Complex Loop Dynamics Underpin Activity, Specificity and Evolvability in the (βα)8 Barrel Enzymes of Histidine and Tryptophan Biosynthesis

Abstract

Enzymes are conformationally dynamic, and their dynamical properties play an important role in regulating their specificity and evolvability. In this context, substantial attention has been paid to the role of ligand-gated conformational changes in enzyme catalysis; however, such studies have focused on tremendously proficient enzymes such as triosephosphate isomerase and orotidine 5’-monophosphate decarboxylase, where the rapid (μs timescale) motion of a single loop dominates the transition between catalytically inactive and active conformations. In contrast, the (βα)8-barrels of tryptophan and histidine biosynthesis, such as the specialist isomerase enzymes HisA and TrpF, and the bifunctional isomerase PriA, are decorated by multiple long loops that undergo conformational transitions on the ms (or slower) timescale. Studying the interdependent motions of multiple slow loops, and their role in catalysis, poses a significant computational challenge. This work combines conventional and enhanced molecular dynamics simulations with empirical valence bond simulations to provide rich detail of the conformational behavior of the catalytic loops in HisA, PriA and TrpF, and the role of their plasticity in facilitating bifunctionality in PriA and evolved HisA variants. In addition, we demonstrate that, similar to other enzymes activated by ligand-gated conformational changes, loops 3 and 4 of HisA and PriA act as gripper loops, facilitating the isomerization of the large bulky substrate ProFAR, albeit now on much slower timescales. This hints at convergent evolution on these different (βα)8-barrel scaffolds. Finally, our work highlights the potential of engineering loop dynamics as a powerful tool to artificially manipulate the diverse catalytic repertoire of TIM-barrel proteins.

Enabling Role of Ligand-Driven Conformational Changes in Enzyme Evolution

“…It is the subject of a recent study on the evolvability of enzymes involved in the biosynthesis of histidine and tryptophan, whose activities have been observed to coexist on a single TIM barrel scaffold. 96,97 Flexible proteins that undergo large ligand-driven conformational changes show short-range dynamic motions at protein loops and at secondary folds attached to these loops. The range of these motions becomes increasingly constrained as enzymes move from flexible open conformations to stiffer closed Michaelis complexes.…”

Q-RepEx: A Python Pipeline to Increase the Sampling of Empirical Valence Bond Simulations

“…The performance of Q-RepEx against conventional EVB simulations was tested on three reactions that we have studied extensively in prior work: the isomerization of substrate Dglyceraldehyde-3-phosphate (GAP) by triosephosphate isomerase from Saccharomyces cerevisiae (yTPI) [32][33][34], the isomerization of substrate ProFAR by PriA from Mycobacterium tuberculosis (MtPriA) [35], and the rate-limiting hydrolysis of the phosphoenzyme intermediate formed during the reaction catalyzed by protein tyrosine phosphatase 1B (PTP1B) [36,37]. Here, we used the same system setup and starting structures as in prior studies for all three systems (PDB IDs 1NEY [38], 3ZS4 [39] and 3I80 [40] for yTPI, MtPriA and PTP1B, respectively), as well as the EVB equilibration procedure outlined in detail in Ref.…”

“…Here, we used the same system setup and starting structures as in prior studies for all three systems (PDB IDs 1NEY [38], 3ZS4 [39] and 3I80 [40] for yTPI, MtPriA and PTP1B, respectively), as well as the EVB equilibration procedure outlined in detail in Ref. [35]. In the case of MtPriA, for simplicity, we selected 10 equilibration trajectories out of the 30 total generated for our prior work (selecting every third trajectory we ran) [35], and used these equilibration endpoints as the starting points for our various Q-RepEx simulations.…”

“…We apply this approach to three model systems, studied by conventional EVB simulations in our prior work, triosephosphate isomerase from Saccharomyces cerevisiae (yTPI) [32][33][34], PriA from Mycobacterium tuberculosis (MtPriA) [35], and protein tyrosine phosphatase 1B (PTP1B) [36,37], and demonstrate that this REMD-EVB framework significantly enhances conformational sampling while reducing computational cost (compared to the need to run a substantially larger number of replicas). Q-RepEx thus provides a useful tool for studies of chemical reactions in complex systems with significant conformational fluctuations along the chemical reaction coordinate, where substantial conformational sampling is critical.…”

Correction to “Loop Dynamics and Enzyme Catalysis in Protein Tyrosine Phosphatases”

“…Since the non-enzymatic reaction is not a good reference state, we have used instead the PTP1B-catalyzed reaction (fit to experimental kinetics) as our reference state, and we use this as a baseline for comparison with YopH, as in our recent studies of other enzymes. 5,6 Here, we have used the pK a difference in solution between the nucleophile and leaving group at each reacting state to calibrate the reaction free energies, yielding values of −1.4 and −10.2 kcal mol −1 to describe the cleavage and hydrolysis steps, respectively. We have also made some minor parameter and simulation protocol updates based on best practices developed moving forward with this project.…”