Pseudo-Luciferase Activity of the SARS-CoV-2 Spike Protein for Cypridina Luciferin.

Enzymatic reactions that involve a luminescent substrate (luciferin) and enzyme (luciferase) from luminous organisms enable a luminescence detection of target proteins and cells with high specificity, albeit that conventional assay design requires a prelabeling of target molecules with luciferase. Here, we report a luciferase-independent luminescence assay in which the target protein directly catalyzes the oxidative luminescence reaction of luciferin. The SARS-CoV-2 antigen (spike) protein catalyzes the light emission of Cypridina luciferin, whereas no such catalytic function was observed for salivary proteins. This selective luminescence reaction is due to the enzymatic recognition of the 3-(1-guanidino)propyl group in luciferin at the interfaces between the units of the spike protein, allowing a specific detection of the spike protein in human saliva without sample pretreatment. This method offers a novel platform to detect virus antigens simply and rapidly without genetic manipulation or antibodies.

Chaperonin GroEL hydrolyses ortho-nitrophenyl ß-galactoside.

We serendipitously found that chaperonin GroEL can hydrolyze ortho-nitrophenyl ß-galactoside (ONPG), a well-known substrate of the enzyme ß-galactosidase. The ONPG hydrolysis by GroEL follows typical enzyme kinetics. Our experiments and molecular docking studies suggest ONPG binding at the ATP binding site of GroEL.

Structural effects of spike protein D614G mutation in SARS-CoV-2.

A single mutation from aspartate to glycine at position 614 has dominated all circulating variants of the severe acute respiratory syndrome coronavirus 2. D614G mutation induces structural changes in the spike (S) protein that strengthen the virus infectivity. Here, we use molecular dynamics simulations to dissect the effects of mutation and 630-loop rigidification on S-protein structure. The introduction of the mutation orders the 630-loop structure and thereby induces global structural changes toward the cryoelectron microscopy structure of the D614G S-protein. The ordered 630-loop weakens local interactions between the 614th residue and others in contrast to disordered structures in the wild-type protein. The mutation allosterically alters global interactions between receptor-binding domains, forming an asymmetric and mobile down conformation and facilitating transitions toward up conformation. The loss of salt bridge between D614 and K854 upon the mutation generally stabilizes S-protein protomer, including the fusion peptide proximal region that mediates membrane fusion. Understanding the molecular basis of D614G mutation is crucial as it dominates in all variants of concern, including Delta and Omicron.

Modified Protein-Water Interactions in CHARMM36m for Thermodynamics and Kinetics of Proteins in Dilute and Crowded Solutions.

Proper balance between protein-protein and protein-water interactions is vital for atomistic molecular dynamics (MD) simulations of globular proteins as well as intrinsically disordered proteins (IDPs). The overestimation of protein-protein interactions tends to make IDPs more compact than those in experiments. Likewise, multiple proteins in crowded solutions are aggregated with each other too strongly. To optimize the balance, Lennard-Jones (LJ) interactions between protein and water are often increased about 10% (with a scaling parameter, λ = 1.1) from the existing force fields. Here, we explore the optimal scaling parameter of protein-water LJ interactions for CHARMM36m in conjunction with the modified TIP3P water model, by performing enhanced sampling MD simulations of several peptides in dilute solutions and conventional MD simulations of globular proteins in dilute and crowded solutions. In our simulations, 10% increase of protein-water LJ interaction for the CHARMM36m cannot maintain stability of a small helical peptide, (AAQAA)3 in a dilute solution and only a small modification of protein-water LJ interaction up to the 3% increase (λ = 1.03) is allowed. The modified protein-water interactions are applicable to other peptides and globular proteins in dilute solutions without changing thermodynamic properties from the original CHARMM36m. However, it has a great impact on the diffusive properties of proteins in crowded solutions, avoiding the formation of too sticky protein-protein interactions.

The inherent flexibility of receptor binding domains in SARS-CoV-2 spike protein.

Spike (S) protein is the primary antigenic target for neutralization and vaccine development for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It decorates the virus surface and undergoes large motions of its receptor binding domains (RBDs) to enter the host cell. Here, we observe Down, one-Up, one-Open, and two-Up-like structures in enhanced molecular dynamics simulations, and characterize the transition pathways via inter-domain interactions. Transient salt-bridges between RBDA and RBDC and the interaction with glycan at N343B support RBDA motions from Down to one-Up. Reduced interactions between RBDA and RBDB in one-Up induce RBDB motions toward two-Up. The simulations overall agree with cryo-electron microscopy structure distributions and FRET experiments and provide hidden functional structures, namely, intermediates along Down-to-one-Up transition with druggable cryptic pockets as well as one-Open with a maximum exposed RBD. The inherent flexibility of S-protein thus provides essential information for antiviral drug rational design or vaccine development.

Unraveling the Coupling between Conformational Changes and Ligand Binding in Ribose Binding Protein Using Multiscale Molecular Dynamics and Free-Energy Calculations.

Conformational changes of proteins upon ligand binding are usually explained in terms of several mechanisms including the induced fit, conformational selection, or their mixtures. Due to the slow time scales, conventional molecular dynamics (cMD) simulations based on the atomistic models cannot easily simulate the open-to-closed conformational transition in proteins. In our previous study, we have developed an enhanced sampling scheme (generalized replica exchange with solute tempering selected surface charged residues: gREST_SSCR) for multidomain proteins and applied it to ligand-mediated conformational changes in the G134R mutant of ribose-binding protein (RBPG134R) in solution. The free-energy landscape (FEL) of RBPG134R in the presence of a ribose at the binding site included the open and closed states and two intermediates, open-like and closed-like forms. Only the open and open-like forms existed in the FEL without a ribose. In the current study, the coupling between the conformational changes and ligand binding is further investigated using coarse-grained MD, multiple atomistic cMD, and free-energy calculations. The ribose is easily dissociated from the binding site of wild-type RBP and RBPG134R in the cMD simulations starting from the open and open-like forms. In contrast, it is stable at the binding site in the simulations from the closed and closed-like forms. The free-energy calculations provide the binding affinities of different structures, supporting the results of cMD simulations. Importantly, cMD simulations from the closed-like structures reveal transitions toward the closed one in the presence of a bound ribose. On the basis of the computational results, we propose a molecular mechanism in which conformational selection and induced fit happen in the first and second halves of the open-to-closed transition in RBP, respectively.

Elucidation of interactions regulating conformational stability and dynamics of SARS-CoV-2 S-protein.

The ongoing COVID-19 pandemic caused by the new coronavirus, SARS-CoV-2, calls for urgent developments of vaccines and antiviral drugs. The spike protein of SARS-CoV-2 (S-protein), which consists of trimeric polypeptide chains with glycosylated residues on the surface, triggers the virus entry into a host cell. Extensive structural and functional studies on this protein have rapidly advanced our understanding of the S-protein structure at atomic resolutions, although most of these structural studies overlook the effect of glycans attached to the S-protein on the conformational stability and functional motions between the inactive down and active up forms. Here, we performed all-atom molecular dynamics simulations of both down and up forms of a fully glycosylated S-protein in solution as well as targeted molecular dynamics simulations between them to elucidate key interdomain interactions for stabilizing each form and inducing the large-scale conformational transitions. The residue-level interaction analysis of the simulation trajectories detects distinct amino acid residues and N-glycans as determinants on conformational stability of each form. During the conformational transitions between them, interdomain interactions mediated by glycosylated residues are switched to play key roles on the stabilization of another form. Electrostatic interactions, as well as hydrogen bonds between the three receptor binding domains, work as driving forces to initiate the conformational transitions toward the active form. This study sheds light on the mechanisms underlying conformational stability and functional motions of the S-protein, which are relevant for vaccine and antiviral drug developments.

Exploring Large Domain Motions in Proteins Using Atomistic Molecular Dynamics with Enhanced Conformational Sampling.

Conformational transitions in multidomain proteins are essential for biological functions. The Apo conformations are typically open and flexible, while the Holo states form more compact conformations stabilized by protein-ligand interactions. Unfortunately, the atomically detailed mechanisms for such open-closed conformational changes are difficult to be accessed experimentally as well as computationally. To simulate the transitions using atomistic molecular dynamics (MD) simulations, efficient conformational sampling algorithms are required. In this work, we propose a new approach based on generalized replica-exchange with solute tempering (gREST) for exploring the open-closed conformational changes in multidomain proteins. Wherein, selected surface charged residues in a target protein are defined as the solute region in gREST simulation and the solute temperatures are different in replicas and exchanged between them to enhance the domain motions. This approach is called gREST selected surface charged residues (gREST_SSCR) and is applied to the Apo and Holo states of ribose binding protein (RBP) in solution. The conformational spaces sampled with gREST_SSCR are much wider than those with the conventional MD, sampling open-closed conformational changes while maintaining RBP domains' stability. The free-energy landscapes of RBP in the Apo and Holo states are drawn along with twist and hinge angles of the two moving domains. The inter-domain salt-bridges that are not observed in the experimental structures are also important in the intermediate states during the conformational changes.

Similarities and Differences between Thymine(6-4)Thymine/Cytosine DNA Lesion Repairs by Photolyases.

Photolyases are ancient enzymes that harvest sunlight to repair DNA pyrimidine lesions such as pyrimidine(6-4)pyrimidone and cyclobutane dimers. Particularly, (6-4) photolyase ((6-4)PHR) plays an important role in maintaining genetic integrity by repairing thymine(6-4)thymine (T(6-4)T) and thymine(6-4)cytosine (T(6-4)C) photolesions. The majority of (6-4)PHR studies have been performed on the basis of the former's activity and assuming the equivalence of the two repair mechanisms, although the latter's activity remains poorly studied. Here, we describe investigations of the repair process of the T(6-4)C dimer using several computational methods from molecular dynamics (MD) simulations to large quantum mechanical/molecular mechanical approaches. Two possible mechanisms, the historically proposed azetidine four-member ring intermediate and the free NH3 formation pathways, were considered. The MD results predicted that important active site histidine residues employed for the repair of the T(6-4)C dimer have protonation states similar to those seen in the (6-4)PHR/T(6-4)T complex. More importantly, despite chemical differences between the two substrates, a similar repair mechanism was identified: His365 protonates NH2, resulting in formation/activation mechanism of a free NH3, inducing NH2 transfer to the 5' base, and ultimately leading to pyrimidine restoration. This reaction is thermodynamically favorable with a rate-limiting barrier of 20.4 kcal mol-1. In contrast, the azetidine intermediate is unfeasible, possessing an energy barrier of 60 kcal mol-1; this barrier is similar to that predicted for the oxetane intermediate in T(6-4)T repair. Although both substrates are repaired with comparable quantum yields, the reactive complex in T(6-4)C was shown to be a 3' base radical with a lower driving force for back electron transfer combined with higher energy barrier for catalysis. These results showed the similarity in the general repair mechanisms between the two substrates while emphasizing differences in the electron dynamics in the repair cycle.

A Pseudohypervalent Sulfur Intermediate as an Oxidative Protective Mechanism in the Archaea Peroxiredoxin Enzyme ApTPx.

Peroxiredoxins (Prxs) are a ubiquitous class of enzymes that have central roles in a number of important physiological processes. Using a multiscale computational approach, we have investigated the mechanism by which the active-site cysteine (Cys50) in the typical 2-Cys Prx from Archaea (ApTPx) is oxidized by H2O2 to sulfenic acid. In addition, its further oxidation to give a sulfinic acid and its possible alternate intramolecular reaction to form an experimentally proposed hypervalent sulfurane were examined. Oxidation of Cys50 by H2O2 to give the sulfenic acid intermediate occurs in one step with a barrier of 82.1 kJ mol-1. A two-step pathway is proposed with a very low barrier of 16.5 kJ mol-1 by which it can subsequently react with an adjacent histidyl (His42) to form the pseudohypervalent sulfurane. This pathway also involves an adjacent aspartyl (Asp45), which helps alternate the protonation state of His42. The sulfurane's Cys50S···NδHis42 interaction was characterized using QTAIM, NCI, and NBO analyses and found to be a noncovalent interaction. Notably, this bond helps orient the Cys50SOH moiety such that it is less susceptible to oxidation by H2O2 to sulfinic acid. Significantly, sulfurane formation is energetically favored to further H2O2 oxidation of Cys50SOH to a sulfinic acid, providing a mechanism by which the active-site Cys50 is protected against overoxidation.

Functional Conversion of CPD and (6-4) Photolyases by Mutation.

Ultraviolet (UV) light from the sun damages DNA by forming a cyclobutane pyrimidine dimer (CPD) and pyrimidine(6-4)pyrimidone photoproducts [(6-4) PP]. Photolyase (PHR) enzymes utilize near-UV/blue light for DNA repair, which is initiated by light-induced electron transfer from the fully reduced flavin adenine dinucleotide chromophore. Despite similar structures and repair mechanisms, the functions of PHR are highly selective; CPD PHR repairs CPD, but not (6-4) PP, and vice versa. In this study, we attempted functional conversion between CPD and (6-4) PHRs. We found that a triple mutant of (6-4) PHR is able to repair the CPD photoproduct, though the repair efficiency is 1 order of magnitude lower than that of wild-type CPD PHR. Difference Fourier transform infrared spectra for repair demonstrate the lack of secondary structural alteration in the mutant, suggesting that the triple mutant gains substrate binding ability while it does not gain the optimized conformational changes from light-induced electron transfer to the release of the repaired DNA. Interestingly, the (6-4) photoproduct is not repaired by the reverse mutation of CPD PHR, and eight additional mutations (total of 11 mutations) introduced into CPD PHR are not sufficient. The observed asymmetric functional conversion is interpreted in terms of a more complex repair mechanism for (6-4) repair, which was supported by quantum chemical/molecular mechanical calculation. These results suggest that CPD PHR may represent an evolutionary origin for photolyase family proteins.

Computational investigations on the catalytic mechanism of maleate isomerase: the role of the active site cysteine residues.

The maleate isomerase (MI) catalysed isomerization of maleate to fumarate has been investigated using a wide range of computational modelling techniques, including small model DFT calculations, QM-cluster approach, quantum mechanical/molecular mechanical approach (QM/MM in the ONIOM formalism) and molecular dynamics simulations. Several fundamental questions regarding the mechanism were answered in detail, such as the activation and stabilization of the catalytic Cys in a rather hydrophobic active site. The two previously proposed mechanisms were considered, where either enediolate or succinyl-Cys intermediate forms. Small model calculations as well as an ONIOM-based approach suggest that an enediolate intermediate is too unstable. Furthermore, the formation of succinyl-Cys intermediate via the nucleophilic attack of Cys76(-) on the substrate C2 (as proposed experimentally) was found to be energetically unfeasible in both QM-cluster and ONIOM approaches. Instead, our results show that Cys194, upon activation via the substrate, acts as a nucleophile and Cys76 acts as an acid/base catalyst, forming a succinyl-Cys intermediate in a concerted fashion. Indeed, the calculated PA of Cys76 is always higher than that of Cys194 before or upon substrate binding in the active site. Furthermore, the mechanism proceeds via multiple steps by substrate rotation around C2-C3 with the assistance of the now negatively charged Cys76, leading to the formation of fumarate. Finally, our calculated barrier is in good agreement with experiment. These findings represent a novel mechanism in the racemase superfamily.

A molecular dynamics and quantum mechanics/molecular mechanics study of the catalytic reductase mechanism of methionine sulfoxide reductase A: formation and reduction of a sulfenic acid.

The catalytic mechanism of MsrA in Mycobacterium tuberculosis, in which S-methionine sulfoxide (Met-O) is reduced to methionine (Met), has been investigated using docking, molecular dynamics (MD) simulations, and ONIOM (quantum mechanics/molecular mechanics) methods. In addition, the roles of specific active site residues, including an aspartyl (Asp87) near the recycling cysteine, tyrosyls (Tyr44 and Tyr92), and glutamyl (Glu52), have been examined, as well as the general effects of the protein and active site on the nature and properties of mechanistic intermediates. The mechanism is initiated by the transfer of a proton from the catalytic cysteine's thiol (Cys13SH) via a bridging water to the R group carboxylate of Glu52. The now anionic sulfur of Cys13 nucleophilically attacks the substrate's sulfur with concomitant transfer of a proton from Glu52 to the sulfoxide oxygen, generating a sulfurane. The active site enhances the proton affinity of the sulfurane oxygen, which can readily accept a proton from the phenolic hydroxyls of Tyr44 or Tyr92 to give a sulfonium cation. Subsequently, Asp87 and the recycling cysteine (Cys154) can facilitate nucleophilic attack of a solvent water at the Cys13S center of the sulfonium to give a sulfenic acid (Cys13SOH) and Met. For the subsequent reduction of Cys13SOH with intramolecular disulfide bond formation, Asp87 can help facilitate nucleophilic attack of Cys154S at the sulfur of Cys13SOH by deprotonating its thiol. This reduction is found likely to occur readily upon suitable positioning of the active site hydrogen bond network and the sulfur centers of both Cys13 and Cys154. The calculated rate-limiting barrier is in good agreement with experiment.

Multi-scale computational enzymology: enhancing our understanding of enzymatic catalysis.

Elucidating the origin of enzymatic catalysis stands as one the great challenges of contemporary biochemistry and biophysics. The recent emergence of computational enzymology has enhanced our atomistic-level description of biocatalysis as well the kinetic and thermodynamic properties of their mechanisms. There exists a diversity of computational methods allowing the investigation of specific enzymatic properties. Small or large density functional theory models allow the comparison of a plethora of mechanistic reactive species and divergent catalytic pathways. Molecular docking can model different substrate conformations embedded within enzyme active sites and determine those with optimal binding affinities. Molecular dynamics simulations provide insights into the dynamics and roles of active site components as well as the interactions between substrate and enzymes. Hybrid quantum mechanical/molecular mechanical (QM/MM) can model reactions in active sites while considering steric and electrostatic contributions provided by the surrounding environment. Using previous studies done within our group, on OvoA, EgtB, ThrRS, LuxS and MsrA enzymatic systems, we will review how these methods can be used either independently or cooperatively to get insights into enzymatic catalysis.

A sulfonium cation intermediate in the mechanism of methionine sulfoxide reductase B: a DFT study.

The hybrid density functional theory method B3LYP in combination with three systematically larger active site models has been used to investigate the substrate binding and catalytic mechanism by which Neisseria gonorrhoeae methionine sulfoxide reductase B (MsrB) reduces methionine-R-sulfoxide (Met-R-SO) to methionine. The first step in the overall mechanism is nucleophilic attack of an active site thiolate at the sulfur of Met-R-SO to form an enzyme-substrate sulfurane. This occurs with concomitant proton transfer from an active site histidine (His480) residue to the substrates oxygen center. The barrier for this step, calculated using our largest most complete active site model, is 17.2 kJ mol(-1). A subsequent conformational rearrangement and intramolecular -OH transfer to form an enzyme-derived sulfenic acid ((Cys495)S-OH) is not enzymatically feasible. Instead, transfer of a second proton from a second histidyl active site residue (His477) to the sulfurane's oxygen center to give water and a sulfonium cation intermediate is found to be greatly preferred, occurring with a quite low barrier of just 1.2 kJ mol(-1). Formation of the final product complex in which an intraprotein disulfide bond is formed with generation of methionine preferably occurs in one step via nucleophilic attack of the sulfur of a second enzyme thiolate ((Cys440)S(-)) at the S(Cys495) center of the sulfonium intermediate with a barrier of 23.8 kJ mol(-1). An alternate pathway for formation of the products via a sulfenic acid intermediate involves enzymatically feasible, but higher energy barriers. The role and impact of hydrogen bonding and active site residues on the properties and stability of substrate and mechanism intermediates and the affects of mutating His477 are also examined and discussed.

Assignment of the 6,6'-dioxo-3,3'-biverdazyl ground state by using broken symmetry and spectroscopy oriented configuration interaction techniques.

The singlet-triplet energy differences (DeltaE(ST)) and the Heisenberg-Dirac-van-Vleck exchange parameter (J) of 6,6'-dioxo-3,3'-biverdazyl (BVD) have been studied by hybrid density functional (HDF), broken symmetry (BS), and spectroscopy oriented configuration interaction (SORCI) methods. Energy surface scans as a function of the dihedral angle between the two verdazyl rings (phi(N2C3C3'N2')) have been performed. The BS computations predict an antiferromagnetic ground state. However, the diradical index (R(BS)) ranges from 97.5 to 99.9%, implying that the interactions between the two unpaired electrons are very weak. To calculate J and DeltaE(ST), the multireference character introduced by these weak spin-spin interactions must be taken into account. Consequently, multireference difference dedicated configuration interaction (MRDDCI) methods, as implemented in the SORCI procedure, are used. The in-plane pi (IPpi), out-of-plane pi (OPpi), and sigma configurations are included in the CI expansions in a balanced fashion. The OPpi-OPpi and OPpi-IPpi overlaps are the predominant factors that influence the J and DeltaE(ST) as a function of phi(N2C3C3'N2') and cause them to peak around 40 and 140 degrees. In these regions, the antiferromagnetic interactions are minimal, and the MRDDCI methods predict a triplet ground state. At phi(N2C3C3'N2') = 0, DeltaE(ST)[MRDDCI3(14,12)] is in excellent agreement with that of 1,1',5,5'-tetramethyl-6,6'-dioxo-3,3'-biverdazyl determined experimentally from electron paramagnetic resonance (EPR) spectroscopy and differs only by 2.3%. Furthermore, DeltaE(ST)[MRDDCI3(14,12)] is consistently smaller than J(Y) as the verdazyl rings rotate with respect to each other. This corroborates the theory that the HDF-BS technique increases the singlet-triplet energy gap and favors the singlet state. Because the SORCI method is specifically designed for large molecules, the present very good results open the way for the computation of the magnetic properties of larger molecules by the SORCI method. To the best of our knowledge, this is the first time that DeltaE(ST) has been computed by the MRDDCI3 method by utilizing such a large CI reference space for a molecule of this size.