Enzymes employ a wide range of protein motions to achieve efficient catalysis of chemical reactions. While the role of collective protein motions in substrate binding, product release, and regulation of enzymatic activity is generally understood, their roles in catalytic steps per se remain uncertain. Here, molecular dynamics simulations, enzyme kinetics, X-ray crystallography, and nuclear magnetic resonance spectroscopy are combined to elucidate the catalytic mechanism of adenylate kinase and to delineate the roles of catalytic residues in catalysis and the conformational change in the enzyme. This study reveals that the motions in the active site, which occur on a time scale of picoseconds to nanoseconds, link the catalytic reaction to the slow conformational dynamics of the enzyme by modulating the free energy landscapes of subdomain motions. In particular, substantial conformational rearrangement occurs in the active site following the catalytic reaction. This rearrangement not only affects the reaction barrier but also promotes a more open conformation of the enzyme after the reaction, which then results in an accelerated opening of the enzyme compared to that of the reactant state. The results illustrate a linkage between enzymatic catalysis and collective protein motions, whereby the disparate time scales between the two processes are bridged by a cascade of intermediate-scale motion of catalytic residues modulating the free energy landscapes of the catalytic and conformational change processes.

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters form a family of molecular motor proteins that couple ATP hydrolysis to substrate translocation across cell membranes. Each nucleotide binding domain of ABC-transporters contains a highly conserved H-loop histidine residue, whose precise mechanistic role in motor functions has remained elusive. By using combined quantum mechanical and molecular mechanical (QM/MM) calculations, we showed that the conserved H-loop residue H662 in E. coli HlyB, a bacterial ABC-transporter, can act first as a general acid and then as a general base to facilitate proton transfer in ATP hydrolysis. Without the assistance of H662, direct proton transfer from the lytic water to ATP results in a substantially higher barrier height. Our findings suggest that the essential function of the H-loop residue H662 is to provide a "chemical linchpin" that shuttles protons between reactants through a relay mechanism, thereby catalyzing ATP hydrolysis in HlyB.

We demonstrate that an external constant electric field is able to modify the secondary structure of a protein and induce a transition from a beta-sheet into a helix-like conformation. This dramatic change is driven by a global rearrangement of the dipole moments at the amide planes. We also predict electric-field-induced modifications of the intermediate states of the protein.

Escherichia coli adenylate kinase (AdK) is a small, monomeric enzyme that synchronizes the catalytic step with the enzyme's conformational dynamics to optimize a phosphoryl transfer reaction and the subsequent release of the product. Guided by experimental measurements of low catalytic activity in seven single-point mutation AdK variants (K13Q, R36A, R88A, R123A, R156K, R167A, and D158A), we utilized classical mechanical simulations to probe mutant dynamics linked to product release, and quantum mechanical and molecular mechanical calculations to compute a free energy barrier for the catalytic event. The goal was to establish a mechanistic connection between the two activities. Our calculations of the free energy barriers in AdK variants were in line with those from experiments, and conformational dynamics consistently demonstrated an enhanced tendency toward enzyme opening. This indicates that the catalytic residues in the wild-type AdK serve a dual role in this enzyme's function�one to lower the energy barrier for the phosphoryl transfer reaction and another to delay enzyme opening, maintaining it in a catalytically active, closed conformation for long enough to enable the subsequent chemical step. Our study also discovers that while each catalytic residue individually contributes to facilitating the catalysis, R36, R123, R156, R167, and D158 are organized in a tightly coordinated interaction network and collectively modulate AdK's conformational transitions. Unlike the existing notion of product release being rate-limiting, our results suggest a mechanistic interconnection between the chemical step and the enzyme's conformational dynamics acting as the bottleneck of the catalytic process. Our results also suggest that the enzyme's active site has evolved to optimize the chemical reaction step while slowing down the overall opening dynamics of the enzyme.

Most of the ongoing projects aimed at the development of specific therapies and vaccines against COVID-19 use the SARS-CoV-2 spike (S) protein as the main target. The binding of the spike protein with the ACE2 receptor (ACE2) of the host cell constitutes the first and key step for virus entry. During this process, the receptor binding domain (RBD) of the S protein plays an essential role, since it contains the receptor binding motif (RBM), responsible for the docking to the receptor. So far, mostly biochemical methods are being tested in order to prevent binding of the virus to ACE2. Here we show, with the help of atomistic simulations, that external electric fields of easily achievable and moderate strengths can dramatically destabilise the S protein, inducing long-lasting structural damage. One striking field-induced conformational change occurs at the level of the recognition loop L3 of the RBD where two parallel beta sheets, believed to be responsible for a high affinity to ACE2, undergo a change into an unstructured coil, which exhibits almost no binding possibilities to the ACE2 receptor. We also show that these severe structural changes upon electric-field application also occur in the mutant RBDs corresponding to the variants of concern (VOC) B.1.1.7 (UK), B.1.351 (South Africa) and P.1 (Brazil). Remarkably, while the structural flexibility of S allows the virus to improve its probability of entering the cell, it is also the origin of the surprising vulnerability of S upon application of electric fields of strengths at least two orders of magnitude smaller than those required for damaging most proteins. Our findings suggest the existence of a clean physical method to weaken the SARS-CoV-2 virus without further biochemical processing. Moreover, the effect could be used for infection prevention purposes and also to develop technologies for in-vitro structural manipulation of S. Since the method is largely unspecific, it can be suitable for application to other mutations in S, to other proteins of SARS-CoV-2 and in general to membrane proteins of other virus types.

The catalytic and allosteric mechanisms of insulin receptor kinase (IRK) are investigated by a combination of ab initio and semiempirical quantum mechanical and molecular mechanical (QM/MM) methods and classical molecular dynamics (MD) simulations. The simulations reveal that the catalytic reaction proceeds in two steps, starting with the transfer of a proton from substrate Tyr to the catalytic Asp1132, followed by the phosphoryl transfer from ATP to substrate Tyr. The enhancement of the catalytic rate of IRK upon phosphorylations in the enzyme's activation loop is found to occur mainly via changes to the free energy landscape of the proton transfer step, favoring the proton transfer in the fully phosphorylated enzyme. In contrast, the effects of the phosphorylations on the phosphoryl transfer are smaller. Equilibrium MD simulations show that IRK phosphorylations affect the protein dynamics of the enzyme before the proton transfer to Asp1132 with only a minor effect after the proton transfer. This finding is consistent with the large change in the proton transfer free energy and the smaller change in the free energy barrier of phosphoryl transfer found by QM/MM simulations. Taken together, the present results provide details on how IRK phosphorylation exerts allosteric control of the catalytic activity via modifications of protein dynamics and free energy landscape of catalytic reaction. The results also highlight the importance of protein dynamics in connecting protein allostery and catalysis to control catalytic activity of enzymes.

First-principles determination of free energy profiles for condensed-phase chemical reactions is hampered by the daunting costs associated with configurational sampling on ab initio quantum mechanical/molecular mechanical (AI/MM) potential energy surfaces. Here, we report a new method that enables efficient AI/MM free energy simulations through mean force fitting. In this method, a free energy path in collective variables (CVs) is first determined on an efficient reactive aiding potential. Based on the configurations sampled along the free energy path, correcting forces to reproduce the AI/MM forces on the CVs are determined through force matching. The AI/MM free energy profile is then predicted from simulations on the aiding potential in conjunction with the correcting forces. Such cycles of correction−prediction are repeated until convergence is established. As the instantaneous forces on the CVs sampled in equilibrium ensembles along the free energy path are fitted, this procedure faithfully restores the target free energy profile by reproducing the free energy mean forces. Due to its close connection with the reaction path-force matching (RP-FM) framework recently introduced by us, we designate the new method as RP-FM in collective variables (RP-FM-CV). We demonstrate the effectiveness of this method on a type-II solution-phase SN 2 reaction, NH 3 + CH 3 Cl (the Menshutkin reaction), simulated with an explicit water solvent. To obtain the AI/MM free energy profiles, we employed the semiempirical AM1/MM Hamiltonian as the base level for determining the string minimum free energy pathway, along which the free energy mean forces are fitted to various target AI/MM levels using the Hartree−Fock (HF) theory, density functional theory (DFT), and the second-order Møller−Plesset perturbation (MP2) theory as the AI method. The forces on the bond-breaking and bond-forming CVs at both the base and target levels are obtained by force transformation from Cartesian to redundant internal coordinates under the Wilson B-matrix formalism, where the linearized FM is facilitated by the use of spline functions. For the Menshutkin reaction tested, our FM treatment greatly reduces the deviations on the CV forces, originally in the range of 12−33 to ∼2 kcal/mol/Å. Comparisons with the experimental and benchmark AI/MM results, tests of the new method under a variety of simulation protocols, and analyses of the solute−solvent radial distribution functions suggest that RP-FM-CV can be used as an efficient, accurate, and robust method for simulating solution-phase chemical reactions.

The isotropic periodic sum (IPS) method was extended to describe long-range electrostatic interactions in combined quantum mechanical and molecular mechanical (QM/MM) calculations. The resulting method, designated QM/MM-IPS, was tested for two ion association processes and a model SN2 reaction in aqueous solution. Potential of mean force (PMF) profiles and radial distribution functions computed from the QM/MM-IPS simulations were compared with those obtained by using the existing QM/MM-Ewald sum and cutoff (QM/MM-Cutoff) methods. In contrast to the QM/MM-Cutoff method, with which PMFs of ion separation tend to display a spurious linear drift, the QM/MM-IPS method successfully eliminates such artifacts, in excellent agreement with the QM/MM-Ewald results. The PMF obtained with the QM/MM-IPS method for the SN2 reaction that transfers an NH3 group between two chloride anions closely resembles that from the QM/MM-Ewald simulations. Compared with QM/MM-Ewald, the QM/MM-IPS method reduces the computational cost by 60-70% when a local region of 12 to 14 Å is used. These results suggest that the QM/MM-IPS method can be used as a reliable and efficient alternative to the QM/MM-Ewald method to incorporate long-range electrostatic effects in simulating solution-phase chemical reactions.