Microcalorimetry reveals multi-state thermal denaturation of G. stearothermophilus tryptophanyl-tRNA synthetase

Mechanistic studies of Geobacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS) afford an unusually detailed description—the escapement mechanism—for the distinct steps coupling catalysis to domain motion, efficiently converting the free energy of ATP hydrolysis into biologically useful alternative forms of information and work. Further elucidation of the escapement mechanism requires understanding thermodynamic linkages between domain configuration and conformational stability. To that end, we compare experimental thermal melting of fully liganded and apo TrpRS with a computational simulation of the melting of its fully liganded form. The simulation also provides important structural cameos at successively higher temperatures, enabling more confident interpretation. Experimental and simulated melting both proceed through a succession of three transitions at successively higher temperature. The low-temperature transition occurs at approximately the growth temperature of the organism and so may be functionally relevant but remains too subtle to characterize structurally. Structural metrics from the simulation imply that the two higher-temperature transitions entail forming a molten globular state followed by unfolding of secondary structures. Ligands that stabilize the enzyme in a pre-transition (PreTS) state compress the temperature range over which these transitions occur and sharpen the transitions to the molten globule and fully denatured states, while broadening the low-temperature transition. The experimental enthalpy changes provide a key parameter necessary to convert changes in melting temperature of combinatorial mutants into mutationally induced conformational free energy changes. The TrpRS urzyme, an excerpted model representing an early ancestral form, containing virtually the entire catalytic apparatus, remains largely intact at the highest simulated temperatures.


INTRODUCTION
Mechanistic studies of B. stearothermophilus tryptophanyl-tRNA synthetase (TrpRS) identified two subtle, and apparently contradictory, ways in which differential conformational stability affects its catalytic cycle. [1][2][3][4] Catalytic assist by Mg 2þ ion, on the one hand, depends entirely on its coupling to domain motions. 3,5,6 On the other hand, the sign of the free energy change for the catalytic conformational change is positive in the presence of the pyrophosphate product, PPi, and negative only in its absence. 7 That deepened understanding, in turn, provides a novel perspective on how biology appears to circumvent the second law of thermodynamics. 4,8,9 The former effect couples assembly of the fully active catalytic apparatus tightly to domain motion; the latter ensures that domain motion requires PPi release, thereby coupling the conformational change reciprocally 10 to the chemistry of ATP utilization. This novel escapement mechanism 3,4 ensures efficient vectorial utilization of ATP 11,12 and is thus a potentially general model for energetic coupling in a broader range of enzymes that transduce NTP hydrolysis free energy. 13,14 Neither front nor back-end gating has been analyzed in detail for any other transducing NTPases, although just as the TrpRS catalytic domain movement is triggered by PPi release, the myosin power stroke appears to be triggered by phosphate release. 13,14 The back-end gating may, thus, be common to many NTPases.
Key to the escapement mechanism are conformational stability changes induced, in turn, by the succession of bound ligands, as chemical events proceed. 2 Untangling different levels of energetic coupling that produce the contradictory gating effects necessarily entails measuring how these stability changes arise from differential ligand binding. That task is facilitated by a set of combinatorial mutants within a packing motif called the D1 switch that mediates shear developed during TrpRS domain motion. 1,15 The D1switch consists of 6-7 residues near the C-terminus of the Rossmann fold N-terminal a-helix that bind the preceding and following b-strands to that helix. It is one of the most widely conserved motifs in the proteome 16 and central to this work.
We stumbled on the D1 switch motif when we attempted to identify residues whose nearest neighbors changed as TrpRS passed through its conformational trajectory during catalysis. 17 Delaunay tessellation decomposes protein packing interactions uniquely into tetrahedra of nearest-neighbor side chains. 18,19 TrpRS crystal structures revealed that the vast majority of the $300 tetrahedra lie within domains that move as rigid bodies and are, thus, structurally invariant. Only about a dozen tetrahedra are structurally dynamic. A series of papers using the PATH algorithm to map the conformational changes during catalysis showed that repacking of aromatic residues in the D1 switch motif-F26, Y33, and F37-constitutes the rate-limiting step in assembling the active site for catalysis upon binding tryptophan and ATP. 1,7,20 Rosetta 21 identified specific mutations of the three aromatic D1 residues plus I4 that should stabilize the excited Pre-TS state relative to the two ground states-Open and Products-on either side. 17 Combinatorial mutagenesis of those residues showed that repacking is coupled by $-5.5 kcal/mol to the active-site Mg 2þ ion. It accounts for the contribution of the metal to transition-state stabilization. A subsequent modular thermodynamic cycle 22 implicated an untwisting domain motion to catalysis by the same coupling free energy. Those studies linked dynamic side chain re-packing in the D1 switch alternately to domain motion and to Mg 2þ -dependent rate acceleration of amino-acid activation. Recent studies of a related thermodynamic cycle measuring the energetic coupling between the HVGH and KMSKS catalytic signatures in the larger leucyl-tRNA synthetase 23 provide complementary evidence that such coupling is common to all class I aaRS. The ensemble of structural, kinetic, and computational studies on the D1 switch afforded a uniquely consistent set of crossvalidating intercorrelations (see Fig. 7 in Ref. 20). Those correlations underlie the TrpRS escapement mechanism and so strongly motivate the present analysis of TrpRS melting behavior.
In this report, we use differential scanning calorimetry (DSC) and computational simulation to compare the melting behavior of fully liganded TrpRS bound to ATP and a non-reactive tryptophan analog stabilizing the crystallographic pre-transition conformational state (1MAU 24 ) with that of the unliganded apo form (1D2R 25 ). Given the differences between experimental (TrpRS dimer; Cp; heat capacity at constant pressure) and computational (TrpRS monomer; Cv; heat capacity at constant volume) methods, there is surprising consistency between the two methods. Moreover, the computational simulations also allow comparison of two structural metrics-the radius of gyration, R g , and a-helical content-that facilitate interpretation. Our principal observations are as follows: (i) Both liganded and apo TrpRS denature via a three-state process involving a molten globular intermediate that forms at temperatures $8 lower than those required to melt a-helical secondary structures. (ii) A broad, unidentified and substantial heat capacity change occurs at approximately the temperature at which the source organism grows optimally and precedes the formation of the molten globule. (iii) A computational free energy surface of a liganded TrpRS monomer exhibits similar multi-step experimental heat capacity changes. That simulation is reversible by definition, because the isolated monomer cannot aggregate. It also furnishes valuable structural metrics and cameo snapshots that aid interpretation of the melting behavior. (iv) The catalytic core known as the urzyme, an experimentally validated model for the ancestral form of class I aminoacyl-tRNA synthetases, containing only the 130 residues necessary to frame the active site, is the last remaining folded structure at high temperatures in the computed free energy surface. Preservation of the tertiary packing of D1 residues at high temperature appears to stabilize the N-terminal a-helix, which has only weak helical propensity.
An accompanying paper 26 describes melting curves obtained by differential scanning fluorometry and far UV circular dichroism for unliganded native and 15 combinatorial TrpRS D1 switch variants. Analysis of unliganded TrpRS described here provide a solid context in which to identify the intrinsic and higher-order structural interactions that allow the D1 switch residues to stabilize or destabilize different conformational states along the structural reaction profile, hence their functional behavior. Our goal is to establish a basis for pursuing two non-trivial longer-range questions: (i) Can we exploit the mutant proteins to correlate stability changes of all three conformations with the steady-state kinetic properties measured previously? 3 (ii) How does coupling between the four mutated residues change along the conformational reaction path, allowing them to exert their highly cooperative contribution to the escapement mechanism? 4

Differential scanning calorimetry
TrpRS solutions (2 lM) were dialyzed overnight against either buffer alone [20 mM HEPES, 50 mM NaCl 2 (pH 7.0)] of the same buffer plus tryptophanamide (50 lM) and ATP (10 mM). In each case, we reserved the dialysate for use as a blank in calorimetry. We loaded 500 ll of each of the four solutions (sample plus blank, liganded vs apo) into the chamber of a TA Instruments Nano DSC 250 microcalorimeter. Thermal scans were performed at 2.98 atm from 10 to 95 C in 5 min intervals. Data from the thermal scans were processed using the TA Nanoanalyze software, v. 3.12 (https://www.tainstruments.com/itcrun-dscrun-nanoanalyze-software/) to subtract the blank profile, normalize the net heat capacity curves, select baselines, and identify peaks. We identified and integrated heat capacity peaks in the profiles using the Voigt Gaussian/Lorentzian hybrid given an onset temperature (Tonset), which gave superior local fit to all curves.

Replica exchange DMD simulations
We make use here of an extended analysis of the free energy surface connecting the pre-transition state (PreTS) and products (Products) conformational states. The replica exchange algorithm efficiently samples the temperature-dependent conformational free energy landscape of a macromolecule by simulating replicas of the equilibrium state at different temperatures and, at predefined time points, exchanging the structures at different temperatures if the difference in their energies is within a threshold. It is reversible by definition because the monomer cannot aggregate.
Our original purpose was to find an independent estimate of the conformational transition-state structure that we could compare with that produced by a minimum action path algorithm. 7 To that end, we projected the simulated trajectory onto the two dominant principal components of the motion to give a 3D function whose z-axis was conformational free energy. That surface was used to map the location of the conformational transition states separating the Pre-transition and Product state conformations to the stationary point of the conformational transition path computed using a minimum action path algorithm and which turned out in excellent agreement with the position of the global saddle point separating the PreTS and Products states in the replica exchange simulation.
We implemented the replica exchange algorithm using the REX/ DMD suite 27-30 that uses Discrete Molecular Dynamics 28,30-32 to simulate replicas at 24 temperatures ranging from $ 175 to $ 405 K separated by a constant interval. DMD approximates atomic interactions by multistep square well potentials 27,33 and uses an Andersen Thermostat to regulate system temperature, while the Lazaridis-Karplus implicit solvation model 34 accounts for the solvation energy. Simulations were set up with the Product state structure, 1I6L, with the PreTS state structure, 1MAU which appears to be the least stable of the crystal structures. 17 We excised the terminal amino acid (R328) from the structures as it does not appear in most crystal structures. Also, since DMD force field does not include parameters for Mg 2þ , we replaced it with Zn 2þ . The two metals have almost exactly the same ionic radii and hydration energies, and they behave almost identically in molecular simulations with empirical force fields. 35 Moreover, Zn 2þ ion parametrizations prove to be the most similar to those of Mg 2þ in potential of mean force profiles used to model ionic interactions with nucleic acids. 36 For these reasons, we do not expect these changes to affect the DMD of Bacillus stearothermophilus tryptophanyl-tRNA synthetase. Differential scanning calorimetry was carried out and processed using TA NanoAnalyze software as described in Methods section. The vertical arrow is to facilitate alignment of two peaks related to the breakdown of non-polar packing (magenta) and secondary structure (red). The gray bars along the x axis indicate the normal growth temperature range for Geobacillus stearothermophilus.
the system in a significant manner. We applied a harmonic constraint (well width ¼ 61 Å ) between ligand atoms and the binding pocket within 3.5 Å of each other to retain ligands in their binding pockets. We applied another weak harmonic constraint (well width ¼ 62 Å ) to atoms which uniquely form native contacts (8 Å ), in either 1I6L or 1MAU, in order to sample conformational spaces adjacent to the two boundary states. Simulations at each temperature ran for 3 Â 10 6 steps ($ 150 ns). Omitting the time to equilibrate (initial 500 000 steps), snapshots were generated every 1000 steps for a total of 2500 snapshots which were then used in all the analyses.
Coordinates from the trajectory furnished a variety of useful metrics to aide interpretation of the melting behavior. The WHAMweighted histogram analysis method-algorithm enable computation of the specific heat capacity, C v , 37 from the energies of structures generated at different temperatures. The atomic coordinates of the TrpRS monomer enable computation of the average radius of gyration, R g from the mean structures at each temperature. Similarly, we used the DSSP algorithm 38,39 as implemented in Pymol 40 with default options to compute the fraction of a-helical residues for each averaged structure.

RESULTS AND DISCUSSION TrpRS melts by a multi-step, ligand-dependent process
Experimental heat capacity changes during TrpRS thermal melting exhibit complex behavior with clear differences between apo [ Fig. 1(a)] and liganded [ Fig. 1(b)] forms. Binding of tryptophanamide and ATP lead to an inhibited state we have termed the pre-transition-state (Pre-TS) conformation because the tryptophanamide and ATP ligands trap it just prior to the chemical step in tryptophan activation. Crystal structures of the apo and Pre-TS states differ substantially 24,25,41 as the anticodon-binding domain rotates toward the catalytic domain by $8 and twists by $3.5 in the PreTS state. The different melting curves must reflect these structural differences.
Melting occurs in three distinguishable stages: initial (T onset ¼ 56, 60 C), intermediate (T onset ¼ 62,69 C) with a major change in heat capacity, and terminating (T onset ¼ 75, 73 C). Pre-TS TrpRS melting is also significantly more cooperative with all changes occurring between 62 and 73 C], compared to 56 and 82 C.
Curiously, although ligand binding normally stabilizes protein structures against thermal melting, the overall melting of the apo form occurs at higher temperature (79 vs 74 C). That observation confirms previous estimates, derived from nucleotide-ligand binding affinity changes between different conformations and from comparing statistical potentials derived from side chain packing analysis which led us to conclude that the Pre-TS state was significantly destabilized by the conformation induced prior to catalysis. 17

Multi-step experimental melting behavior matches that observed in a computational free energy surface of a liganded TrpRS monomer
To validate the thermodynamic interpretations of TrpRS melting behavior, we take advantage here of detailed correspondencesand differences-between the experimental melting curves (Fig. 1) and those derived from a computational temperature-dependent conformational free energy surface of the liganded, wild-type TrpRS monomer PreTS complex. That surface (Fig. 2) is reversible by definition, 42 because the isolated monomer cannot aggregate. It was constructed previously using Replica Exchange Discrete Molecular Dynamics (REX/DMD 29,42,43 ) simulations to confirm the computational transition states identified by a minimum action path analysis. 1,7,20 Figure 2 shows heat capacity changes, C v , as a function of temperature. That plot shares important characteristics with the experimental melting curves. All three show three main transitions; they have the same relative peak heights, integrated areas, and relative widths. They differ only in the temperatures at which they occur. We discuss possible reasons for this discrepancy further in the subsequent section discussing influences on the melting.

FIG. 2.
Computational traces of structural and thermodynamic changes associated with fully liganded monomeric TrpRS thermal denaturation. Gray shading highlights successive maxima in the specific heat capacity, C v , (blue curve). Temperatures in C are included above the x axis to facilitate comparison with the experimental melting curves in Fig. 1. Each transition corresponds to increases in volume, as indicated by the radius of gyration (R g , green curve, bounded by its uncertainties in gray). The 14% increase in R g in the second transition corresponds to that (15-20%) expected from formation of a molten globule. 44 Similarly, the loss in helical content (red curve) occurs in two stages, the more dramatic of which coincides with the second transition with an R g value increased by 20%. The two, principal, symmetric peaks occur at temperatures corresponding to those measured by Thermofluor (65 vs 69 C) and ellipticity (89 vs 80 C). Vertical dashed lines align discontinuities in both structural parameters (black lines highlight transitions of interest) with the stationary points of the C v trace.
Structural Dynamics ARTICLE pubs.aip.org/aip/sdy Thus, the correspondence between experimental Thermofluor and CD melting curves (Fig. 1) and computational occurrences of these two transition events (Fig. 2) is significantly detailed.

Structural details associated with the simulated melting facilitate interpretation
Structural metrics-radius of gyration, R g , and percentage helix-computed from the mean coordinates of the DMD trajectories accompany the simulated heat capacity changes obtained during the DMD simulation. Temperature-dependences of both metrics are consistent with the maxima in the C v plot: Heat capacity maxima are stationary points where the protein absorbs and loses excess heat from and to the system in equal amounts, as it undergoes conformation changes, so heat capacity changes transiently remain constant with respect to system temperature. In keeping with stationary behavior, local maxima in the C v traces for the two major transitions, coincide with discontinuities in the slopes of both structural metrics, each changing faster before the C v maximum and more slowly afterward (black tracings, Fig. 2).
The average R g increases by 14% during the second transition, corresponding to the increase expected for a molten globule transition (15%). 44 R g increases again by 20% at 87 C. At the 67 C transition temperature for the second peak of the specific heat capacity curve, 70% of the helices present in the native protein remain intact. At 87 C, the third transition temperature, only 28% of the native helices remain intact. Each heat capacity peak, therefore, corresponds to a structurally different transition in both metrics and thus to a different unfolding process.
The initial peak in the C v trace is broader, has a smaller amplitude, and involves minimal changes in either structural metric. It is evident mainly because it initiates the increasing variance of R g , which occurs almost exclusively with the formation of the molten globule. As 60 C coincides with the optimal growth temperature for Geobacillus stearothermophilus, it is possible that this local maximum in C v has biological significance. However, temperature-dependent activity curves for native TrpRS 45 are not informative on this point.
The dimer interface and the presence of active-site ligands strongly influence TrpRS melting Figure 3 compares transitions in experimental and simulated melting curves. The computational melting transitions distribute substantially more widely than the experimental ones in Fig. 1. The dimer interface buries $2160 Å 2 of surface area in the PreTS state. That value suggests a rather low dimer dissociation constant 10 À8 < K D < 10 À6 . 46 We previously used scanning force microscopy to count monomers and dimers at successively higher dilutions to measure the strength of the dimer interface. 47 Those estimates, 6.6 6 4 Â 10 À9 for unliganded and 1.1 6 1 Â 10 À8 M for a complex with a nonhydrolysable aminoacyl-5 0 -adenylate product analog, are at the lower end of that range. Thus, considerable binding free energy is bound into the dimer dissociation constant, which probably induces a considerable difference between the melting behavior of monomer and dimer. Although we have no ready means to verify the assumption, we reason that much of the difference between the three melting temperatures in Fig. 3 may arise because we simulated the TrpRS monomer, not the dimer, and that the dimer interface also compresses the separation between transitions.
Consistent with that assumption, Pre-TS ligands compress the experimental traces still further to a narrower temperature range over which the three melting events occur (dashed lines above the scale vs solid lines above the temperature scale). Simulation of the monomer shows the same three melting events seen in the DSC of the dimer, but they are separated much more widely in temperature. Structural cartoons through the middle and high temperature thermal events both entail significant volume increases, supporting identification of the middle transition as formation of a molten globule.
We assembled structural snapshots over the course of the exchange trajectory from each replica at given temperatures, noted in Fig. 3. The average structures at each temperature were analyzed for native helical content using DSSP as indicated in Fig. 1. Notably, melting is localized to both the connecting peptide 1 (CP1) and anticodon-binding (ABD) domains (deep purple surfaces). The transition at 65 C can reasonably be assigned to molten globule formation because tertiary structures of those two domains are expanded, but not unfolded.

FIG. 3. TrpRS melting transitions.
Experimental differential scanning calorimetry is above and replica exchange DMD simulation below the temperature scale.
Dashed lines indicate fully liganded molecules. Computational traces are for the monomer, not the dimer as above the scale. Bottom row shows Pymol cartoons of mean structures observed for the ensemble of 24 replicas at the indicated temperatures. These temperature-dependent transitions in the overall trajectory help guide the interpretations of the melting events. We identified native secondary structures (forest green) using the DSSP criterion used to identify native a-helices in Fig. 2; denatured segments are deep purple. Gray vertical bars indicate molten globular and helix-melting transitions.
Structural Dynamics ARTICLE pubs.aip.org/aip/sdy The TrpRS urzyme appears to unfold last Ding et al. 48 suggested the persistence of a core 3D structure in unfolded forms of many proteins. Remarkably, for TrpRS, that core, Fig. 4, coincides closely with its urzyme (ur ¼ primordial, authentic, original þ enzyme 49 ), a model for the ancestral enzyme that itself appears to be a catalytically active molten globule. 50 The TrpRS urzyme was constructed by fusing two disjoint segments with similar secondary structures-N-and C-terminal b-a-b crossover connections. 51 One of the core helices that remains intact at 94 C in the computational profile is the long helix (154-165) from the C-terminal segment (salmon). The sequence of that segment has an unusually high fundamental helix propensity as determined by the physics-based and unbiased database-independent Agadir algorithm. 52 In contrast, the helix in the N-terminal segment, has a far lower propensity, but its first eight residues nevertheless remain helical at 368 K, suggesting that extrinsic forces sustain it. Further discussion in the accompanying paper 26 considers what these extrinsic forces might be.
Hilvert's work with chorismate mutase 53,54 identified an efficient, potentially widespread coupling between folding and catalysis-i.e., high transition-state affinity. In light of those findings, it is notable that the last part of the TrpRS monomer to melt in the REX/DMD free energy simulations closely approximates the TrpRS urzyme, a construct studied as a model for the evolution of aminoacyl-tRNA synthetases (Fig. 4). At 94 C, the CP1 and ABD domains, as well as the long-C-terminal alpha helix are all fully unfolded. Thus, the modules that remain intact at the highest temperatures are also likely the most ancient part of the protein which, when excerpted, retains a full range of catalytic activities. 49,51,55 The structural integrity of the TrpRS urzyme at high temperature is consistent with the experimental observation that the urzyme itself seems to function as a molten globule, 50 whose only well-folded state is its complex with the transition state for amino acid activation.

CONCLUSIONS
G. stearothermophilus tryptophanyl-tRNA synthetase is notable because multidisciplinary mechanistic studies have worked out reciprocally coupled gates making catalysis conditional to domain motion and, paradoxically, making domain motion conditional on catalysis. Maximizing the efficiency of converting the free energy of ATP hydrolysis to useful forms of work and information requires both gating functions. 4,10 This paper describes thermal melting studies pursuant to attributing thermodynamic aspects of gating to specific amino acid residues in the D1 switch, a molecular switching motif that imposes multi-state behavior. The accompanying paper 26 outlines a paradigm for using high throughput differential scanning fluorimetry to derive thermodynamic estimates for inter-residue energetic coupling, potentially in a ligand-dependent framework.

AUTHOR DECLARATIONS Conflict of Interest
The authors have no conflicts to disclose.

Author Contributions
Srinivas Niranj Chandrasekaran: Conceptualization (equal); Data curation (equal); Investigation (equal); Methodology (equal); Project  49,51 ) is superimposed on the averaged structure from the ensemble at 94 C (sand, sky, and salmon). The first crossover of the urzyme is colored sky, and the second is colored Salmon in keeping with the colors in (b). Teal sticks are the four D1 switch residues. The urzyme remains largely intact whereas most of the rest of the monomer is unfolded. (b) TrpRS helix propensities computed using the Agadir server 52 for different temperatures. Only three of the helices observed in the TrpRS structure (residues 154-166; residues 253-261; and residues 303-324) have peak propensities exceeding 2.5 (dashed line) at the highest temperature used in our experiments (368 K). The Rossmann fold (gray) and anticodon-binding (ABD; black) domains are interleaved; the first helix participates in both domains, as its Nterminus moves as a rigid body with the ABD. This interleaving likely contributes to the high cooperativity of the helix melting transition monitored by h 221. 26 The two colored segments together comprise the urzyme.

DATA AVAILABILITY
The data that support the findings of this study are available from the corresponding author upon reasonable request.