Loop Dynamics and Enzyme Catalysis in Protein Tyrosine Phosphatases.

Protein tyrosine phosphatases (PTPs) play an important role in cellular signaling and have been implicated in human cancers, diabetes, and obesity. Despite shared catalytic mechanisms and transition states for the chemical steps of catalysis, catalytic rates within the PTP family vary over several orders of magnitude. These rate differences have been implied to arise from differing conformational dynamics of the closure of a protein loop, the WPD-loop, which carries a catalytically critical residue. The present work reports computational studies of the human protein tyrosine phosphatase 1B (PTP1B) and YopH from Yersinia pestis, for which NMR has demonstrated a link between their respective rates of WPD-loop motion and catalysis rates, which differ by an order of magnitude. We have performed detailed structural analysis, both conventional and enhanced sampling simulations of their loop dynamics, as well as empirical valence bond simulations of the chemical step of catalysis. These analyses revealed the key residues and structural features responsible for these differences, as well as the residues and pathways that facilitate allosteric communication in these enzymes. Curiously, our wild-type YopH simulations also identify a catalytically incompetent hyper-open conformation of its WPD-loop, sampled as a rare event, previously only experimentally observed in YopH-based chimeras. The effect of differences within the WPD-loop and its neighboring loops on the modulation of loop dynamics, as revealed in this work, may provide a facile means for the family of PTP enzymes to respond to environmental changes and regulate their catalytic activities.

Evolutionary repurposing of a sulfatase: A new Michaelis complex leads to efficient transition state charge offset.

The recruitment and evolutionary optimization of promiscuous enzymes is key to the rapid adaptation of organisms to changing environments. Our understanding of the precise mechanisms underlying enzyme repurposing is, however, limited: What are the active-site features that enable the molecular recognition of multiple substrates with contrasting catalytic requirements? To gain insights into the molecular determinants of adaptation in promiscuous enzymes, we performed the laboratory evolution of an arylsulfatase to improve its initially weak phenylphosphonate hydrolase activity. The evolutionary trajectory led to a 100,000-fold enhancement of phenylphosphonate hydrolysis, while the native sulfate and promiscuous phosphate mono- and diester hydrolyses were only marginally affected (≤50-fold). Structural, kinetic, and in silico characterizations of the evolutionary intermediates revealed that two key mutations, T50A and M72V, locally reshaped the active site, improving access to the catalytic machinery for the phosphonate. Measured transition state (TS) charge changes along the trajectory suggest the creation of a new Michaelis complex (E•S, enzyme-substrate), with enhanced leaving group stabilization in the TS for the promiscuous phosphonate (ßleavinggroup from -1.08 to -0.42). Rather than altering the catalytic machinery, evolutionary repurposing was achieved by fine-tuning the molecular recognition of the phosphonate in the Michaelis complex, and by extension, also in the TS. This molecular scenario constitutes a mechanistic alternative to adaptation solely based on enzyme flexibility and conformational selection. Instead, rapid functional transitions between distinct chemical reactions rely on the high reactivity of permissive active-site architectures that allow multiple substrate binding modes.

Managing Coronavirus Disease 2019 Spread With Voluntary Public Health Measures: Sweden as a Case Study for Pandemic Control.

BACKGROUND: The coronavirus disease 19 (COVID-19) pandemic has spread globally, causing extensive illness and mortality. In advance of effective antiviral therapies, countries have applied different public health strategies to control spread and manage healthcare need. Sweden has taken a unique approach of not implementing strict closures, instead urging personal responsibility. We analyze the results of this and other potential strategies for pandemic control in Sweden. METHODS: We implemented individual-based modeling of COVID-19 spread in Sweden using population, employment, and household data. Epidemiological parameters for COVID-19 were validated on a limited date range; where substantial uncertainties remained, multiple parameters were tested. The effects of different public health strategies were tested over a 160-day period, analyzed for their effects on intensive care unit (ICU) demand and death rate, and compared with Swedish data for April 2020. RESULTS: Swedish mortality rates are intermediate between rates for European countries that quickly imposed stringent public health controls and those for countries that acted later. Models most closely reproducing reported mortality data suggest that large portions of the population voluntarily self-isolate. Swedish ICU use rates remained lower than predicted, but a large fraction of deaths occurred in non-ICU patients. This suggests that patient prognosis was considered in ICU admission, reducing healthcare load at a cost of decreased survival in patients not admitted. CONCLUSIONS: The Swedish COVID-19 strategy has thus far yielded a striking result: mild mandates overlaid with voluntary measures can achieve results highly similar to late-onset stringent mandates. However, this policy causes more healthcare demand and more deaths than early stringent control and depends on continued public will.

Harnessing Conformational Plasticity to Generate Designer Enzymes.

Recent years have witnessed an explosion of interest in understanding the role of conformational dynamics both in the evolution of new enzymatic activities from existing enzymes and in facilitating the emergence of enzymatic activity de novo on scaffolds that were previously non-catalytic. There are also an increasing number of examples in the literature of targeted engineering of conformational dynamics being successfully used to alter enzyme selectivity and activity. Despite the obvious importance of conformational dynamics to both enzyme function and evolvability, many (although not all) computational design approaches still focus either on pure sequence-based approaches or on using structures with limited flexibility to guide the design. However, there exist a wide variety of computational approaches that can be (re)purposed to introduce conformational dynamics as a key consideration in the design process. Coupled with laboratory evolution and more conventional existing sequence- and structure-based approaches, these techniques provide powerful tools for greatly expanding the protein engineering toolkit. This Perspective provides an overview of evolutionary studies that have dissected the role of conformational dynamics in facilitating the emergence of novel enzymes, as well as advances in computational approaches that allow one to target conformational dynamics as part of enzyme design. Harnessing conformational dynamics in engineering studies is a powerful paradigm with which to engineer the next generation of designer biocatalysts.

Evolution of chalcone isomerase from a noncatalytic ancestor.

The emergence of catalysis in a noncatalytic protein scaffold is a rare, unexplored event. Chalcone isomerase (CHI), a key enzyme in plant flavonoid biosynthesis, is presumed to have evolved from a nonenzymatic ancestor related to the widely distributed fatty-acid binding proteins (FAPs) and a plant protein family with no isomerase activity (CHILs). Ancestral inference supported the evolution of CHI from a protein lacking isomerase activity. Further, we identified four alternative founder mutations, i.e., mutations that individually instated activity, including a mutation that is not phylogenetically traceable. Despite strong epistasis in other cases of protein evolution, CHI's laboratory reconstructed mutational trajectory shows weak epistasis. Thus, enantioselective CHI activity could readily emerge despite a catalytically inactive starting point. Accordingly, X-ray crystallography, NMR, and molecular dynamics simulations reveal reshaping of the active site toward a productive substrate-binding mode and repositioning of the catalytic arginine that was inherited from the ancestral fatty-acid binding proteins.

Publisher Correction: Evolution of chalcone isomerase from a noncatalytic ancestor.

In the version of this article originally published, the number for the equal contributions footnote was missing for Miriam Kaltenbach and Jason R. Burke in the author list. The error has been corrected in the PDF and print versions of this article.

Modeling the Alkaline Hydrolysis of Diaryl Sulfate Diesters: A Mechanistic Study.

Phosphate and sulfate esters have important roles in regulating cellular processes. However, while there has been substantial experimental and computational investigation of the mechanisms and the transition states involved in phosphate ester hydrolysis, there is far less work on sulfate ester hydrolysis. Here, we report a detailed computational study of the alkaline hydrolysis of diaryl sulfate diesters, using different DFT functionals as well as mixed implicit/explicit solvation with varying numbers of explicit water molecules. We consider the impact of the computational model on computed linear free-energy relationships (LFER) and the nature of the transition states (TS) involved. We obtain good qualitative agreement with experimental LFER data when using a pure implicit solvent model and excellent agreement with experimental kinetic isotope effects for all models used. Our calculations suggest that sulfate diester hydrolysis proceeds through loose transition states, with minimal bond formation to the nucleophile and bond cleavage to the leaving group already initiated. Comparison to prior work indicates that these TS are similar in nature to those for the alkaline hydrolysis of neutral arylsulfonate monoesters or charged phosphate diesters and fluorophosphates. Obtaining more detailed insights into the transition states involved assists in understanding the selectivity of enzymes that hydrolyze these reactions.

Uncovering the Role of Key Active-Site Side Chains in Catalysis: An Extended Brønsted Relationship for Substrate Deprotonation Catalyzed by Wild-Type and Variants of Triosephosphate Isomerase.

We report results of detailed empirical valence bond simulations that model the effect of several amino acid substitutions on the thermodynamic (ΔG°) and kinetic activation (ΔG⧧) barriers to deprotonation of dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (GAP) bound to wild-type triosephosphate isomerase (TIM), as well as to the K12G, E97A, E97D, E97Q, K12G/E97A, I170A, L230A, I170A/L230A, and P166A variants of this enzyme. The EVB simulations model the observed effect of the P166A mutation on protein structure. The E97A, E97Q, and E97D mutations of the conserved E97 side chain result in ≤1.0 kcal mol-1 decreases in the activation barrier for substrate deprotonation. The agreement between experimental and computed activation barriers is within ±1 kcal mol-1, with a strong linear correlation between ΔG⧧ and ΔG° for all 11 variants, with slopes ß = 0.73 (R2 = 0.994) and ß = 0.74 (R2 = 0.995) for the deprotonation of DHAP and GAP, respectively. These Brønsted-type correlations show that the amino acid side chains examined in this study function to reduce the standard-state Gibbs free energy of reaction for deprotonation of the weak α-carbonyl carbon acid substrate to form the enediolate phosphate reaction intermediate. TIM utilizes the cationic side chain of K12 to provide direct electrostatic stabilization of the enolate oxyanion, and the nonpolar side chains of P166, I170, and L230 are utilized for the construction of an active-site cavity that provides optimal stabilization of the enediolate phosphate intermediate relative to the carbon acid substrate.

GTP Hydrolysis Without an Active Site Base: A Unifying Mechanism for Ras and Related GTPases.

GTP hydrolysis is a biologically crucial reaction, being involved in regulating almost all cellular processes. As a result, the enzymes that catalyze this reaction are among the most important drug targets. Despite their vital importance and decades of substantial research effort, the fundamental mechanism of enzyme-catalyzed GTP hydrolysis by GTPases remains highly controversial. Specifically, how do these regulatory proteins hydrolyze GTP without an obvious general base in the active site to activate the water molecule for nucleophilic attack? To answer this question, we perform empirical valence bond simulations of GTPase-catalyzed GTP hydrolysis, comparing solvent- and substrate-assisted pathways in three distinct GTPases, Ras, Rab, and the Gαi subunit of a heterotrimeric G-protein, both in the presence and in the absence of the corresponding GTPase activating proteins. Our results demonstrate that a general base is not needed in the active site, as the preferred mechanism for GTP hydrolysis is a conserved solvent-assisted pathway. This pathway involves the rate-limiting nucleophilic attack of a water molecule, leading to a short-lived intermediate that tautomerizes to form H2PO4- and GDP as the final products. Our fundamental biochemical insight into the enzymatic regulation of GTP hydrolysis not only resolves a decades-old mechanistic controversy but also has high relevance for drug discovery efforts. That is, revisiting the role of oncogenic mutants with respect to our mechanistic findings would pave the way for a new starting point to discover drugs for (so far) "undruggable" GTPases like Ras.

Role of Ligand-Driven Conformational Changes in Enzyme Catalysis: Modeling the Reactivity of the Catalytic Cage of Triosephosphate Isomerase.

We have previously performed empirical valence bond calculations of the kinetic activation barriers, Δ G‡calc, for the deprotonation of complexes between TIM and the whole substrate glyceraldehyde-3-phosphate (GAP, Kulkarni et al. J. Am. Chem. Soc. 2017 , 139 , 10514 - 10525 ). We now extend this work to also study the deprotonation of the substrate pieces glycolaldehyde (GA) and GA·HPi [HPi = phosphite dianion]. Our combined calculations provide activation barriers, Δ G‡calc, for the TIM-catalyzed deprotonation of GAP (12.9 ± 0.8 kcal·mol-1), of the substrate piece GA (15.0 ± 2.4 kcal·mol-1), and of the pieces GA·HPi (15.5 ± 3.5 kcal·mol-1). The effect of bound dianion on Δ G‡calc is small (≤2.6 kcal·mol-1), in comparison to the much larger 12.0 and 5.8 kcal·mol-1 intrinsic phosphodianion and phosphite dianion binding energy utilized to stabilize the transition states for TIM-catalyzed deprotonation of GAP and GA·HPi, respectively. This shows that the dianion binding energy is essentially fully expressed at our protein model for the Michaelis complex, where it is utilized to drive an activating change in enzyme conformation. The results represent an example of the synergistic use of results from experiments and calculations to advance our understanding of enzymatic reaction mechanisms.

Enzyme Architecture: Modeling the Operation of a Hydrophobic Clamp in Catalysis by Triosephosphate Isomerase.

Triosephosphate isomerase (TIM) is a proficient catalyst of the reversible isomerization of dihydroxyacetone phosphate (DHAP) to d-glyceraldehyde phosphate (GAP), via general base catalysis by E165. Historically, this enzyme has been an extremely important model system for understanding the fundamentals of biological catalysis. TIM is activated through an energetically demanding conformational change, which helps position the side chains of two key hydrophobic residues (I170 and L230), over the carboxylate side chain of E165. This is critical both for creating a hydrophobic pocket for the catalytic base and for maintaining correct active site architecture. Truncation of these residues to alanine causes significant falloffs in TIM's catalytic activity, but experiments have failed to provide a full description of the action of this clamp in promoting substrate deprotonation. We perform here detailed empirical valence bond calculations of the TIM-catalyzed deprotonation of DHAP and GAP by both wild-type TIM and its I170A, L230A, and I170A/L230A mutants, obtaining exceptional quantitative agreement with experiment. Our calculations provide a linear free energy relationship, with slope 0.8, between the activation barriers and Gibbs free energies for these TIM-catalyzed reactions. We conclude that these clamping side chains minimize the Gibbs free energy for substrate deprotonation, and that the effects on reaction driving force are largely expressed at the transition state for proton transfer. Our combined analysis of previous experimental and current computational results allows us to provide an overview of the breakdown of ground-state and transition state effects in enzyme catalysis in unprecedented detail, providing a molecular description of the operation of a hydrophobic clamp in triosephosphate isomerase.

Simulating the reactions of substituted pyridinio-N-phosphonates with pyridine as a model for biological phosphoryl transfer.

Phosphoryl transfer reactions can proceed through several plausible mechanisms, and the potential for both solvent and substrate-assisted pathways (involving proton transfer to the phosphoryl oxygens) complicates both experimental and computational interpretations. To avoid this problem, we have used electronic structure calculations to probe the mechanisms of the reactions of pyridinio-N-phosphonates with pyridine. These compounds avoid the additional complexity introduced by proton transfer between the nucleophile and the leaving group, while also serving as a valuable model for biological P-N cleavage. Through a comparative study of a range of substrates of varying basicity, we demonstrate a unified concerted mechanism for the phosphoryl transfer reactions of these model compounds, proceeding through a dissociative transition state. Finally, a comparison of these transition states with previously characterized transition states for related compounds provides a more complete model for non-enzymatic phosphoryl transfer, which is a critical stepping stone to being able to fully understand phosphoryl transfer in biology.

Capturing the Role of Explicit Solvent in the Dimerization of RuV (bda) Water Oxidation Catalysts.

A ground-breaking empirical valence bond study for a soluble transition-metal complex is presented. The full reaction of catalyst monomers approaching and reacting in the RuV oxidation state were studied. Analysis of the solvation shell in the reactant and along the reaction coordinate revealed that the oxo itself is hydrophobic, which adds a significant driving force to form the dimer. The effect of the solvent on the reaction between the prereactive dimer and the product was small. The solvent seems to lower the barrier for the isoquinoline (isoq) complex while it is increased for pyridines. By comparing the reaction in the gas phase and solution, the proposed π-stacking interaction of the isoq ligands is found to be entirely driven by the water medium.

Promiscuity in the Enzymatic Catalysis of Phosphate and Sulfate Transfer.

The enzymes that facilitate phosphate and sulfate hydrolysis are among the most proficient natural catalysts known to date. Interestingly, a large number of these enzymes are promiscuous catalysts that exhibit both phosphatase and sulfatase activities in the same active site and, on top of that, have also been demonstrated to efficiently catalyze the hydrolysis of other additional substrates with varying degrees of efficiency. Understanding the factors that underlie such multifunctionality is crucial both for understanding functional evolution in enzyme superfamilies and for the development of artificial enzymes. In this Current Topic, we have primarily focused on the structural and mechanistic basis for catalytic promiscuity among enzymes that facilitate both phosphoryl and sulfuryl transfer in the same active site, while comparing this to how catalytic promiscuity manifests in other promiscuous phosphatases. We have also drawn on the large number of experimental and computational studies of selected model systems in the literature to explore the different features driving the catalytic promiscuity of such enzymes. Finally, on the basis of this comparative analysis, we probe the plausible origins and determinants of catalytic promiscuity in enzymes that catalyze phosphoryl and sulfuryl transfer.

Recent advances in QM/MM free energy calculations using reference potentials.

BACKGROUND: Recent years have seen enormous progress in the development of methods for modeling (bio)molecular systems. This has allowed for the simulation of ever larger and more complex systems. However, as such complexity increases, the requirements needed for these models to be accurate and physically meaningful become more and more difficult to fulfill. The use of simplified models to describe complex biological systems has long been shown to be an effective way to overcome some of the limitations associated with this computational cost in a rational way. SCOPE OF REVIEW: Hybrid QM/MM approaches have rapidly become one of the most popular computational tools for studying chemical reactivity in biomolecular systems. However, the high cost involved in performing high-level QM calculations has limited the applicability of these approaches when calculating free energies of chemical processes. In this review, we present some of the advances in using reference potentials and mean field approximations to accelerate high-level QM/MM calculations. We present illustrative applications of these approaches and discuss challenges and future perspectives for the field. MAJOR CONCLUSIONS: The use of physically-based simplifications has shown to effectively reduce the cost of high-level QM/MM calculations. In particular, lower-level reference potentials enable one to reduce the cost of expensive free energy calculations, thus expanding the scope of problems that can be addressed. GENERAL SIGNIFICANCE: As was already demonstrated 40 years ago, the usage of simplified models still allows one to obtain cutting edge results with substantially reduced computational cost. This article is part of a Special Issue entitled Recent developments of molecular dynamics.

The Competing Mechanisms of Phosphate Monoester Dianion Hydrolysis.

Despite the numerous experimental and theoretical studies on phosphate monoester hydrolysis, significant questions remain concerning the mechanistic details of these biologically critical reactions. In the present work we construct a linear free energy relationship for phosphate monoester hydrolysis to explore the effect of modulating leaving group pKa on the competition between solvent- and substrate-assisted pathways for the hydrolysis of these compounds. Through detailed comparative electronic-structure studies of methyl phosphate and a series of substituted aryl phosphate monoesters, we demonstrate that the preferred mechanism is dependent on the nature of the leaving group. For good leaving groups, a strong preference is observed for a more dissociative solvent-assisted pathway. However, the energy difference between the two pathways gradually reduces as the leaving group pKa increases and creates mechanistic ambiguity for reactions involving relatively poor alkoxy leaving groups. Our calculations show that the transition-state structures vary smoothly across the range of pKas studied and that the pathways remain discrete mechanistic alternatives. Therefore, while not impossible, a biological catalyst would have to surmount a significantly higher activation barrier to facilitate a substrate-assisted pathway than for the solvent-assisted pathway when phosphate is bonded to good leaving groups. For poor leaving groups, this intrinsic preference disappears.

Why nature really chose phosphate.

Phosphoryl transfer plays key roles in signaling, energy transduction, protein synthesis, and maintaining the integrity of the genetic material. On the surface, it would appear to be a simple nucleophile displacement reaction. However, this simplicity is deceptive, as, even in aqueous solution, the low-lying d-orbitals on the phosphorus atom allow for eight distinct mechanistic possibilities, before even introducing the complexities of the enzyme catalyzed reactions. To further complicate matters, while powerful, traditional experimental techniques such as the use of linear free-energy relationships (LFER) or measuring isotope effects cannot make unique distinctions between different potential mechanisms. A quarter of a century has passed since Westheimer wrote his seminal review, 'Why Nature Chose Phosphate' (Science 235 (1987), 1173), and a lot has changed in the field since then. The present review revisits this biologically crucial issue, exploring both relevant enzymatic systems as well as the corresponding chemistry in aqueous solution, and demonstrating that the only way key questions in this field are likely to be resolved is through careful theoretical studies (which of course should be able to reproduce all relevant experimental data). Finally, we demonstrate that the reason that nature really chose phosphate is due to interplay between two counteracting effects: on the one hand, phosphates are negatively charged and the resulting charge-charge repulsion with the attacking nucleophile contributes to the very high barrier for hydrolysis, making phosphate esters among the most inert compounds known. However, biology is not only about reducing the barrier to unfavorable chemical reactions. That is, the same charge-charge repulsion that makes phosphate ester hydrolysis so unfavorable also makes it possible to regulate, by exploiting the electrostatics. This means that phosphate ester hydrolysis can not only be turned on, but also be turned off, by fine tuning the electrostatic environment and the present review demonstrates numerous examples where this is the case. Without this capacity for regulation, it would be impossible to have for instance a signaling or metabolic cascade, where the action of each participant is determined by the fine-tuned activity of the previous piece in the production line. This makes phosphate esters the ideal compounds to facilitate life as we know it.

The conformation of a catalytic loop is central to GTPase activity on the ribosome.

The translational GTPases hydrolyze GTP on the ribosome at several stages of the protein synthesis cycle. Because of the strong conservation of their catalytic center, these enzymes are expected to operate through a universal hydrolysis mechanism, in which a critical histidine residue together with the sarcin-ricin loop of the large ribosomal subunit is necessary for GTPase activation. Here we examine different possible pathways for GTP hydrolysis by EF-Tu through extensive computer simulations. We show that a conformational change of the peptide plane preceding this histidine has a decisive effect on the energetics of the reaction. This transition was predicted earlier by us and has recently been confirmed experimentally. It is found to promote early proton transfer from water to the γ-phosphate group of GTP, followed by nucleophilic attack by hydroxide ion. The calculated reaction energetics is in good agreement with available kinetic data, for both wild-type and mutant versions of EF-Tu, and indicates that the latter may enforce a change in mechanism toward more concerted pathways.