavy-duty black vinyl case resists scuffs and tears. Foam-padded bottom protects against drops. Inner pocket for manual and diskettes.
Network 4 Operating Software. 26-2773 $230.00 The Network 4 Shared Learning Svstem will add flexibility and economy to your computer lab or classroom. Our new Network 4 System incporates some of the most advanced networking techonology available to make shared computer learning possible for large groups. Using a twisted pair cable, the Network 4 lets you connect up to 63 TRS-80 Model III or Model 4 computers to a central host computer with a hard disk drive. The student stations can then share the hard disk, each station operating virtually as a stand-alone system. Each user can quickly access a set of teacher-designated programs and data from the hard disk. Information can be sent from station to statsion without using the hard disk and reports can be printed on a printer at the host computer.
How it Works. The storage capacity of the host computer’s hard disk drive is divided into “volumes” to serve as diskettes for storage by the individual stations. Students in the network can use up to four of these volumes at any time for class activities. Teachers use the system manager’s program to create student “accounts.” These accounts allow students various levels of access to the teacher-specified volumes.
Network 4 System Requirements. A Network 4 system requires a Model 4 host (requires Master Upgrade Kit), a 15-megabyte hard disk, Network 4 Operating Software, cables and 64K Model 4 student stations. Model III or Model 4 systems can be upgraded with a Student Station Upgrade Kit.
Network 4: Master Upgrade Kit 26-1136 $299.95 Requires Installation (not included).
Network 4: Student Station Upgrade Kit 26-1137 $299.95 Requires Installation (not included).
Network 4: 100-Foot Trunk Cable 26-1218 $16.95 Includes termination.
Network 4: 500-Foot Trunk Cable 26-1214 $69.95 Includes termination.
Network 4: 1000-Foot Trunk Cable 26-1215 $119.95 Includes termination.
Target PlannerCalc 26‑1512 $99.95 Now you don’t have to learn to program to use a spreadsheet! PlannerCalc’s easy-to-use design eliminates the need for an extensive user’s manual. Create your own financial models by entering and processing data by column, row, or individual location. The program offers you a wide variety of mathematical operations, including conditional commands. And best of all PlannerCalc is a spreadsheet program that uses plain English formulas instead of the more complex algebraic formulas. For example, an obscure equation line “A=500” cabe be written as “LINE 1 WIDGETS = 500.” And a “Help” screen that details an operation is available by simply pressing the question mark key. You can vary the width of columns from 2 to 30 characters and split the display horizontally or vertically for easy data comparison.
pfs:report 26‑1517 $99.95 Produces reports from files created with pfs:file.
pfd:file 26‑1518 $124.95 Create and organize your records according to your own specifications! With pfs:filem you get an easy-to-use personal filing program that works like a paper filing system but without the paper! You decide how your records are to appear by simply organizing information on “forms” that you design right on your display screen. Form layouts can have as much or as little structure as you wish. The advantage? Store, retrieve and review information in a fraction of the time it would take with a conventional filing system. Once you’ve created the basic form, all you do is fill in the blanks. Enter data in any order that you like. Retrieval is not limited by the order in which the information is stored. And you can change the original form design without having to re-enter the data. pfs:file can be used in various applications: business, professional, home and education.
[B25] This versatile management and engineering planning tool takes advantage of Model 4’s 128K memory capacity and 80 x 24 display screen to handle large spreadsheet analysis problems. You simply define the formulas, enter the figures and VisiCalc performs all calculations automatically. Change any value or formula, and VisiCalc instantly updates every related number! You can view countless ‘what if … ? ‘ situations in seconds. Enhancements include an entry editor to modify formulas and labels without reentering them, and logical and relational operators to perform testing and branching. Optional printer allows printing of any portion of your worksheet.
Formation Forms Management System 26‑1527 $199.95 Now you can quickly and easily complete such forms as insurance clalms and loan applications. It’s the perfect solution for businesses that continuously filling out preprinted forms. To start, you first “create” the form on the display by typing in the questions and setting the print positions of the answers. Then each time you use this form. you’ll merely type in the answers on the display screen. Your answers are then printed on the preprinted form in perfect position!
$199.00 An incredibly easy-to-use decision support tool. Multiplan lets you consolidate related worksheets so that information is transferred between them automatically. All prompts are full length words or phrases. Plain English names can be assigned to any column, cell or area. If you have any questions, an On-Line Reference Guide knows where you are in Multiplan and offers information related to your problem. With Multiplan, up to eight display windows can be created on the screen simultaneously. Sorting can be performed in alphabetical or numerical order. Vary individual column widths, center numbers and text within a column and align decimals. Format your own reports, too. Multiplan can also directly utilize your current VisiCalc files. Includes tutorial manual.
Apartment Management System 26‑1538 $299.95 An efflclent and easy-to-use system designed to provide property owners and managers with rapid and accurate monitoring of apartment complexes. Maintains a history of prior payments for each apartment. Divides monthly rental charges into user-defined categories. Produces many financial and problem reports on reauest. Provides a printout of all transactions that can be used as an accounting audit trail. Provides month-end income totals. Maintains a history or repair orders and other notes regarding maintenance on apartments. Stores up to 450 apartment units on each diskette. Prints mailing labels. Requires Model 4 with three floppy drives or Hard Disk.
W-2 Writer 26‑1539 $49.95 For use with the Payroll system. Using the information already collected on the Pavroll data diskette, W-2 Writer saves vou time by printing W-2 forms customized to fit your needs.
General Ledger 26‑1540 $199.95 Makes small business accounting procedures faster and easier. Maintains accurate records of all bookkeeping transactions and provides a balanced ledfer. Use General Ledger alone or interface with the Accounts Receivable, Accounts Payable and Payroll programs for a complete, interactive accounting system. Handles up to 100 accounts and 1150 monthly transactions. Generates easy-to-read financial reports, including Trial Balance (with or without a worksheet), Income Statement, Balance Sheet, General Ledger, Account List with all accounts (or a specified range of accounts and their current-period and year-to-date balances. Posted Transactions Report and Unposted Transactions Report for the current period. Computes up to five levels of totals and two levels of headings for financial statements. Provides each accoun’ts end-of-period balance for the previous fiscal year’s 12 periods, as well as each account’s end-of-period balance for all complete periods of the current fiscal year. Lets you compare current year figures with previous year figures on the same balance sheet or income statement.
Accounts Receivable 26‑1541 $199.95 A balance forward system which provides rapid and accurate monitoring of sales and incoming funds. Interfaces with General Ledger – all transactions are summarized and the totals are automatically posted to General Ledger. Offers seven standard General Ledger accounts, plus 10 user-definable accounts. handles up to 2000 transactions per month with up to 150 customers on file. Reports include Unposed Transactions, Posted Transactions, Customer List, General Ledger Account List, and Aging Report on selected customer accounts. offers optional use of minimum payment amount on statements, and optional late charge processing (compounding option also available). You can print mailing labels, statements, an Aged Trial Balance for audit purposes and a General Ledger report.
Accounts Payable 26‑1542 $199.95 Automatically posts transactions and payments to General Ledger program. Handles up to 1100 transactions, 75 vendors, five General Ledger expense distributions per invoice and 14 invoices per check. Includes four General Ledger accounts, plus 10 user-definable accounts. Calculates due dates, discount dates and discount amounts by vendor payment terms, Prints Uposted Transactions List, Transaction Posting Report, Vendor List, Open Items List, Cash Requirements Report, Vendor Mailing Labels, G/L Account List, G/L Distribution Report, Check Review Report (for verification before check printing), Checks, Check Register and Payment Posting Report.
Payroll 26‑1543 $199.95 Tracks earnings, deductions and taxes for each employee. as well as employer contributions such as FICA, FUI, SUI and more. Interfaces with the General Ledger program. You can customize your system in accordance with local, state and federal tax laws. Processes up to 80 employees, each with up to nine earnings and nine deductions categories. Handles payroll tasks for up to three states simultaneously. Earning/Deduction types allow changing amounts for employees, taxed or exempt. Also define categories for FLSA and EEOC reporting, keep historical records by period, quarter and year, and more.
Invoice Writer 26‑1544 $49.95 Provides a rapid and accurate method of creating invoices using the up-to-date information contained in the Accounts Receivable system. Automatically computes taxes and discount rates that are defined by you. Requires Accounts Receivable.
Inventory Control 26‑1545 $199.95 Designed to make inventory control easler and more accurate. Produces a reorder report showing items below assigned reorder level plus an on-hand listing of items. Prints ten major reports, plus nine custom reports designed by you. Prints purchase orders and labels. Allows physical inventory count to be taken by cycle and lets you automatically reorder stock. Provides customized reports using the SORT utility. Processes up to 100 vendors, 50 purchase orders, 1200 inventory items, and 350 purchase order line items.
Enhanced VisiCalc 26‑1569 $99.95 This versatile management and engineering planning tool takes advantage of Model 4’s 128K memory capacity and 80 x 24 display screen to handle large problems. You simply define the formulas, enter the figures and VisiCalc performs all calculations automatically. Change any value or formula, and VisiCalc instantly updates every related number! You can view countless “what if. . . ?” situations in seconds. Enhancements include an entry editor to modify formulas and labels without reentering them, and logical and relational operators to perform testing and branching. Optional printer allows printing of any portion of your worksheet.
SuperSCRIPSIT 26‑1595 $199.00 Includes all the word processin features of Model 4 SCRIPSIT and more. SuperCCRIPSIT supports proportional-spaced printing, multiple line spacing, underline, boldface, super and subscripts, multiple columns, full 80 x24 display format and reverse video to aid in editing.
Disk Scripsit 26‑1596 $99.95 A low-cost. easy-to-use word processing program for Model 4. Lets you prepare memos. letters, reports and manuscripts right on the screen – displays a full screen of text in an 80-character by 24-line format. Multiple edit features let you delete, insert and move blocks of text. Then print as many correction-free copies as you wish with an optional printer. lncludes all the standard features that have made SCRIPSIT an outstanding word processing program for years. There’s a “global search” mode to find phrases or sentences that you want to alter or delete. Also features reformatting, hyphenation, centering, headers, footers, justification, margin and tab settings, automatic page numbering and more. With comprehensive learning manual and quick reference card.
Videotex Plus 26‑1598 $49.95 Videotex Plus is composed of three major modules: the primary communication program, a specialized program for use with “store and forward” information services and a module that lets you create and edit log-on sequences and save them on diskette for later use with the special automatic log-on procedure.
SCRIPSIT Spelling Dictionary 26‑1600 $99.95 Automatically checks text prepared with SuperSCRIPSIT for misspellings. Contains 73,000 words. Add your own user list.
Profile 4 Plus Filing System 26‑1635 $249.95 Create your own personallzed electronic filing system! Profile 4 Plus offers you many possibilities for manuipulating data you enter. You can store almost any type of information, then retreieve it at the touch of a key. Managers can keep track of price lists, business inventories, vendor and customer records, personal projects and more! And this enhanced version of Profile for the Model 4 has many new features that make it easy to develop your own specialized system. A full-screen Define Files program makes it easy to create and change fields, even after the file is expanded. Sort and select records by any field in any segment of the file. And sorting is extended over all available disk space – not just RAM. Features indexing for faster sorting and selection. Sort by up to five fields – in ascending or decending order -to a maximum of 85 characters. And you can select records using up to 16 dlfferent search criteria. Mass operations are accessed via indexes. as well as the regular scan operation. Indexes for report and label printing, and SuperSCRIPSIT and VisiCalc selections. A built-in math package can perform arithmetic operations – such as calculating taxes, extensions and totlas – among several fields and then update another field automatically. Stores up to 700 records of 255 characters in length on a 2-disk data base. 2900 with 4 disks. Create up to 36 different reports and 36 different sets of mailing labels for each file. Design reports and labels up to 200 characters across and 99 print lines down. Up to ten different screen formats for each file. Up to 36 SuperSCRIPSIT and VisiCalc spin-off file formats per file for reports and form letters. Comes with easy-to-understand tutorial manual.
TK!Solver 26‑1630 $299.95 Makes it easy to solve mathematical problems. You simply enter one or more equations and then the program solves them. Ail the tools needed for problem-solving are built into the program, including matnematical functions, facilities for converting units of measurement and the ability to produce graphs and tables (with optional printer). TK!Solver can solve a broad range of problems using models and equations with variables of known and unknown values. Given a model and known values of variables, the TK!Solver program solves for as many unknown values as is possible. The program’s problem-solving ability is powerful and versatile enough to apply to many fields, from fianncial planning to architectural design to chemical analysis. You can also use it in conjunction with the SolverPacks below for special applications. Requires 128K Model 4.
Finance Solverpack 26‑1631 $99.95 For use with TK!Solver for problems in financial analysis. The topics include conpound interest, net present value, level debt service, cost of equity capital, analysis of financial statements, stock option pricing, bond swaps, convertible debt and bond refunding decisions. Requires TK!Solver.
Science Solverpack 26‑1632 $99.95 Covers a wide range of tipocs in introductory science at a level comparable to American secondary schools. Covers topics in chemistry, biology, physics, thermodynamics and population studies. Requires TK!Solver.
Mechanical Engineering SolverPack 26‑1633 $99.95 The topics include analysis of elsatic beam bending, springs, area moment of inertia, Mohr’s circle, rotational systems, heat transfer, fluid flow and hydralics. Requires TK!Solver.
13 Ghosts 26‑1927 $19.95 Armed only with your “Ghost Blaster”, you’re out to de-spook a Ghost Town. You’ll encounter a skeleton that’s immune to your blast and a “laughing ghost” with an annoying knack for waking up dangerous ghoul friends. You’ll be lucky to get out alive!
Computer Diplomacy 26‑1957 $19.95 A realistic game of strategy and international politics. You command one of the seven great powers of Europe prior to World War I. You seek to control the continent through careful planning and by negotiating alliances with other players. For up to seven players.
Disk $19.95 A licensed version of the original arcade game. Get your frogs safely across the road. Then guide them to their lily pads on the other side of the river. Optional computer-generated music requires mini amplifier.
Assembly Language Development System 26‑2012 $149.00 Powerful package that’s a necessity for the assembly language programmer. With full screen Editor, Assembler with sorted cross-reference listing, Linker for creating an absolute file, atuomatic File Transfer, and Debug modules. offers numerous important and helpful features and includes TRSDOS 6 and Model III TRSDOS 1.3 support.
TRSDOS 6 Training Course 26‑2019 $749.95 A comprehensive course in using TRSDOS 6 on your Model 4.
BFBLIB BASIC Routines Diskette 26‑2021 $19.95 Supercharge your programs with these 121 subroutines, functions and utilities described in BASIC Faster and Better & Other Mysteries. Modify routines or use “as is”.
Construction Estimator 26‑2025 $49.95 Provides builders, appraisers. real estate developers and insurance adjustors with time-saving and competitive building information. Includes three templates for use with VisiCalc on one diskette: Quantity Suwey (cost) Method. Square Foot Method and Unit-in-Place Method. Estimates construction costs. property values and individual component costs. Compares material and lumber suppliers, material and cost differences, plus actual and estimated costs. Automatically calculates cost difference between suppliers, percent of monies expended, completion percentages, savings realized by item. individual item extensions, cost savings realized by item, individual item extensions, cost savings and/or cost overages. Features automatic recalculation of entries and customization of worksheet to your specific needs. Provides for tax calculations, sales commissions, loan points, bank draws. Home Owner Warranty. advance requested, plus many personalized categories. Gives you more cost-effective and efficient use of your bidding time.
Graphics-90 26‑2027 $39.95 A complete graphics and animation program. Now you can easily create your own games in BASIC that are equal to many machine-language games! Create state-of-the-art pictures and animation sequences with your Model III or 4 and display the images in random or sequential order. Print graphics on any Radio Shack dot-matric printer with bit-image capability. Two disk drives are recommended for best graphics.
COBOL-4 26‑2203 $199.95 Based on the ANSI ’74 standard, COBOL is an English-oriented programming language designed primarily for developing business applications on computers. Features single-step compiler multi-key ISAM, COBOL editor and DEBUG> A listing of the program is generated showing the original source statements, error information, data allocation, interactive DEBUG information and optional cross-reference of all program labels and data items. Includes 297-page manual.
BASCOM-4 26‑2210 $199.00 Feature packed for fast compilation of Model 4 BASIC programs. BASIC compiler lets you execute faster than the same interpreted programs, and require less memory. Includes compiler, linker routine library and run-time package. Source-code security is provided.
Alcor Pascal 26‑2212 $249.95 Alcor Pascal is a full Pascal implementation. For speed and efficiency, the compiler generates a pcode (pseudo code) that can be interpreted, or further translated into machine instructions for execution directly by the hardware. For added space, an optional pcode optimizer reduces a program by 10-30%. For added speed, an optional native code generator converts the p-code to machine language. Resulting code executes between three and five fimes taster than the pcode. Object code is portable, so you can compile programs on one system and execute them on another. Includes compiler, interpreter, linking loader for building libraries of commonly-used routines, full-screen text editor. large library of routines, extensive string routine library, and a documentation package which includes a Pascal tutorial with 500-line data base program.
CP/M Plus 26‑2216 $149.95 A valuable addition to Model 4, CP/M Plus is designed for the business user. It supports thousands of existing CP/M programs, including popular word processing, spreadsheet analysis and database management programs. CP/M Plus is easy to learn and even offers a HELP facility. It’s upward compatible with CP/M 2.2, but has many new features – including a high-performance file system. In addition, CP/M Plus automatically logs in a disk when it detects a media change. For easy file selection, time and date stamping options allow the user to record in the disk directory two entries for time and date stamps for each file. No additional hardware required.
CBASIC 26‑2217 $99.95 A super-efficient business language that complements application development under CP/M Plus. CBASIC works as a complier/interpreter that uses random access memory more efficiently. That means more memory for the application program. Source code is compiled by CBASIC into an intermediate file composed of psuedocode (p-code) instructions. During program execution, the CBASIC run-time monitor interprets each P-CODE instruction and performs the operation. CBASIC provides two types of files for maximum flexibitlty: fixed record length files and stream input/output files. CBASIC also includes native code compilation, relocatable code, convenient linkage to assembly programs, string lengths to 32,000 bytes, debugging capabilities, cross reference lister, support for multi-user operating systems and more.
FORTRAN-4 26‑2219 $99.95 Write programs in FORTRAN using EDIT-80, a powerful editor with commands for interline and intraline editing. Compiler reads your FORTRAN source program from diskette, translates it into relocatable object code and saves it on diskette. Linker lets you input the relocatable object program, execute it, and save it on diskette as a TRSDOS command file. Assembler lets you create relocation assembly language subroutines that you can link to your FORTRAN program. Includes 250 page manual.
TRS-80 Model 4 C Programming System 26‑2230 $249.95 A complete implementation of C language for program development. The library routines are Unix-compatible, making it easy to transport programs from other computers to the Model 4 – or vice versa. Includes a powerful, programmable full-screen text editor. You can map editor commands to the Model 4 keyboard as desired and define your own editor commands. The TRS-80 C compiler generates a very efficient and compact object code. TRS-80 C programs will execute 10 to 50 times faster than BASIC programs. The compact size of the object code lets you develop large programs without overlays or chaining. Also compatible with TRS-80 Pascal. You can call functions or procedures written in Pascal from a TRS-80 C program or vice versa.
TRSDOS 6.2 26‑316 $39.95 Operates 20% faster than TRSDOS 6.1 Features a HELP utility for all commands. Features user-definable system calls and many technical enhancements. Includes replacement pages for the Technical Reference Manual.
TRSDOS 6.2 Utilities 26‑315 $39.95 Includes Quick Format and Backup that lets you make a backup in less than one minute. The BASIC Answer which allows use of labels instead of line numbers in BASIC while programming; Mod 324 which simplifiesconversion of Model III BASIC programs for Model 4 BASIC; UNKILL to recover killed porgrames; BSORT for sorting from BASIC with tax variables; and COMP which compares two disks or files for a match. Requires TRSDOS 6.2.
DoubleDuty 26‑2231 $69.95 Divides your computer’s 128K memory into three complete and and independent partitions. Partition one and two each operate like a 64K Model 4. You can load and execute any Model 4 program designed to run on a 64K system into each partition. You can also run BASIC or TRSDOS commands in either partition. In addition. you can use Partition 3 to execute TRSDOS library commands. It’s like having two separate Model 4’s! Since most Model 4 programs require no more than 64K, DoubleDuty takes advantage of your system’s increased memory capacity. And because DoubleDuty takes up almost no memory itself, it is able to more than double the system’s operating potential. For example, you could load the SCRIPSIT word processing program in Partition 1, and an electronic filing program in Partition 2. While typing a document in SCRIPSIT, you discover that you need data stored in the file program – you simply move to the file program, find the information, and return to SCRIPSIT. You may reference the TRSDOS directory for information – all without disturbing the your document. Designed to sue your fill memory capacity for convenience and speed. Requires 128K Model 4 or a Model III modified with Model 4 Upgrade Kit and the Model 4 64K RAM Kit. DoubleDuty may not be compatible with all Model 4 programs. Communications may not be used with DoubleDuty.
Font Editing Package 26‑5334 $24.95 Use your TRS-80 Model 4 to create your own type styles that can be printed with the DMP-2100P. Easy-to-use package includes loading and editing programs, plus utilities to customize your own drivers. Starter fonts include Letter Gothic, Bold PS, Italics PS, Italics Micro, Modern Micro, Cursive Script, Olde English, Games (chessboard and men; card suits), and Shadow (8-pitch block characters).
Keyboard Cover 26‑1342 $12.95 Protect your Model III or 4 keyboard from dust and dirt when not in use.
CR-510 Card Reader 26‑1266 $1,595.00 [RSC-12] Administrators, teachers and students can benefit from this TRS-80 input device. Use with Radio Shack School Adminuistrative Softwar Product Series to expedite administrative chores, grade, attendance and scheduling functions. The CR-510 provides single, demand or continuous feed operation and is controlled through manual switches or software. Cards must be at least 6 inches in length, and can be either marked or punched (with standard keypunch). Special-oprder Card Holder allows cards up to 14 inches to be read. Included with the CR-510 is a comprehensive owner’s manual, diagnostic test card, packet of 200 general purpose cards, and diskette with COBOL and BASIC driver routines. Requires TRS-80 computer with RS-232C port. Cables available separately.
Model 4 Technical Reference Manual 26‑2110 $24.95 Provides technical information and theory of operation on Model 4 hardware. Includes vital information for the assembly language software developer or user, including SVC calls, file formats and much more.
Model 4/4P Disk System Owner’s Manual 26‑2117 $17.95 Covers initial setup, BASIC and applications programs. Also includes documentation for TRSDOS Version 6 commands.
Model III/4 Owner’s Manual 26‑2112 $5.95 Provides detailed information on Model III and Model 4 operation. Including BASIC language. 259 pages.
Looking for pinout of the RS232 port on the bottom of a TRS80 Model 4, and the parallel port on the some machine.
There are two ports (edge connectors) on the bottom, one printer, the other serial.
Would you happen to have that info?
Fun perusing your website. I am a TRS80 Model IVD user (active)with MFM Hard drive in custom build set up. I aquired the inventory of 2 RS vendors years ago thus have a life long supply of Hardware. Software, we’ll I write my own in BASIC to run my office. MY biggest concern is a printer. Several years ago I found a replacement printer (DWP 410 …which is one of the fastest daisy wheel printers you’ll ever see) while searching the web and it is still huumming along. I recently had to re-ink the ribbons and I was successful.
Anyone is welcome to contact me if you need help tracting down “working” parts (hardware, software and OS).
I have a model 4P and what I believe is a Radio Shack 5MB external disk (not the white one, black).
ROM’s, software or hardware or is all that’s needed a cable?
Feel free to email me at caprio (at) uxpro.com if you know how to do this.
I have a 4P that I would donate to a hobbiest. All I want is the cost of shipping. It was operational when it was put in storage. I may have disks somewhere? If I can find them I’d throw them in.
Amazing stuff, owned the silver Co-Co II and the model IV.
…just a crazy (nutbar) dream… Have a great day fans!
I just bought a TRS-80 model 4.
I formatted a disk in drive#1 while keeping TRSDOS in drive #0.
After writing a program in BASIC, I was unable to save it to a disk in the destination drive#1??
Do I need to specify the drive (if so, how?); I typed Save “name of file”.BAS, but it did not work….
I was also unable to make back-up disks of TRSDOS??
Do you have any copies of TRSDOS that I can buy??
Wenn die Energie umgewandelt wird, dann kann sie nicht in den Fettdepots gespeichert werden.
wieder etwas zugenommen und bin jetzt bei etwa 68 Kilo, was mich wirklich stört.
Years ago I purchased a model 4 serial number 0001932. Unfortunately the case was cracked and the crt cathode damaged. I am in the process of re-building this 128k computer. Since the serial number is so low, I would like to find a replacement tube and analog video board. This computer has the board wired to the crt so I assume I would need both.
Whom could I contact for these parts?