Analysis of the molecular determinants for furin cleavage of the spike protein S1/S2 site in defined strains of the prototype coronavirus murine hepatitis virus (MHV).

We analyzed the spike protein S1/S2 cleavage of selected strains of a prototype coronavirus, mouse hepatitis virus (MHV) by the cellular protease furin, in order to understand the structural requirements underlying the sequence selectivity of the scissile segment. The probability of cleavage of selected MHV strains was first evaluated from furin cleavage scores predicted by the ProP computer software, and then cleavage was measured experimentally with a fluorogenic peptide cleavage assay consisting of S1/S2 peptide mimics and purified furin. We found that in vitro cleavability varied across MHV strains in line with predicted results-but with the notable exception of MHV-A59, which was not cleaved despite a high score predicted for its sequence. Using the known X-Ray structure of furin in complex with a substrate-like inhibitor as an initial structural reference, we carried out molecular dynamics (MD) simulations to learn the modes of binding of the peptides in the furin active site, and the suitability of the complex for initiation of the enzymatic cleavage. We identified the 3D structural requirements of the furin active site configuration that enable bound peptides to undergo cleavage, and the way in which the various strains tested experimentally are fulfilling these requirements. We find that despite some flexibility in the organization of the peptide bound to the active site of the enzyme, the presence of a histidine at P2 of MHV-A59 fails to properly orient the sidechain of His194 of the furin catalytic triad and therefore produces a distortion that renders the peptide/complex structural configuration in the active site incompatible with requirements for cleavage initiation. The Ser/Thr in P1 of MHV-2 and MHV-S has a similar effect of distorting the conformation of the furin active site residues produced by the elimination of the canonical salt-bridge formed by arginine in P1 position. This work informs a study of coronavirus infection and pathogenesis with respect to the function of the viral spike protein, and suggests an important process of viral adaptation and evolution within the spike S1/S2 structural loop.

BTG1 mutation yields supercompetitive B cells primed for malignant transformation.

Multicellular life requires altruistic cooperation between cells. The adaptive immune system is a notable exception, wherein germinal center B cells compete vigorously for limiting positive selection signals. Studying primary human lymphomas and developing new mouse models, we found that mutations affecting BTG1 disrupt a critical immune gatekeeper mechanism that strictly limits B cell fitness during antibody affinity maturation. This mechanism converted germinal center B cells into supercompetitors that rapidly outstrip their normal counterparts. This effect was conferred by a small shift in MYC protein induction kinetics but resulted in aggressive invasive lymphomas, which in humans are linked to dire clinical outcomes. Our findings reveal a delicate evolutionary trade-off between natural selection of B cells to provide immunity and potentially dangerous features that recall the more competitive nature of unicellular organisms.

Analysis of the molecular determinants for furin cleavage of the spike protein S1/S2 site in defined strains of the prototype coronavirus murine hepatitis virus (MHV).

We have analyzed the spike protein S1/S2 cleavage site of selected strains of MHV by the cellular protease furin, in order to understand the structural requirements underlying the sequence selectivity of the scissile segment. The probability of cleavage of the various MHV strains was first evaluated from furin cleavage scores predicted by the ProP computer software, and then cleavage was measured experimentally with a fluorogenic peptide cleavage assay consisting of S1/S2 peptide mimics and purified furin. We found that in vitro cleavability varied across MHV strains in line with predicted results-but with the notable exception of MHV-A59, which was not cleaved despite a high score predicted for its sequence. Using the known X-Ray structure of furin in complex with a substrate-like inhibitor as an initial structural reference, we carried out molecular dynamics (MD) simulations to learn the modes of binding of the peptides in the furin active site, and the suitability of the complex for initiation of the enzymatic cleavage. We thus identified the 3D structural requirements of the furin active site configuration that enable bound peptides to undergo cleavage, and the way in which the various strains tested experimentally are fulfilling these requirements. We find that despite some flexibility in the organization of the peptide bound to the active site of the enzyme, the presence of a histidine at P2 of MHV-A59 fails to properly orient the sidechain of His194 of the furin catalytic triad and therefore produces a distortion that renders the peptide/complex structural configuration in the active site incompatible with requirements for cleavage initiation. The Ser/Thr in P1 of MHV-2 and MHV-S has a similar effect of distorting the conformation of the furin active site residues produced by the elimination of the canonical salt-bridge formed by arginine in P1 position. This work informs a study of coronavirus infection and pathogenesis with respect to the function of the viral spike protein, and suggests an important process of viral adaptation and evolution within the spike S1/S2 structural loop.

The allosteric mechanism leading to an open-groove lipid conductive state of the TMEM16F scramblase.

TMEM16F is a Ca2+-activated phospholipid scramblase in the TMEM16 family of membrane proteins. Unlike other TMEM16s exhibiting a membrane-exposed hydrophilic groove that serves as a translocation pathway for lipids, the experimentally determined structures of TMEM16F shows the groove in a closed conformation even under conditions of maximal scramblase activity. It is currently unknown if/how TMEM16F groove can open for lipid scrambling. Here we describe the analysis of ~400 µs all-atom molecular dynamics (MD) simulations of the TMEM16F revealing an allosteric mechanism leading to an open-groove, lipid scrambling competent state of the protein. The groove opens into a continuous hydrophilic conduit that is highly similar in structure to that seen in other activated scramblases. The allosteric pathway connects this opening to an observed destabilization of the Ca2+ ion bound at the distal site near the dimer interface, to the dynamics of specific protein regions that produces the open-groove state to scramble phospholipids.

Molecular determinants of pH sensing in the proton-activated chloride channel.

In response to acidic pH, the widely expressed proton-activated chloride (PAC) channel opens and conducts anions across cellular membranes. By doing so, PAC plays an important role in both cellular physiology (endosome acidification) and diseases associated with tissue acidosis (acid-induced cell death). Despite the available structural information, how proton binding in the extracellular domain (ECD) leads to PAC channel opening remains largely unknown. Here, through comprehensive mutagenesis and electrophysiological studies, we identified several critical titratable residues, including two histidine residues (H130 and H131) and an aspartic acid residue (D269) at the distal end of the ECD, together with the previously characterized H98 at the transmembrane domain-ECD interface, as potential pH sensors for human PAC. Mutations of these residues resulted in significant changes in pH sensitivity. Some combined mutants also exhibited large basal PAC channel activities at neutral pH. By combining molecular dynamics simulations with structural and functional analysis, we further found that the ß12 strand at the intersubunit interface and the associated "joint region" connecting the upper and lower ECDs allosterically regulate the proton-dependent PAC activation. Our studies suggest a distinct pH-sensing and gating mechanism of this new family of ion channels sensitive to acidic environment.

Conformational transitions in BTG1 antiproliferative protein and their modulation by disease mutants.

B cell translocation gene 1 (BTG1) protein belongs to the BTG/transducer of ERBB2 (TOB) family of antiproliferative proteins whose members regulate various key cellular processes such as cell cycle progression, apoptosis, and differentiation. Somatic missense mutations in BTG1 are found in ∼70% of a particularly malignant and disseminated subtype of diffuse large B cell lymphoma (DLBCL). Antiproliferative activity of BTG1 has been linked to its ability to associate with transcriptional cofactors and various enzymes. However, molecular mechanisms underlying these functional interactions and how the disease-linked mutations in BTG1 affect these mechanisms are currently unknown. To start filling these knowledge gaps, here, using atomistic molecular dynamics (MD) simulations, we explored structural, dynamic, and kinetic characteristics of BTG1 protein, and studied how various DLBCL mutations affect these characteristics. We focused on the protein region formed by α2 and α4 helices, as this interface has been reported not only to serve as a binding hotspot for several cellular partners but also to harbor sites for the majority of known DLBCL mutations. Markov state modeling analysis of extensive MD simulations revealed that the α2-α4 interface in the wild-type (WT) BTG1 undergoes conformational transitions between closed and open metastable states. Importantly, we show that some of the mutations in this region that are observed in DLBCL, such as Q36H, F40C, Q45P, E50K (in α2), and A83T and A84E (in α4), either overstabilize one of these two metastable states or give rise to new conformations in which these helices are distorted (i.e., kinked or unfolded). Based on these results, we conclude that the rapid interconversion between the closed and open conformations of the α2-α4 interface is an essential component of the BTG1 functional dynamics that can prime the protein for functional associations with its binding partners. Disruption of the native dynamic equilibrium by DLBCL mutants leads to the ensemble of conformations in BTG1 that are unlikely structurally and/or kinetically to enable productive functional interactions with the binding proteins.

Simulation of pH-Dependent Conformational Transitions in Membrane Proteins: The CLC-ec1 Cl-/H+ Antiporter.

Intracellular transport of chloride by members of the CLC transporter family involves a coupled exchange between a Cl- anion and a proton (H+), which makes the transport function dependent on ambient pH. Transport activity peaks at pH 4.5 and stalls at neutral pH. However, a structure of the WT protein at acidic pH is not available, making it difficult to assess the global conformational rearrangements that support a pH-dependent gating mechanism. To enable modeling of the CLC-ec1 dimer at acidic pH, we have applied molecular dynamics simulations (MD) featuring a new force field modification scheme-termed an Equilibrium constant pH approach (ECpH). The ECpH method utilizes linear interpolation between the force field parameters of protonated and deprotonated states of titratable residues to achieve a representation of pH-dependence in a narrow range of physiological pH values. Simulations of the CLC-ec1 dimer at neutral and acidic pH comparing ECpH-MD to canonical MD, in which the pH-dependent protonation is represented by a binary scheme, substantiates the better agreement of the conformational changes and the final model with experimental data from NMR, cross-link and AFM studies, and reveals structural elements that support the gate-opening at pH 4.5, including the key glutamates Gluin and Gluex.

Localization atomic force microscopy.

Understanding structural dynamics of biomolecules at the single-molecule level is vital to advancing our knowledge of molecular mechanisms. Currently, there are few techniques that can capture dynamics at the sub-nanometre scale and in physiologically relevant conditions. Atomic force microscopy (AFM)1 has the advantage of analysing unlabelled single molecules in physiological buffer and at ambient temperature and pressure, but its resolution limits the assessment of conformational details of biomolecules2. Here we present localization AFM (LAFM), a technique developed to overcome current resolution limitations. By applying localization image reconstruction algorithms3 to peak positions in high-speed AFM and conventional AFM data, we increase the resolution beyond the limits set by the tip radius, and resolve single amino acid residues on soft protein surfaces in native and dynamic conditions. LAFM enables the calculation of high-resolution maps from either images of many molecules or many images of a single molecule acquired over time, facilitating single-molecule structural analysis. LAFM is a post-acquisition image reconstruction method that can be applied to any biomolecular AFM dataset.

Diversity of mechanisms in Ras-GAP catalysis of guanosine triphosphate hydrolysis revealed by molecular modeling.

The mechanism of the deceptively simple reaction of guanosine triphosphate (GTP) hydrolysis catalyzed by the cellular protein Ras in complex with the activating protein GAP is an important issue because of the significance of this reaction in cancer research. We show that molecular modeling of GTP hydrolysis in the Ras-GAP active site reveals a diversity of mechanisms of the intrinsic chemical reaction depending on molecular groups at position 61 in Ras occupied by glutamine in the wild-type enzyme. First, a comparison of reaction energy profiles computed at the quantum mechanics/molecular mechanics (QM/MM) level shows that an assignment of the Gln61 side chain in the wild-type Ras either to QM or to MM parts leads to different scenarios corresponding to the glutamine-assisted or the substrate-assisted mechanisms. Second, replacement of Gln61 by the nitro-analog of glutamine (NGln) or by Glu, applied in experimental studies, results in two more scenarios featuring the so-called two-water and the concerted-type mechanisms. The glutamine-assisted mechanism in the wild-type Ras-GAP, in which the conserved Gln61 plays a decisive role, switching between the amide and imide tautomer forms, is consistent with the known experimental results of structural, kinetic and spectroscopy studies. The results emphasize the role of the Ras residue Gln61 in Ras-GAP catalysis and explain the retained catalytic activity of the Ras-GAP complex towards GTP hydrolysis in the Gln61NGln and Gln61Glu mutants of Ras.

Allosteric Control of N-Acetyl-Aspartate Hydrolysis by the Y231C and F295S Mutants of Human Aspartoacylase.

We present the results of molecular modeling of conformational changes in the Y231C and F295S mutants of human aspartoacylase (hAsp), which allow us to propose a mechanism of allosteric regulation of enzyme activity of these protein variants. The hAsp enzyme hydrolyzes one of the most abundant amino acid derivatives in the brain, N-acetyl-aspartate. It is important to understand the reasons for diminishing activity of the mutated enzymes, which is crucial for Canavan disease patients bearing the mutated gene. We explore a model which suggests operation of hAsp in the dimer form with two dynamically inequivalent subunits. Large-scale molecular dynamics simulations reveal that the replacements Y231C and F295S at the periphery of the protein shift the equilibrium between hAsp conformations with the open and closed gates to the enzyme active site buried inside the protein. Application of the dynamical network analysis and the Markov state model approach allows us to strengthen this conclusion and provide a detailed description of dynamically induced structural changes of the protein. The decreased availability of the active site for substrate molecules in the mutated enzymes explains their diminishing activity observed in clinical experiments.

Three Faces of N-Acetylaspartate: Activator, Substrate, and Inhibitor of Human Aspartoacylase.

Hydrolysis of N-acetylaspartate (NAA), one of the most concentrated metabolites in brain, catalyzed by human aspartoacylase (hAsp) shows a remarkable dependence of the reaction rate on substrate concentration. At low NAA concentrations, sigmoidal shape of kinetic curve is observed, followed by typical rate growth of the enzyme-catalyzed reaction, whereas at high NAA concentrations self-inhibition takes place. We show that this rate dependence is consistent with a molecular model, in which N-acetylaspartate appears to have three faces in the enzyme reaction, acting as activator at low concentrations, substrate at moderate concentrations, and inhibitor at high concentrations. To support this conclusion we identify binding sites of NAA at the hAsp dimer including those on the protein surface (activating sites) and at the dimer interface (inhibiting site). Using the Markov state model approach we demonstrate that population of either activating or inhibiting site shifts the equilibrium between the hAsp dimer conformations with the open and closed gates leading to the enzyme active site buried inside the protein. These conclusions are in accord with the calculated values of binding constants of NAA at the hAsp dimer, indicating that the activating site with a higher affinity to NAA should be occupied first, whereas the inhibiting site with a lower affinity to NAA should be occupied later. Application of the dynamical network analysis shows that communication pathways between the regulatory sites (activating or inhibiting) and the gates to the active site do not interfere. These considerations allow us to develop a kinetic mechanism and to derive the equation for the reaction rate covering the entire NAA concentration range. Perfect agreement between theoretical and experimental kinetic data provides strong support to the proposed catalytic model.

Role of Protein Dimeric Interface in Allosteric Inhibition of N-Acetyl-Aspartate Hydrolysis by Human Aspartoacylase.

The results of molecular modeling suggest a mechanism of allosteric inhibition upon hydrolysis of N-acetyl-aspartate (NAA), one of the most abundant amino acid derivatives in brain, by human aspartoacylase (hAsp). Details of this reaction are important to suggest the practical ways to control the enzyme activity. Search for allosteric sites using the Allosite web server and SiteMap analysis allowed us to identify substrate binding pockets located at the interface between the subunits of the hAsp dimer molecule. Molecular docking of NAA to the pointed areas at the dimer interface predicted a specific site, in which the substrate molecule interacts with the Gly237, Arg233, Glu290, and Lys292 residues. Analysis of multiple long-scaled molecular dynamics trajectories (the total simulation time exceeded 1.5 µs) showed that binding of NAA to the identified allosteric site induced significant rigidity to the protein loops with the amino acid side chains forming gates to the enzyme active site. Application of the protein dynamical network algorithms showed that substantial reorganization of the signal propagation pathways of intersubunit communication in the dimer occurred upon allosteric NAA binding to the remote site. The modeling approaches provide an explanation to the observed decrease of the reaction rate of NAA hydrolysis by hAsp at high substrate concentrations.

Reaction Mechanism of Guanosine Triphosphate Hydrolysis by the Vision-Related Protein Complex Arl3-RP2.

Complexes of small GTPases with GTPase-activating proteins have been intensively studied with the main focus on the complex of H-Ras with p120GAP (Ras-GAP). The detailed mechanism of GTP hydrolysis is still unresolved. To clarify it, we calculated the energy profile of GTP hydrolysis in the active site of a recently characterized vision-related member of this family, the Arl3-RP2 complex. The mechanism suggested in this study retains the main features of GTP hydrolysis by the Ras-GAP complex, but the relative energies of the corresponding intermediates are different and an additional intermediate exists in the Arl3-RP2 complex compared with the Ras-GAP. These differences arise from small deviations in the catalytic arginine conformation of the active site. In the Arl3-RP2 complex, the first two intermediates, corresponding to the Pγ-Oßγ bond cleavage and the glutamine-assisted proton transfer, are almost isoenergetic with the ES complex. Numerical simulations of the kinetic curves demonstrate that the concentrations of these intermediates are comparable with that of ES during the reaction. The calculated IR spectra reveal specific vibrational bands, corresponding to these intermediates. These specific features of the Arl3-RP2 complex open the opportunity to identify spectroscopically two more reaction intermediates in GTP hydrolysis in addition to the ES and EP complexes.

Modeling the Complete Catalytic Cycle of Aspartoacylase.

The complete catalytic cycle of aspartoacylase (ASPA), a zinc-dependent enzyme responsible for cleavage of N-acetyl-l-aspartate, is characterized by the methods of molecular modeling. The reaction energy profile connecting the enzyme-substrate (ES) and the enzyme-product (EP) complexes is constructed by the quantum mechanics/molecular mechanics (QM/MM) method assisted by the molecular dynamics (MD) simulations with the QM/MM potentials. Starting from the crystal structure of ASPA complexed with the intermediate analogue, the minimum-energy geometry configurations and the corresponding transition states are located. The stages of substrate binding to the enzyme active site and release of the products are modeled by MD calculations with the replica-exchange umbrella sampling technique. It is shown that the first reaction steps, nucleophilic attack of a zinc-bound nucleophilic water molecule at the carbonyl carbon and the amide bond cleavage, are consistent with the glutamate-assisted mechanism hypothesized for the zinc-dependent hydrolases. The stages of formation of the products, acetate and l-aspartate, and regeneration of the enzyme are characterized for the first time. The constructed free energy diagram from the reactants to the products suggests that the enzyme regeneration, but not the nucleophilic attack of the catalytic water molecule, corresponds to the rate-determining stage of the full catalytic cycle of ASPA.

Slow-binding inhibition of acetylcholinesterase by an alkylammonium derivative of 6-methyluracil: mechanism and possible advantages for myasthenia gravis treatment.

Inhibition of human AChE (acetylcholinesterase) and BChE (butyrylcholinesterase) by an alkylammonium derivative of 6-methyluracil, C-547, a potential drug for the treatment of MG (myasthenia gravis) was studied. Kinetic analysis of AChE inhibition showed that C-547 is a slow-binding inhibitor of type B, i.e. after formation of the initial enzyme·inhibitor complex (Ki=140 pM), an induced-fit step allows establishment of the final complex (Ki*=22 pM). The estimated koff is low, 0.05 min(-1) On the other hand, reversible inhibition of human BChE is a fast-binding process of mixed-type (Ki=1.77 µM; Ki'=3.17 µM). The crystal structure of mouse AChE complexed with C-547 was solved at 3.13 Å resolution. The complex is stabilized by cation-π, stacking and hydrogen-bonding interactions. Molecular dynamics simulations of the binding/dissociation processes of C-547 and C-35 (a non-charged analogue) to mouse and human AChEs were performed. Molecular modelling on mouse and human AChE showed that the slow step results from an enzyme conformational change that allows C-547 to cross the bottleneck in the active-site gorge, followed by formation of tight complex, as observed in the crystal structure. In contrast, the related non-charged compound C-35 is not a slow-binding inhibitor. It does not cross the bottleneck because it is not sensitive to the electrostatic driving force to reach the bottom of the gorge. Thus C-547 is one of the most potent and selective reversible inhibitors of AChE with a long residence time, τ=20 min, longer than for other reversible inhibitors used in the treatment of MG. This makes C-547 a promising drug for the treatment of this disease.