Influenza hemagglutinin drives viral entry via two sequential intramembrane mechanisms.

Enveloped viruses enter cells via a process of membrane fusion between the viral envelope and a cellular membrane. For influenza virus, mutational data have shown that the membrane-inserted portions of the hemagglutinin protein play a critical role in achieving fusion. In contrast to the relatively well-understood ectodomain, a predictive mechanistic understanding of the intramembrane mechanisms by which influenza hemagglutinin drives fusion has been elusive. We used molecular dynamics simulations of fusion between a full-length hemagglutinin proteoliposome and a lipid bilayer to analyze these mechanisms. In our simulations, hemagglutinin first acts within the membrane to increase lipid tail protrusion and promote stalk formation and then acts to engage the distal leaflets of each membrane and promote stalk widening, curvature, and eventual fusion. These two sequential mechanisms, one occurring before stalk formation and one after, are consistent with our experimental measurements of single-virus fusion kinetics to liposomes of different sizes. The resulting model also helps explain and integrate previous mutational and biophysical data, particularly the mutational sensitivity of the fusion peptide N terminus and the length sensitivity of the transmembrane domain. We hypothesize that entry by other enveloped viruses may also use sequential processes of acyl tail exposure, followed by membrane curvature and distal leaflet engagement.

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.

Cryptic genetic variation shapes the adaptive evolutionary potential of enzymes.

Genetic variation among orthologous proteins can cause cryptic phenotypic properties that only manifest in changing environments. Such variation may impact the evolvability of proteins, but the underlying molecular basis remains unclear. Here, we performed comparative directed evolution of four orthologous metallo-ß-lactamases toward a new function and found that different starting genotypes evolved to distinct evolutionary outcomes. Despite a low initial fitness, one ortholog reached a significantly higher fitness plateau than its counterparts, via increasing catalytic activity. By contrast, the ortholog with the highest initial activity evolved to a less-optimal and phenotypically distinct outcome through changes in expression, oligomerization and activity. We show how cryptic molecular properties and conformational variation of active site residues in the initial genotypes cause epistasis, that could lead to distinct evolutionary outcomes. Our work highlights the importance of understanding the molecular details that connect genetic variation to protein function to improve the prediction of protein evolution.

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.

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.

Cooperativity and flexibility in enzyme evolution.

Enzymes are flexible catalysts, and there has been substantial discussion about the extent to which this flexibility contributes to their catalytic efficiency. What has been significantly less discussed is the extent to which this flexibility contributes to their evolvability. Despite this, recent years have seen an increasing number of both experimental and computational studies that demonstrate that cooperativity and flexibility play significant roles in enzyme innovation. This review covers key developments in the field that emphasize the importance of enzyme dynamics not just to the evolution of new enzyme function(s), but also as a property that can be harnessed in the design of new artificial enzymes.

Extending the Nonbonded Cationic Dummy Model to Account for Ion-Induced Dipole Interactions.

Modeling metalloproteins often requires classical molecular dynamics (MD) simulations in order to capture their relevant motions, which in turn necessitates reliable descriptions of the metal centers involved. One of the most successful approaches to date is provided by the "cationic dummy model", where the positive charge of the metal ion is transferred toward dummy particles that are bonded to the central metal ion in a predefined coordination geometry. While this approach allows for ligand exchange, and captures the correct electrostatics as demonstrated for different divalent metal ions, current dummy models neglect ion-induced dipole interactions. In the present work, we resolve this weakness by taking advantage of the recently introduced 12-6-4 type Lennard-Jones potential to include ion-induced dipole interactions. We revise our previous dummy model for Mg2+ and demonstrate that the resulting model can simultaneously reproduce the experimental solvation free energy and metal-ligand distances without the need for artificial restraints or bonds. As ion-induced dipole interactions become particularly important for highly charged metal ions, we develop dummy models for the biologically relevant ions Al3+, Fe3+, and Cr3+. Finally, the effectiveness of our new models is demonstrated in MD simulations of several diverse (and highly challenging to simulate) metalloproteins.

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.

Probing the mechanisms for the selectivity and promiscuity of methyl parathion hydrolase.

Diverse organophosphate hydrolases have convergently evolved the ability to hydrolyse man-made organophosphates. Thus, these enzymes are attractive model systems for studying the factors shaping enzyme functional evolution. Methyl parathion hydrolase (MPH) is an enzyme from the metallo-ß-lactamase superfamily, which hydrolyses a wide range of organophosphate, aryl ester and lactone substrates. In addition, MPH demonstrates metal-ion-dependent selectivity patterns. The origins of this remain unclear, but are linked to open questions about the more general role of metal ions in functional evolution and divergence within enzyme superfamilies. Here, we present detailed mechanistic studies of the paraoxonase and arylesterase activities of MPH complexed with five different transition metal ions, and demonstrate that the hydrolysis reactions proceed via similar pathways and transition states. However, while it is possible to discern a clear structural origin for the selectivity between different substrates, the selectivity between different metal ions appears to lie instead in the distinct electrostatic properties of the metal ions themselves, which causes subtle changes in transition state geometries and metal-metal distances at the transition state rather than significant structural changes in the active site. While subtle, these differences can be significant for shaping the metal-ion-dependent activity patterns observed for this enzyme.This article is part of the themed issue 'Multiscale modelling at the physics-chemistry-biology interface'.

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.

Substrate and Enzyme Specificity of the Kinetic Isotope Effects Associated with the Dioxygenation of Nitroaromatic Contaminants.

Compound-specific isotope analysis (CSIA) is a promising approach for tracking biotransformation of organic pollutants, but isotope fractionation associated with aromatic oxygenations is only poorly understood. We investigated the dioxygenation of a series of nitroaromatic compounds to the corresponding catechols by two enzymes, namely, nitrobenzene and 2-nitrotoluene dioxygenase (NBDO and 2NTDO) to elucidate the enzyme- and substrate-specificity of C and H isotope fractionation. While the apparent (13)C- and (2)H-kinetic isotope effects of nitrobenzene, nitrotoluene isomers, 2,6-dinitrotoluene, and naphthalene dioxygenation by NBDO varied considerably, the correlation of C and H isotope fractionation revealed a common mechanism for nitrobenzene and nitrotoluenes. Similar observations were made for the dioxygenation of these substrates by 2NTDO. Evaluation of reaction kinetics, isotope effects, and commitment-to-catalysis based on experiment and theory showed that rates of dioxygenation are determined by the enzymatic O2 activation and aromatic C oxygenation. The contribution of enzymatic O2 activation to the reaction rate varies for different nitroaromatic substrates of NBDO and 2NTDO. Because aromatic dioxygenation by nonheme iron dioxygenases is frequently the initial step of biodegradation, O2 activation kinetics may also have been responsible for the minor isotope fractionation reported for the oxygenation of other aromatic contaminants.

Promiscuity and electrostatic flexibility in the alkaline phosphatase superfamily.

Catalytic promiscuity, that is, the ability of single enzymes to facilitate the turnover of multiple, chemically distinct substrates, is a widespread phenomenon that plays an important role in the evolution of enzyme function. Additionally, such pre-existing multifunctionality can be harnessed in artificial enzyme design. The members of the alkaline phosphatase superfamily have served extensively as both experimental and computational model systems for enhancing our understanding of catalytic promiscuity. In this Opinion, we present key recent computational studies into the catalytic activity of these highly promiscuous enzymes, highlighting the valuable insight they have provided into both the molecular basis for catalytic promiscuity in general, and its implications for the evolution of phosphatase activity.

The structure of cyclolinopeptide A in chloroform refined by RDC measurements.

Three-dimensional structures of molecules traditionally assigned from nuclear Overhauser effects and vicinal coupling constants are recently complemented by measurements of residual dipolar couplings. Residual dipolar couplings measured in a stretched poly(dimethylsiloxane) gel were used to determine the structure of cyclolinopeptide A in chloroform solution at -50 °C. After structure refinement, conformational details of main cluster were discussed in relation to crystal and nuclear Overhauser effect derived structures.

Molecular Dynamics Simulation of Nitrobenzene Dioxygenase Using AMBER Force Field.

Molecular dynamics simulation of the oxygenase component of nitrobenzene dioxygenase (NBDO) system, a member of the naphthalene family of Rieske nonheme iron dioxygenases, has been carried out using the AMBER force field combined with a new set of parameters for the description of the mononuclear nonheme iron center and iron-sulfur Rieske cluster. Simulation results provide information on the structure and dynamics of nitrobenzene dioxygenase in an aqueous environment and shed light on specific interactions that occur in its catalytic center. The results suggest that the architecture of the active site is stabilized by key hydrogen bonds, and Asn258 positions the substrate for oxidation. Analysis of protein-water interactions reveal the presence of a network of solvent molecules at the entrance to the active site, which could be of potential catalytic importance.

A DFT study of the cis-dihydroxylation of nitroaromatic compounds catalyzed by nitrobenzene dioxygenase.

The mechanism of cis-dihydroxylation of nitrobenzene and 2-nitrotoluene catalyzed by nitrobenzene 1,2-dioxygenase (NBDO), a member of the naphthalene family of Rieske non-heme iron dioxygenases, was studied by means of the density functional theory method using four models of the enzyme active site. Different possible reaction pathways for the substrate dioxygenation initiated either by the Fe(III)-OOH or HO-Fe(V)═O attack on the aromatic ring were considered and the computed activation barriers compared with the Gibbs free energy of activation for the oxygen-oxygen cleavage leading to the formation of the iron(V)-oxo species from its ferric hydroperoxo precursor. The mechanism of the substrate cis-dihydroxylation leading to the formation of a cis-dihydrodiol was then investigated, and the most feasible mechanism was found to be starting with the attack of the high-valent iron-oxo species on the substrate ring yielding a radical intermediate, which further evolves toward the final product.

A New Maximum Likelihood Approach for Free Energy Profile Construction from Molecular Simulations.

A novel variational method for construction of free energy profiles from molecular simulation data is presented. The variational free energy profile (VFEP) method uses the maximum likelihood principle applied to the global free energy profile based on the entire set of simulation data (e.g from multiple biased simulations) that spans the free energy surface. The new method addresses common obstacles in two major problems usually observed in traditional methods for estimating free energy surfaces: the need for overlap in the re-weighting procedure and the problem of data representation. Test cases demonstrate that VFEP outperforms other methods in terms of the amount and sparsity of the data needed to construct the overall free energy profiles. For typical chemical reactions, only ~5 windows and ~20-35 independent data points per window are sufficient to obtain an overall qualitatively correct free energy profile with sampling errors an order of magnitude smaller than the free energy barrier. The proposed approach thus provides a feasible mechanism to quickly construct the global free energy profile and identify free energy barriers and basins in free energy simulations via a robust, variational procedure that determines an analytic representation of the free energy profile without the requirement of numerically unstable histograms or binning procedures. It can serve as a new framework for biased simulations and is suitable to be used together with other methods to tackle with the free energy estimation problem.

A theoretical study of carbon-carbon bond formation by a Michael-type addition.

A theoretical study of the Michael-type addition of 1,3-dicarbonyl compounds to α,ß-unsaturated carbonyl compounds has been performed in the gas phase by means of the AM1 semiempirical method and by density functional theory (DFT) calculations within the B3LYP and M06-2X hybrid functionals. A molecular model has been selected to mimic the role of a base, which is traditionally used as a catalyst in Michael reactions, an acetate moiety to modulate its basicity, and point charges to imitate the stabilization of the negative charge developed in the substrate during the reaction when taking place in enzymatic environments. Results of the study of six different reactions obtained at the three different levels of calculations show that the reaction takes place in three steps: in the first step the α proton of the acetylacetone is abstracted by the base, then the nucleophilic attack on the ß-carbon of the α,ß-unsaturated carbonyl compound takes place generating the negatively charged enolate intermediate, and finally the product is formed through a proton transfer back from the protonated base. According to the energy profiles, the rate limiting step corresponds to the abstraction of the proton or the carbon-carbon bond formation step, depending on substituents of the substrates and method of calculation. The effect of the substituents on the acidity of the α proton of the acetylacetone and the steric hindrance can be analyzed by comparing these two separated steps. Moreover, the result of adding a positive charge close to the center that develops a negative charge during the reaction confirms the catalytic role of the oxyanion hole proposed in enzyme catalysed Michael-type additions. Stabilization of the intermediate implies, in agreement with the Hammond postulate, a reduction of the barrier of the carbon-carbon bond formation step. Our results can be used to predict the features that a new designed biocatalyst must present to efficiently accelerate this fundamental reaction in organic synthesis.

Measurements of heavy-atom isotope effects using 1H NMR spectroscopy.

A novel method for measuring heavy-atom KIEs for magnetically active isotopes using (1)H NMR is presented. It takes advantage of the resonance split of the protons coupled with the heavy atom in the (1)H spectrum. The method is validated by the example of the (13)C-KIE on the hydroamination of styrene with aniline, catalyzed by phosphine-ligated palladium triflates.

Molecular dynamics study of the hydration of the hydroxyl radical at body temperature.

Classical molecular dynamics (MD) simulation of ˙OH in liquid water at 37 °C has been performed using flexible models of the solute and solvent molecules. We derived the Morse function describing the bond stretching of the radical and the potential for ˙OH-H(2)O interactions, including short-range interactions of hydrogen atoms. Scans of the potential energy surface of the ˙OH-H(2)O complex have been performed using the DFT method with the B3LYP functional and the 6-311G(d,p) basis set. The DFT-derived partial charges, ±0.375e, and the equilibrium bond-length, 0.975 Å, of ˙OH resulted in the dipole moment of 1.76 D. The radical-water radial distribution functions revealed that ˙OH is not built into the solvent structure but it rather occupies distortions or cavities in the hydrogen-bonded network. The solvent structure at 37 °C has been found to be the same as that of pure water. The hydration cage of the radical comprises 13-14 water molecules. The estimated hydration enthalpy -42 ± 5 kJ mol(-1) is comparable with the experimental value -39 ± 6 kJ mol(-1) for 25 °C. Inspection of hydrogen bonds showed the importance of short-range interaction of hydrogen atoms and indicated that neglect of the angular condition greatly overestimates the number of the H-acceptor radical-water bonds. The mean number ̅n = 0.85 of radical-water H-bonds has been calculated using geometric definition of H-bond and ̅n = 0.62 has been obtained when the energetic condition, E(da)≤-8 kJ mol(-1), was additionally considered. The continuous lifetimes of 0.033 ps and 0.024 ps have been estimated for the radical H-donor and the H-acceptor bonds, respectively. Within statistical uncertainty the radical self-diffusion coefficient, (2.9 ± 0.6) × 10(-9) m(2) s(-1), is the same as (3.1 ± 0.5) × 10(-9) m(2) s(-1) calculated for water in solution and in pure solvent. To the best of our knowledge, this is the first study of the ˙OH(aq) properties at a biologically relevant body temperature.

A DFT Study of the Kinetic Isotope Effects on the Competing SN2 and E2 Reactions between Hypochlorite Anion and Ethyl Chloride.

Kinetic isotope effects (KIEs) on the two alternative reactions, SN2 and E2, between hypochlorite anion and ethyl chloride in water have been studied theoretically using B3LYP and M06-2X functionals. It has been found that the latter one yields more correct geometries and energetics. Although, in the qualitative sense, KIEs obtained using both DFT functionals are in agreement, interpretation of some of them, like (18)O-KIE in the present case, leads to different mechanistic conclusions.