Abstract
Protein structures are dynamic and can explore a large conformational landscape1,2. Only some of these structural substates are important for protein function (i.e. ligand binding, catalysis and regulation)3–5. How evolution shapes the structural ensemble to optimize a specific function is poorly understood>3,4. One of the constraints on the evolution of proteins is the stability of the folded ‘native’ state. Despite this, 44% of the human proteome contains intrinsically disordered (ID) peptide segments >30 residues in length6, the majority of which have no known function7–9. Here we show that the entropic force produced by an ID carboxy-terminus (ID-tail) shifts the conformational ensemble of human UDP-α-D-glucose-6-dehydrogenase (hUGDH) toward a substate with a high affinity for an allosteric inhibitor. The function of the ID-tail does not depend on its sequence or chemical composition. Instead, the affinity enhancement can be accurately predicted based on the length of the ID segment and is consistent with the entropic force generated by an unstructured peptide attached to the protein surface10–13. Our data show that the unfolded state of the ID-tail rectifies the dynamics and structure of hUGDH to favor inhibitor binding. Because this entropic rectifier does not have any sequence or structural constraints, it is an easily acquired adaptation. This model implies that evolution selects for disordered segments to tune the energy landscape of proteins, which may explain the persistence of ID in the proteome.
ID segments can exhibit complex functions such as ligand binding, the scaffolding of multi-protein complexes and mediating allosteric regulation14–18. However, many ID segments are assumed to be nonfunctional and are often removed from proteins to facilitate structural studies. For example, the 30-residue disordered C-terminus of hUGDH (residues 465–494) is often removed with no apparent impact on kinetic parameters19. Here, we show that this C-terminal segment (called the ‘ID-tail’) plays a novel role in the allosteric mechanism of hUGDH. hUGDH catalyzes the NAD+-dependent oxidation of UDP-α-D-glucose (UDP-Glc) to UDP-α-D-glucuronic acid19 and is regulated by the allosteric feedback inhibitor UDP-α-D-Xylose (UDP-Xyl)20,21. Three hUGDH dimers associate to form an inactive hexamer (E*)22–26 (Fig. 1a, b). The binding of substrate induces an allosteric switch (T131-loop/α6 helix) in the E* hexamer to isomerize into the active state (E)22,23,26,27 (Fig. 1a, c-e). The allosteric inhibitor UDP-Xyl competes with UDP-Glc for the active sites, and upon binding, triggers the allosteric switch to produce the inhibited state (EΩ)22,24,25,27. This inhibition mechanism is atypical in that the active site also functions as an allosteric site to control the structure and activity of the hexamer22–27 (Fig. 1a, c-e). The EΩ state has a high affinity for UDP-Xyl and a low affinity for UDP-Glc22,27. Because of this, the allosteric transition of the inhibited EΩ hexamer to the E state can be observed as cooperativity in substrate saturation curves22,27. We compared the structure and activity of full-length hUGDH (hUGDHFL) to a construct lacking the ID-tail (hUGDHΔID). The structures of E* states of hUGDHFL and hUGDHΔID were solved in isomorphous crystal lattices and revealed no significant differences (Extended Data Fig. 1a-c). hUGDHFL and hUGDHΔID also have a similar kcat and Km for both substrate and coenzyme, consistent with earlier reports19 (Extended Data Table 2). In contrast, the allosteric response is sensitive to the ID-tail; deletion of the ID-tail reduces the affinity for UDP-Xyl by an order of magnitude (Fig. 1f). The fact that inhibited hUGDHΔID still binds UDP-Glc cooperatively shows that the deletion of the ID-tail reduces UDP-Xyl affinity but does not prevent the formation of the EΩ hexamer (Fig. 1f and Extended Data Fig. 2a, b).
Both the ID-tail and the α6 helix of the allosteric switch are located in the hexamer-building interface between adjacent dimers, suggesting that these two elements may work together to increase UDP-Xyl affinity (Fig. 1b). We used the allostery quenching A136M substitution to see if the ID-tail functions independently of the allosteric switch; this substitution has been shown to lock the allosteric switch and the hexamer in the low UDP-Xyl affinity, E state22. Inhibition studies show no significant difference in UDP-Xyl affinity between hUGDHFL-A136M and hUGDHΔID-A136M, which suggests that the ID-tail requires a functional allosteric switch and the EΩ state to enhance the affinity for UDP-Xyl (Fig. 1f).
The location of the α6 helix in the hexamer-building interface suggests that the oligomeric structure might be important for the function of the ID-tail (Fig. 1b). In fact, sedimentation velocity analyses shows that hUGDHΔID E* hexamer is slightly less stable than the hUGDHFL E* hexamer, which might explain the reduced UDP-Xyl affinity (Extended Data Fig. 3a). We tested the role of the hexamer using the M11 interfacial loop substitution, which prevents hexamer formation and stabilizes the dimer (hUGDHFL-dimer and hUGDHΔID-dimer)27. UDP-Xyl binds to the hUGDHFL-dimer with a 7-fold higher affinity than hUGDHΔID-dimer, which shows that the ID-tail enhanced affinity does not require the hexamer (Fig. 1f and Supplementary Information Section 1).
The ID-tail is highly conserved in vertebrate UGDHs (Fig. 2a). We examined the importance of primary structure by randomizing the native sequence to create two distinct ID-tails (hUGDHR1 and hUGDHR2) (Fig. 2b). Surprisingly, the hUGDHFL, hUGDHR1 and hUGDHR2 constructs have similar affinities for UDP-Xyl (Fig. 2c). Next, all six prolines in the ID-tail were substituted with serine (hUGDH-Pro) (Fig. 2b). Because serine and proline both promote disorder28,29, this substitution will conserve the unfolded state while disrupting any possible proline-specific interactions. Analysis of hUGDH-Pro shows that the prolines do not contribute to UDP-Xyl affinity (Fig. 2c). Since all of the above constructs conserve the positive charge of the native ID-tail (pI = 10.1), we created a negatively charged ID-tail (pI = 4.4) using lysine to serine substitutions (hUGDH-Lys) (Fig. 2b). Despite the charge switch, there is still no significant change in UDP-Xyl affinity (Fig. 2c). Finally, we show that the ID-tail can be replaced with polyserine (hUGDHSer) without significantly changing UDP-Xyl affinity (Fig. 2b, c). Thus, the conserved primary structure is not required for UDP-Xyl affinity but may have been selected for an additional, unrelated function in vivo (Fig. 2a). The absence of sequence constraints argues against any mechanism where the ID-tail specifically interacts with the inhibitor or the protein.
Next, we considered the possibility that the ID-tail might enhance UDP-Xyl affinity through a sequence-independent interaction involving the polypeptide backbone. Because the 6 prolines in the hUGDHFL, hUGDHR1 and hUGDHR2 ID-tails sample 16 unique positions throughout the sequence without altering UDP-Xyl affinity, it is unlikely that a backbone specific interaction is important for function (Fig. 2b, c). Still, if there is a backbone specific interaction, then a plot of affinity versus ID-tail length would reveal a jump discontinuity when the critical segment is removed. Inhibition studies comparing hUGDHFL, hUGDHΔID, and three new constructs with varying length ID-tails (hUGDH2×FL, hUGDH0.5×FL, and hUGDH0.26×FL, hUGDH0.13×FL) show that the affinity can be modeled as a simple exponential decay (Fig. 2b, d). We confirmed that this saturable effect is sequence independent using corresponding polyserine ID-tails (hUGDHSer, hUGDH0.5×Ser, hUGDH0.26×Ser, hUGDH0.13×Ser) and the scrambled R1 construct (hUGDHR1, hUGDH0.5×R1, hUGDH0.26×R1, hUGDH0.13×R1) (Fig. 2d). The fact that the native, polyserine and R1 variable length ID-tails give the same results in the dynamic range of Figure 2d should dispel any concerns of a sequence-specific mechanism. It is notable that hUGDH0.13×FL, hUGDH0.13×Ser and hUGDH0.13×R1 still enhance UDP-Xyl binding affinity; the conformations of these short, four-residue ID-tails are tightly constrained within a surface pocket, which should stabilize any weak structure (Fig. 3a). Still, none of the E, E* and EΩ hUGDHFL crystal structures (42 unique chains) show evidence of an ordered interaction within the pocket (Extended Data Fig. 1 and22,24–26).
The data presented thus far are strong evidence that the high affinity binding of UDP-Xyl is a function of the unfolded state of the ID-tail. An unstructured polymer tethered to a surface generates an entropic force at the point of attachment10–12 that can be strong enough to distort lipid bilayers30 and alter protein stability13. This force originates from the volume exclusion effects of the surface, which reduce the conformational entropy of the attached polymer (Fig. 3b). Since the entropy of the polymer increases with distance from the surface, the entropic force converges to a maximum value as the chain length increases10–12. The unfavorable change in free energy produced by constraining an unstructured, non-interacting peptide (ΔGconstrained) is:
(Equation 1) |
where Ω1 is the sum of all possible states of an unconstrained peptide and Ω2 is the subset of states constrained by the protein surface and the adjacent ID-tail. Using Monte Carlo sampling of coarse grained, sterically allowed ϕ,ψ bins, we calculated the fraction of constrained conformations for various ID-tail lengths (see Methods, Fig. 3b and Extended Data Fig. 5). For this simulation, the adjacent ID-tail was held in a fixed conformation (Extended Data Fig. 5). If the conformational entropy of the ID-tail contributes to the change in UDP-Xyl affinity, then we would expect Ω2/Ω1 and the affinity constant Ki to show similar behavior with increasing length of the tail. Despite the simplicity of the Monte Carlo model, the simulations confirm that Ω2/Ω1 converges as the ID-tail length increases (Fig. 3c).
Previous studies have showed that the entropic force generated by a tethered polymer can alter protein stability13. We have carried out thermal denaturation studies of hUGDH dimers (chosen to avoid complications arising from hexamer dissociation), and we find that the high affinity hUGDHFL-dimer (Ki = 0.17 μM) is less stable than the low affinity hUGDHΔID-dimer (Ki = 1.23 μM) (Fig. 3d). The destabilizing effect of the ID-tail should also be reflected in the structure and dynamics of hUGDH. To examine these changes at the peptide level, we compared the hydrogen/deuterium exchange (HDX) rates of hUGDHFL-dimer and hUGDHΔID-dimer using mass spectrometry. As expected, the fragment corresponding to the ID-tail is fully exchanged in less than 120 s, which is consistent with a disordered peptide31 (Extended Data Fig. 6a). The ID-tail increases the HDX rates of several segments in NAD+ binding domain, with largest increases being observed in the allosteric switch and an adjacent peptide (Fig. 3e-g). An increase in HDX rates for a buried peptide like the allosteric switch and the surrounding segments indicates an increase in the overall dynamics of the domain. This is significant, because the binding of UDP-Xyl induces the allosteric switch and surrounding core residues to change conformation and repack into the high affinity EΩ state22,27 (Fig. 1a, c-e). The ID-tail also decreases the HDX rates of several segments in the dimerization and sugar binding domains, suggesting that these areas become more structured (Fig. 3e, g). The largest decrease is seen for the α9 helix of the dimerization domain (residues 222–240). This helix is largely solvent inaccessible in crystal structures, which suggests that the ID-tail reduces the overall dynamics of the dimer interface (Fig. 3g). Overall, the data show that the cost of constraining the ID-tail destabilizes a low affinity substate, which biases the conformational ensemble toward a structurally and dynamically distinct high affinity substate. A simple exponential fit of the Ω2/Ω1 ratios in Figure 3c shows that the energetic cost of constraining the ID-tail converges to approximately 2.4 kcal mol−1 (Equation 1). Thus, our simple Monte Carlo model supports the argument that entropic confinement effects generate sufficiently strong forces to explain the maximum expected gain in UDP-Xyl binding affinity of −1.45 kcal/mol (Figs. 2d, 3c, and Extended Data Fig. 5). More rigorous calculations on other systems using simpler polymer models (and simpler confinement geometries), also find confinement free energy costs of this same magnitude32,33.
If the ID-tail favors the dynamics associated with the repacking of the allosteric switch into the EΩ state, then we would expect to see a difference in the activation (E* to E) and inhibition kinetics (E* to EΩ) (Fig. 1a). Pre-steady state analysis of progress curves shows that the ID-tail slows the rate of activation hysteresis (E* to E) by 39% (Fig. 4a). Next, we examined the UDP-Xyl induced isomerization of hUGDH to the EΩ state. Transient-state analysis of UDP-Xyl binding kinetics revealed a three-phase exponential decay of hUGDH time-resolved tryptophan fluorescence, and the data were globally fit by computer simulation (see Methods and Extended Data Fig. 7a-e). The same kinetic model produced the best fit for both hUGDHFL and hUGDHΔID and predicts UDP-Xyl affinities that are consistent with our steady state inhibition studies (Extended Data Figure 7):
(Equation 2) |
According to this model, UDP-Xyl binds to the E* state and induces two, sequential isomerizations. Based on the allosteric model, we had expected a single isomerization from E* to the EΩ state (Fig. 1a). We are calling the additional transient E†, which represents an intermediate between the E* to the EΩ states. The ID-tail changes the kinetic parameters of each transient observed in the time-resolved fluorescence (Extended Data Figure 7e). The largest effect of the ID-tail is a 4.4-fold enhancement of the initial UDP-Xyl binding step, corresponding to a −0.9 kcal mol−1 gain in affinity (Fig. 4b). The kinetic model predicts an overall favorable gain in binding affinity of −1.3 kcal mol−1, which agrees well with the observed gain of −1.39 kcal mol−1 (Fig. 4b and Extended Data Table 2). The different stabilities of the corresponding hUGDHFL and hUGDHΔID transients combined with the fact that the ID-tail slows activation hysteresis and accelerates inhibition kinetics, supports our conclusion that the ID-tail alters the energy landscape to favor UDP-Xyl inhibition (Fig. 4d).
Collectively, our data supports a model in which the entropic force of the ID-tail rectifies the energy landscape of hUGDH to favor a substate with a high affinity for UDP-Xyl. We can now interpret the exponential curve in Figure 2d as follows:
(Equation 3) |
i) hUGDH exists as an ensemble of low affinity (Kiunbiased) and high affinity (Kibiased) substates; ii) the ID-tail functions as a length-dependent (l) entropic rectifier that biases (k) the distribution toward the high affinity substate; iii) the observed UDP-Xyl affinity results from a fractional summation of the low and high affinity substates at a given ID-tail length (Fig. 4d). The fit to Equation 3 produces a Kibiased of 0.46 ± 0.18 μM UDP-Xyl, which corresponds to a maximum favorable gain in binding energy of approximately −1.45 kcal mol-1. The lack of sequence constraints implies that the entropic force of any ID-segment is capable of shaping the conformational ensemble of a protein. In fact, an N-terminal hexahistidine affinity tag has been shown to alter the internal dynamics of a myoglobin34. Thus, the persistence of low complexity ID-segments in the proteome may reflect the selection for entropic rectifiers that can tune the function of a protein by shaping the native-state ensemble.
METHODS:
Protein expression, purification, and quantification of hUGDH constructs.
All hUGDH sequences were synthesized and cloned into pET-15b vectors (Norclone). Sequences contained an N-terminal hexahistidine affinity tag adjacent to a tobacco etch virus (TEV) cleavage site. The expression and purification of hUGDH constructs were conducted under identical conditions as previously described22–27. Following purification, the N-terminal hexahistidine tag was cleaved with TEV protease. An additional immobilized metal affinity column (IMAC) was used to obtain the pure, His tag free protein. Unless otherwise noted, all proteins were dialyzed into a storage buffer [25 mM Tris pH 8.0 and 50 mM NaCl] and concentrated to ≥ 20 mg/mL. Proteins were quantified in dilution replicates (N ≥ 6) using their respective molar extinction coefficients that is based on their specific amino acid composition35.
Protein crystallization, data collection, and structure solution.
To crystallize the E* conformation of hUGDHΔID, the protein (10.4 mg/mL) was dialyzed into 20 mM MES pH 5.6, 150 mM NaCl and crystallized at 20 °C using free interface diffusion in a 1.0 mm capillary containing 5 μL of 10.4 mg/mL enzyme and 200 μL of precipitant solution (100 mM MES pH 6.2, 100 mM MgCl2, and 16% PEG 3350). Crystals were cryoprotected in the precipitant solution supplemented with 18% glycerol and then plunged into liquid nitrogen. A 2.64 Å resolution data set was collected on the 22-ID beamline (SER-CAT) at the Argonne National Lab using an MAR 300 mm CCD detector. The data were processed in space group C2 using XDS36 and 5% of the data were set aside for cross-validation37. The crystal parameters and data collection statistics are summarized in Extended Data Table 1. The structure was solved by molecular replacement using the PHENIX software suite38 and hUGDH (PDB entry: 3TF5) as a search model. The structure was then subjected to iterative cycles of manual rebuilding using COOT39 and automated refinement using PHENIX with both NCS restraints38,40. B-factors were refined using TLS as implemented in PHENIX. Refinement statistics41,42 are summarized in Extended Data Table 1.
The EΩ hUGDHFL was crystallized in the presence of 5 mM UDP-xylose and 10 mM adenosine di-phosphate at 25 °C using the hanging drop vapor diffusion method. 1 uL of protein was mixed in a 1:1 ratio with reservoir solution (0.1 M HEPES pH 7.2, 14% 1–6-hexanediol, and 10% PEG 3350). Crystals were cryoprotected in the precipitant solution supplemented with 20% glycerol and then plunged into liquid nitrogen. A 2.0 Å resolution data set was collected on the 21-ID beamline (SER-CAT) at the Argonne National Laboratory (Argonne, IL) using a MAR 300 mm CCD detector. The data set was processed using XDS36 and 5% of the data were set aside for cross validation37. The data collection statistics are listed in Extended Data Table 1. The EΩ hUGDHFL structure was solved by molecular replacement using the Protein Data Bank (PDB) entry 2Q3E as a search model in PHENIX38, and refined as described above. Refinement statistics41,42 are summarized in Extended Data Table 1.
Steady state kinetics.
All steady state kinetic assays were conducted as previously described22–27. Briefly, assays contained either 100 nM hUGDH [FL, FL-A136M, ΔID, ΔID-A136M, R1, R2, -Pro, -Lys, 0.13×FL, 0.26×FL, 0.5×FL, 2×FL, 0.13×Ser, 0.26×Ser, 0.5×Ser, Ser, 0.13×R1, 0.26×R1, or 0.5×R1] or 500 nM hUGDH [FL-dimer, ΔID-dimer] in a standard reaction buffer [50 mM HEPES pH 7.5, 50 mM NaCl, and 5 mM EDTA] with either saturating amounts of NAD+ or UDP-Glucose (purchased from Sigma). Substrate and enzyme were incubated separately at 25 °C for 5 minutes, and then reactions were initiated by rapid mixing of both solutions. Progress curves were obtained by continuously monitoring NADH production at 340 nm (molar absorptivity coefficient of 6220 M−1cm−1) on an Agilent 8453 UV/Vis spectrometer equipped with a Peltier temperature controller (25 °C). hUGDH progress curves display hysteresis, thus the observed initial velocity (vi) represents a transient and does not satisfy steady state conditions. To obtain steady state initial velocities (vss), progress curves prior to the depletion of 10% substrate were fit to Frieden’s equation43 as in previous studies22,27,43:
(Equation 4) |
where τ is the relaxation time of the lag, and the length of the lag is eτ. The vss was used for determination of hUGDH steady state kinetic parameters. Data were fit using nonlinear regression analysis in PRISM (GraphPad Software Inc., San Diego, CA).
Because the hUGDHFL-A136M, hUGDHΔID-A136M, hUGDHFL-dimer and hUGDHΔID-dimer constructs do not exhibit hysteresis, the observed initial velocity was used for the determination of steady state parameters as previously described22. UDP-Glc substrate saturation curves were fit to Equation 5.
(Equation 5) |
As previously reported22,23,27, the NAD+ saturation curves of the hUGDH hexameric enzyme display negative cooperativity and were fit to sigmoidal rate equation (Equation 6) :
(Equation 6) |
The determination of the Ki for the allosteric inhibitor UDP-Xylose has been previously described22,27. Briefly, data were globally fit to the model for competitive inhibition with cooperativity (Equation 7)using PRISM.
(Equation 7) |
KM, kcat, and Ki were shared parameters in global fitting, while h was fit locally to each curve. The hUGDH dimers (hUGDHFL-dimer and hUGDHΔID-dimer) exhibited mixed inhibition with respect to both UDP-Glc and NAD+, and were globally fit to (Equation 8).
(Equation 8) |
Here, Ki refers the competitive inhibition component, and αKi gives the noncompetitive contribution. KM, kcat, α and Ki were shared parameters global fitting.
Sedimentation velocity.
Sedimentation velocity analysis was conducted as previously described22–27. Briefly, hUGDH constructs were dialyzed >12 hr at 4 °C into (25 mM HEPES pH 7.5 and 150 mM KCl) and diluted to a final concentration of 9 μM. In ligand-bound studies, hUGDH constructs were dialyzed with comparable amounts of either substrate (UDP-Glc) or allosteric inhibitor (UDP-Xyl) for > 24 h. Samples were loaded into cells equipped with 12 mm double-sector Epon centerpieces and quartz windows. The cells were then loaded into an An60 Ti rotor and equilibrated to 20 °C for 1 h. Sedimentation velocity data were collected at 50,000 rpm in an Optima XLA analytical ultracentrifuge for 8–12 h. Data were recorded at 280 nm in radial step sizes of 0.003 cm. SEDNTERP44 was used to estimate the partial specific volume of all hUGDH constructs, and the buffer density (1.00726 g/mL) and viscosity (0.01018 P). SEDFIT45 was used to model and fit all data. Data were modeled as a continuous sedimentation coefficient (c(s)) distribution. The baseline, meniscus, frictional coefficient, and systematic time-invariant, and radial invariant noise were fit46. HYDROPRO47 was used to predict s-values based on crystal structures. The expected drag from the ID-tail was estimated by calculating the expected s-values from crystal structures with and without modeled, energy minimized ID-tails. The data fits for all experiments can be found in Extended Data Figure 3.
Evolutionary rate analysis.
79 UGDH sequences from vertebrates were used for analysis after removing redundancy at the organism level (only one UGDH sequence used per organism). The protein sequences were aligned using MUSCLE48, and rates of evolution at each alignment position was calculated under the JTT model49 using MEGA7 (log-likelihood method)50. The rates were normalized such that the average rate of evolution was 1.0 across the entire protein. Residue positions evolving faster than average show a rate greater than 1.0. In Extended Data Fig. 4, only the rates at alignment positions where the human UGDH did not have an indel were used.
Monte Carlo sampling.
The free energy cost of tethering an unstructured, non-interacting peptide to an impenetrable surface depends on the ratio of all constrained and unconstrained states:
(Equation 1) |
Where R is the gas constant, T is temperature, Ω1 is the number of all possible states of an unconstrained, self-avoiding peptide and Ω2 is the number of the Ω1 states that do not conflict with the constraint imposed by the protein surface. To simplify, we used polyserine peptides, ignored sidechain entropy and used a hard sphere potential along with 166 coarse grained ϕ,ψ bins to calculate Ω1 and Ω2. Each bin represents a 10°×10° range of ϕ,ψ values of peptide conformations in the ‘allowed’ region of the original Ramachandran map (Extended Data Figure 5a, b). This calculation is nontrivial for large polymers, and an exhaustive grid search of all conformations was only conducted for the 3- and 4-residue ID-tails (Extended Data Figure 5c). We used the following Monte Carlo procedure to estimate the fraction of surface-constrained conformations (Ω2/Ω1) for each ID-tail. To determine the self-avoiding Ω1 mesostates, we randomly assigned one of the 166 ϕ,ψ bins to each ϕ,ψ torsion angle in the ID-tail and then looked for steric clashes within the conformer using the ‘outer limit’ for atomic clashes as described in the original Ramachandran map51. Next, each of Ω1 mesostates was analyzed for steric clashes with the surface or the adjacent ID-tail (Extended Data Figure 5d-l). Prior to the simulation, hydrogens were added to the hexamer structure using reduce program52, and an adjacent ID-tail was modeled in an extended conformation and fixed during the simulation (Extended Data Figure 5d-f). The simulation was stopped when a minimum of 124,000 self-avoiding conformers were analyzed and the ratio of surface-constrained conformations (Ω2/Ω1) reached convergence (Extended Data Figure 5c). The convergence threshold was defined as a change in the cumulative ratio of less than 10−5 within a window of 5000 trials. All runs reached convergence except for the 10-mer simulations, which only converged to 2 decimal places (Extended Data Figure 5c-l). We estimated the accuracy in our Monte Carlo simulations by comparing the results to the full grid search of the 3- and 4- residue ID-tails (Extended Data Figure 5c).
Thermodynamic shift assay.
Solutions of hUGDH [FL-dimer or ΔID-dimer] at 0.1 mg/mL were prepared with 5X SYPRO Orange ThemoFluor (ThermoFischer) in the standard reaction buffer (50 mM HEPES pH 7.5, 50 mM NaCl, and 5 mM EDTA). Samples were then briefly spun and allowed to equilibrate for 20 minutes. The thermal denaturation experiments were conducted in replicates (N ≥ 3) and data was acquired using a Bio-Rad MiniOpticon Real-Time qPCR machine. A fluorescence excitation spectrum wavelength between 470–505 nm and an emission spectrum between 540–570 nm were used. The fluorescence emission for each solution was recorded every 30 seconds as the temperature was increased from 25 to 80 °C (ramp speed of 0.5 °C/sec). Baselines were subtracted from the raw data using the buffer control experiments. Data were fit to a Boltzmann sigmoidal curve (Equation 8) to obtain the apparent TM values53.
(Equation 9) |
Hydrogen-Deuterium Exchange Mass Spectrometry.
Studies have shown that Hydrogen-Deuterium Exchange (HDX) is an appropriate probe for protein dynamics and can illuminate differences between wild-type and mutant proteins54,55. HDX is a powerful tool for foot-printing the solvent accessible regions of a protein56, and was utilized in this study to compare structural and dynamic changes between the dimerized versions of hUGDH (hUGDHFL-dimer and hUGDHΔID-dimer).
Proteins were expressed and purified in the Wood Lab as previously described22–27. Proteins were then flash frozen and shipped overnight on dry ice to the Gross Lab at Washington University in St. Louis for HDX MS analysis. Protein solutions (2 μL) were continuously labeled at 25 °C by adding 20 μL of 10 mM HEPES buffer that contained 99.9% deuterium oxide (pD = 7.4). Samples were quenched by adding 33 μL of 8 M Guanidine Hydrochloride and 100 mM TCEP (final pH = 3.0) at 30 s, 1 min, 2 min, 15 min, 1 hr, and 2 hr time points57,58. One minute after quenching, samples were flash frozen in liquid nitrogen and stored for less than 36 hours at −80 °C. Control samples contained 10 mM HEPES in water rather than deuterium oxide. Each sample was thawed seconds prior to LC-MS analysis. On-line protein digestion was performed with a custom-packed pepsin column (2 mm x 20 mm) at a flow rate of 200 μL/min in 0.1% trifluoroacetic acid. For desalting, a Zorbax Eclipse XDB-C-8 trap column (2.1 × 15mm, 3.5 μm) was used to trap peptic peptides for 3 min. Following this, peptides were separated using a Hypersil Gold C-18 analytical column (2.1 × 50 mm, 2.5 μm), 4–80% gradient of acetonitrile with 0.1% formic acid (B), and a 100 μL/min flow rate. Peptides were detected using a LTQ XL Orbitrap mass spectrometer (Thermo Fisher Scientific), with a mass resolving power of 50000, m/z 400. Additional parameters were spray voltage of 5 kV, capillary temperature of 275 °C, capillary voltage of 49 V, and a tube lens of 163 V. All experiments were conducted in quadruplicate.
As a prelude to HDX, protein mapping was conducted by identifying pepsin-digested peptides. Product-ion mass spectra were collected in the data-dependent mode, picking the six most abundant ions from selected MS/MS. Peptides were identified using Mascot (Matrix Science, London, UK). Following HDX, mass spectra were analyzed with HDX Examiner (Sierra Analytics, Modesto, CA). Percent deuterium (%D) uptake was plotted against time for hUGDHFL-dimer and hUGDHΔID-dimer. To magnify slight, yet significant changes in uptake, the cumulative differences in HDX for hUGDHFL-dimer versus hUGDHΔID-dimer were calculated. These values were plotted alongside 3 times the error propagation for all measurements of both variants for each peptide, after the data and error were normalized - divided by the number of time points considered for each data point (Extended Data Fig. 6). The propagation error for each peptide is equal to the square root of the sum of all squared standard deviation values for collective time-dependent measurements of hUGDHFL-dimer and hUGDHΔID-dimer. The cumulative %D uptake was compared to 3 times the propagation error. Differences that were greater than 3 times the propagation error were noted as regions of change affected by the presence of the ID-tail. We chose to normalize the data to be more inclusive of peptides with low intensity that are found at most time points. In like manner, we have excluded those peptides that have avoided detection for more than two time points.
Stopped-Flow Analysis of hUGDH Hysteresis.
The allosteric activation (E* to E) of hUGDH can be observed as a lag (hysteresis) in progress curves22,27 (See Extended Data Figure 7f for examples). The allosteric activation rates for hUGDHFL (N ≥ 6) and hUGDHΔID (N ≥ 6) were monitored at 25 °C using an Applied Photophysics SX20 stopped flow spectrophotometer. Enzyme solutions contained 500 nM hUGDHFL or hUGDHΔID in the standard reaction buffer [50 mM HEPES pH 7.5, 50 mM NaCl, and 5 mM EDTA]. This solution was rapidly mixed with an equal volume of standard reaction buffer that contained both substrate and cofactor. The mixed solution contained 250 nM hUGDHFL or hUGDHΔID, with saturating amounts of both substrate and cofactor. The progress of the reaction was monitored by NADH production, with the absorbance reading at 340 nm being acquired every 10–15 ms. Progress curves were fit to Equation 4 to determine the length of the lag in enzyme activation (E* to E). The mean and standard deviation of the hysteretic lags were derived from 6 or more progress curves.
Transient State Kinetics of UDP-Xyl Binding.
Stopped-flow fluorescence studies were conducted at 25 °C using an Applied Photophysics SX20 stopped flow spectrophotometer with a dead time of ~1.2 ms. Syringes were loaded with 500 nM of hUGDHFL or hUGDHΔID and variable concentrations of UDP-Xyl, and then rapidly mixed. The change in intrinsic tryptophan fluorescence was continuously monitored using an excitation wavelength of 290 nm and an emission filter with a cutoff below 320 nm (Extended Data Fig. 7). Fluorescence decay curves were averaged from experimental replicates (N ≥ 4) for each concentration in the series. Raw data was corrected for the inner filter effect using the molar absorptivity at both the excitation and emission of UDP-Xyl59. Data were globally fit using computer simulation with KinTek Global Kinetic Explorer program (KinTek Corp., Austin, TX)60,61. Multiple input models based on the known structural states were tested, and the best fit model was determined using confidence contour analysis62. Microscopic rate constants and errors are reported in Extended Data Fig. 7e. Fit data and confidence contours can be found in Extended Data Fig. 7a-d.
Extended Data
Extended Data Table 1|.
Data collection | ||
---|---|---|
Protein Data Bank Entry | 5W4X | 5VR8 |
E* hUGDHΔID | EΩ hUGDHFL | |
Space group | C2 | P1211 |
Unit cell dimensions a,b,c (β) | 178.19, 114.07, 97.24 | 89.08, 196.49, 111.26 |
(116.9°) | (111.9°) | |
Completeness (%) | 99.9 (91.1 )a | 93.2 (60.0)a |
No. reflections | 324,675 | 2,730,154 |
Redundancy | 6.4 (6.1) | 12.3(10.3) |
II σ(I) | 21.9 (1.5) | 14.9 (2.5) |
CC1/2b | 99.9 (64.9) | 99.7(79.3) |
Rmeas(%)c | 6.5 (122.5) | 13.2(89.3) |
Refinement | ||
Resolution (Å) | 2.65 | 2.00 |
Rwork / Rtree | 0.19/0.23 | 0.16/0.19 |
No. atoms: Protein / Ligand / Water | 10887/33/36 | 21584/394/1097 |
B-factors (Å2): Protein / Ligand / Water | 89.9/97.4/64.3 | 33.2/27.1/32.3 |
Stereochemical Ideality | ||
Bond lengths (Å2) | 0.004 | 0.008 |
Bond angles (°) | 0.75 | 0.91 |
φ,Ψ Preferred (%)d | 98.98 | 97.8 |
φ,ΨAdditionally allowed (%) | 1.02 | 2.2 |
φ,ΨDisallowed region (%) | 0.0 | 0.0 |
Extended Data Table 2|.
hUGDH |
KM (UDP-GIc, μM) |
Kcat
b (s-1) |
KiUDX (UDP-Xyl,μM) |
αUDGC | Δ ΔGe
(kcal•mol-1) |
# of Data Pointsh |
---|---|---|---|---|---|---|
ΔID | 17.8 ±0.9 | 0.7 ±0.01 | 5.44 ± 0.55 | ------ | 0.00 | 42 |
FL | 12.7 ±0.6 | 0.8 ±0.01 | 0.52 ± 0.04 | ------ | −1.39 | 38 |
R1 | 12.9 ±1.0 | 0.8 ±0.01 | 0.60 ±0.06 | ------ | −1.31 | 59 |
0.13×R1 | 12.8 ± 1.2 | 1.0 ±0.01 | 2.59 ±0.24 | ------ | −0.44 | 40 |
0.26×R1 | 12.4 ± 1.0 | 1.0 ±0.01 | 1.81 ±0.18 | ------ | −0.65 | 42 |
0.5×R1 | 11.1 ±0.8 | 1.0 ±0.01 | 1.09 ±0.08 | ------ | −0.95 | 47 |
R2 | 43.7 ± 3.6 | 0.7 ±0.01 | 0.78 ±0.07 | ------ | −1.15 | 50 |
−Lys | 30.1 ± 1.9 | 0.5 ±0.01 | 0.29 ±0.03 | ------ | −1.73 | 39 |
−Pro | 13.1 ±0.9 | 0.9 ±0.01 | 0.72 ±0.07 | ------ | −1.20 | 26 |
0.13×FL | 18.8 ±0.9 | 1.0 ±0.01 | 2.76 ±0.15 | ------ | −0.40 | 46 |
0.26×FL | 18.3 ±0.7 | 0.8 ±0.01 | 1.99 ± 0.12 | ------ | −0.60 | 42 |
0.5×FL | 18.8 ±0.9 | 0.9 ±0.01 | 1.12 ± 0.08 | ------ | −0.94 | 50 |
2×FL | 15.2 ±0.7 | 0.6 ±0.01 | 0.30 ± 0.02 | ------ | −1.72 | 43 |
0.13×Ser | 16.9 ± 1.0 | 0.9 ±0.01 | 2.67 ± 0.24 | ------ | −0.42 | 49 |
0.26×Ser | 18.4 ± 1.0 | 0.9 ±0.01 | 1.76 ±0.18 | ------ | −0.67 | 43 |
0.5×Ser | 17.4 ± 1.3 | 0.8 ±0.01 | 1.09 ± 0.10 | ------ | −0.95 | 49 |
Ser | 17.8 ±1.0 | 0.7 + 0.01 | 0.60 ±0.05 | ------ | −1.31 | 53 |
ΔID-dimer | 286 ± 27 | 0.1 ±0.01 | 1.23 + 0.15d | 22 ± 12 | 0.00f | 36 |
FL-dimer | 83.2 ±2.2 | 0.1 ±0.01 | 0.17 ±0.01d | 36 ±5 | −1.17f | 50 |
ΔID-A136M | 9.9 ± 0.6 | 0.3 ±0.01 | 4.20 ±0.51 | ------ | 0.00g | 30 |
FL-A136M | 8.5 ± 0.6 | 0.7 ±0.01 | 4.41 ±0.37 | ------ | 0.03g | 55 |
Kinetic parameters and associated standard errors (±) for all constructs were derived from global analyses of data in Extended Data Fig. 2
One catalytic turnover of UDP-GlcA produces two molecules of NADH per cycle
α describes the mode of mixed inhibition (Equation 8). An α > 1 in the UDP-Glc saturation curves shows that UDP-Xyl binds preferentially to the allosteric binding, and secondarily to the coenzyme binding site
Competitive Ki from the fit to the mixed inhibition Equation 8
Change in UDP-Xyl binding free energy (kcal•mol−1) of hUGDH constructs relative to hUGDHΔID (ΔΔG = RT ln()).
Change in UDP-Xyl binding free energy relative to the hUGDHΔID-dimer
Change in UDP-Xyl binding free energy relative to the hUGDHΔID-A136M
The number of independent data points used in global analysis (see Methods).
Extended Data Table 3|.
hUGDH |
Km (NAD+, mM) |
K0.5b (NAD+, mM) |
Hill (h) |
Kcatc (S-1) |
UDX (Ki, μM) |
αnadd | # of Data Pointse |
---|---|---|---|---|---|---|---|
FL | ------------- | 0.8 ± 0.20 | 0.8 ± 0.1 | 0.9 ± 0.08 | ------------- | ------------- | 18 |
ΔID | ------------- | 0.3 ±0.06 | 0.6 ± 0.1 | 0.7 ±0.03 | ------------- | ------------- | 12 |
FL-dimer | 2.0 ± 0.26 | ------------- | ------------- | 0.1 ± 0.01 | 2.1 ±0.4 | 0.9 ±0.2 | 37 |
ΔID-dimer | 3.2 ±0.10 | ------------- | ------------- | 0.2 ±0.01 | 3.6 ±0.8 | 0.6 ±0.2 | 47 |
R1 | ------------- | 0.4 ± 0.03 | 0.9 ±0.1 | 0.7 ±0.01 | ------------- | ------------- | 17 |
R2 | ------------- | 0.8 ±0.14 | 0.7 ± 0.1 | 0.7 ±0.01 | ------------- | ------------- | 15 |
−Lys | ------------- | 2.9 ±0.61 | 0.8 ± 0.1 | 0.6 ±0.04 | ------------- | ------------- | 10 |
−Pro | ------------- | 0.5 ±0.06 | 0.6 ± 0.1 | 1.2 ±0.03 | ------------- | ------------- | 12 |
0.13×FL | ------------- | 0.4 ±0.03 | 0.7 ± 0.1 | 1.0 ±0.03 | ------------- | ------------- | 13 |
0.26×FL | ------------- | 0.2 ± 0.03 | 0.8 ± 0.2 | 0.8 ±0.05 | ------------- | ------------- | 11 |
0.5×FL | ------------- | 0.3 ±0.03 | 0.9 ±0.1 | 0.9 ±0.02 | ------------- | ------------- | 12 |
2×FL | ------------- | 1.4 ±0.31 | 0.8 ±0.1 | 0.9 ±0.01 | ------------- | ------------- | 18 |
0.13×Ser | ------------- | 0.9 ±0.24 | 0.7 ±0.1 | 1.2 ±0.10 | ------------- | ------------- | 12 |
0.26×Ser | ------------- | 1.0 ± 0.27 | 0.7 ± 0.1 | 1.1 ± 0.09 | ------------- | ------------- | 15 |
0.5×Ser | ------------- | 1.2 ±0.34 | 0.7 ± 0.1 | 1.1 ± 0.09 | ------------- | ------------- | 13 |
Ser | 1.3 ± 0.19 | 0.7 ± 0.1 | 1.0 ± 0.04 | ------------- | ------------- | 13 |
Kinetic parameters and associated standard errors (±) for all constructs were derived from global analyses of data in Extended Data Fig. 2
Hexameric hUGDH displays negative cooperativity with NAD+ binding, which indicates a mix of high affinity and low affinity sites23–28. In previous work, we showed that the native hUGDHFL K0.5 of 0.8 mM NAD+ corresponds to a mix of high and low affinity sites of (KM of 88 μM and 1.8 mM, respectively)23. This is consistent with the published Kd of 30 μM for the coenzyme24.
One catalytic turnover of UDP-GlcA produces two molecules of NADH per cycle
α describes the mode of mixed inhibition (Equation 8). An α < 1 in the NAD+ saturation curves show that UDP-Xyl binds preferentially to the allosteric binding, and secondarily to the coenzyme binding site.
The number of independent data points used in nonlinear regression (see Methods).
Supplementary Material
Acknowledgements:
The authors would like to thank Professors Andrew P. Karplus, Brian W. Matthews, and Savvas N. Savvides, and the members of the Z.A.W. laboratory for helpful discussions. We also thank the reviewers for their contributions to the peer review of our manuscript, especially those of reviewer #3, who suggested that the ID-tail would generate an entropic force. Finally, we thank Dr. Richard Wang of Norclone for his heroic efforts in producing the R1 truncation constructs in record time. This work was supported by the NIH National Institute of General Medicine grants R01GM114298 awarded to Z.A.W. and P41GM103422 awarded to M.L.G.
Footnotes
The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper. Correspondence and requests for materials should be addressed to Z.A.W. (zaw@uga.edu). Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
DATA AVAILABILITY STATEMENT: The structure factors and coordinates described in this manuscript have been deposited and released (PDB entries: 5W4X and 5VR8). All data generated or analyzed in this study can be found within the Extended Data Files and the provided Source Data.
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