Bruno L. Victor

Superoxide reduction by Nanoarchaeum equitans neelaredoxin, an enzyme lacking the highly conserved glutamate iron ligand

Superoxide reductases (SORs) are antioxidant enzymes present in many prokaryotes, either anaerobes or microaerophiles, which detoxify superoxide by reducing it to hydrogen peroxide. The reaction mechanism involves the diffusion-limited encounter of superoxide with the reduced iron site and concomitant formation of an Fe3+–(hydro)peroxo adduct that, upon protonation, leads to the formation of hydrogen peroxide. By the end of this process, a glutamate residue coordinates the ferric ion, acting as a sixth ligand. Although this residue is able to shuttle protons to the intermediate at low pH, it seems to have a minor relevance to the overall reduction mechanism. Nevertheless, this ligand is conserved in most SORs known thus far, with the notable exception of neelaredoxin from Nanoarchaeum equitans. The protein of this organism was cloned and overexpressed, and its spectroscopic characterization revealed distinct pH-equilibrium properties in comparison with those of glutamate-containing SORs. A three-dimensional model of this protein was generated in an effort to identify structural properties that could explain these distinct features. Pulse radiolysis measurements showed that the efficiency of this enzyme in reducing superoxide is comparable to that of glutamate-containing SORs, thus definitely ruling out the requirement for such a ligand in the reduction mechanism.

Docking and electron transfer studies between rubredoxin and rubredoxin:oxygen oxidoreductase

The interaction and electron transfer (ET) between rubredoxin (Rd) and rubredoxin:oxygen oxidoreductase (ROO) from Desulfovibrio gigas is studied by molecular modelling techniques. Experimental kinetic assays using recombinant proteins show that the Rd reoxidation by ROO displays a bell-shaped dependence on ionic strength, suggesting a non-trivial electrostatic dependence of the interaction between these two proteins. Rigid docking studies reveal a prevalence for Rd to interact, in a very specific way, with the surface of the ROO dimer near its FMN cofactors. The optimization of the lowest energy complexes, using molecular dynamics simulation, shows a very tight interaction between the surface of the two proteins, with a high probability for Rd residues (but not the iron centre directly) to be in direct contact with the FMN cofactors of ROO. Both electrostatics and van der Waals interactions contribute to the final energy of the complex. In these complexes, the major contributions for complex formation are polar interactions between acidic residues of Rd and basic residues of ROO, plus substantial non-polar interactions between different groups. Important residues for this process are identified. ET estimates (using the Pathways model), in the optimized lowest energy complexes, suggest that these configurations are efficient for transferring electrons. The experimental bell-shaped dependence of kinetics on ionic strength is analysed in view of the molecular modelling results, and hypotheses for the molecular basis of this phenomenon are discussed.

Predicting the Thermodynamics and Kinetics of Helix Formation in a Cyclic Peptide Model

The peptide Ac-(cyclo-2,6)-R[KAAAD]-NH2 (cyc-RKAAAD) is a short cyclic peptide known to adopt a remarkably stable single turn α-helix in water. Due to its simplicity and the availability of thermodynamic and kinetic experimental data, cyc-RKAAAD poses as an ideal model for evaluating the aptness of current molecular dynamics (MD) simulation methodologies to accurately sample conformations that reproduce experimentally observed properties. In this work, we extensively sample the conformational space of cyc-RKAAAD using microsecond-timescale MD simulations. We characterize the peptide conformational preferences in terms of secondary structure propensities and, using Cartesian-coordinate principal component analysis (cPCA), construct its free energy landscape, thus obtaining a detailed weighted discrimination between the helical and nonhelical subensembles. The cPCA state discrimination, together with a Markov model built from it, allowed us to estimate the free energy of unfolding (-0.57 kJ/mol) and the relaxation time (∼0.435 μs) at 298.15 K, which are in excellent agreement with the experimentally reported values (-0.22 kJ/mol and 0.42 μs, Serrano, A. L.; Tucker, M. J.; Gai, F. J. Phys. Chem. B, 2011, 115, 7472-7478.). Additionally, we present simulations conducted using two enhanced sampling methods: replica-exchange molecular dynamics (REMD) and bias-exchange metadynamics (BE-MetaD). We compare the free energy landscape obtained by these two methods with the results from MD simulations and discuss the sampling and computational gains achieved. Overall, the results obtained attest to the suitability of modern simulation methods to explore the conformational behavior of peptide systems with a high level of realism.

Dioxygen and nitric oxide pathways and affinity to the catalytic site of rubredoxin:oxygen oxidoreductase from Desulfovibrio gigas

Rubredoxin:oxygen oxidoreductase (ROO) is the terminal oxidase of a soluble electron transfer chain found in Desulfovibrio gigas. This protein belongs to the flavodiiron family and was initially described as an oxygen reductase, converting this substrate to water and acting as an oxygen-detoxifying system. However, more recent studies evidenced also the ability for this protein to act as a nitric oxide reductase, suggesting an alternative physiological role. To clarify the apparent bifunctional nature of this protein, we performed molecular dynamics simulations of the protein, in different redox states, together with O2 and NO molecules in aqueous solution. The two small molecules were parameterized using free-energy calculations of the hydration process. With these simulations we were able to identify specific protein paths that allow the diffusion of both these molecules through the protein towards the catalytic centers. Also, we have tried to characterize the preference of ROO towards the presence of O2 and/or NO at the active site. By using free-energy simulations, we did not find any significant preference for ROO to accommodate both O2 and NO. Also, from our molecular dynamics simulations we were able to identify similar diffusion profiles for both O2 and NO molecules. These two conclusions are in good agreement with previous experimental works stating that ROO is able to catalyze both O2 and NO.

Structural Analysis of Thermus thermophilus HB27 Mannosyl-3-phosphoglycerate Synthase Provides Evidence for a Second Catalytic Metal Ion and New Insight into the Retaining Mechanism of Glycosyltransferases

Mannosyl-3-phosphoglycerate synthase is a glycosyltransferase involved in the two-step synthetic pathway of mannosylglycerate, a compatible solute that accumulates in response to salt and/or heat stresses in many microorganisms thriving in hot environments. The three-dimensional structure of mannosyl-3-phosphoglycerate synthase from Thermus thermophilus HB27 in its binary complex form, with GDP-α-d-mannose and Mg2+, shows a second metal binding site, about 6 Å away from the mannose moiety. Kinetic and mutagenesis studies have shown that this metal site plays a role in catalysis. Additionally, Asp167 in the DXD motif is found within van der Waals contact distance of the C1′ atom in the mannopyranose ring, suggesting its action as a catalytic nucleophile, either in the formation of a glycosyl-enzyme intermediate according to the double-displacement SN2 reaction mechanism or in the stabilization of the oxocarbenium ion-like intermediate according to the DN*ANss (SNi-like) reaction mechanism. We propose that either mechanism may occur in retaining glycosyltransferases with a GT-A fold, and, based on the gathered structural information, we identified an extended structural signature toward a common scaffold between the inverting and retaining glycosyltransferases.

Self-assembly molecular dynamics simulations shed light into the interaction of the influenza fusion Peptide with a membrane bilayer.

Influenza virus is one of the most devastating human pathogens. In order to infect host cells, this virus fuses its membrane with the host membrane in a process mediated by the glycoprotein hemagglutinin. During fusion, the N-terminal region of hemagglutinin, which is known as the fusion peptide (FP), inserts into the host membrane, promoting lipid mixing between the viral and host membranes. Therefore, this peptide plays a key role in the fusion process, but the exact mechanism by which it promotes lipid mixing is still unclear. To shed light into this matter, we performed molecular dynamics (MD) simulations of the influenza FP in different environments (water, dodecylphosphocholine (DPC) micelles, and a dimyristoylphosphatidylcholine (DMPC) membrane). While in pure water the peptide lost its initial secondary structure, in simulations performed in the presence of DPC micelles it remained stable, in agreement with previous experimental observations. In simulations performed in the presence of a preassembled DMPC bilayer, the peptide became unstructured and was unable to insert into the membrane as a result of technical limitations of the method used. To overcome this problem, we used a self-assembly strategy, assembling the membrane together with the peptide. These simulations revealed that the peptide can adopt a membrane-spanning conformation, which had not been predicted by previous MD simulation studies. The peptide insertion had a strong effect on the membrane, lowering the bilayer thickness, disordering nearby lipids, and promoting lipid tail protrusion. These results contribute to a better understanding of the role of the FP in the fusion process.

The Catalase Activity of Catalase-Peroxidases Is Modulated by Changes in the pKa of the Distal Histidine.

The unusual Met-Tyr-Trp adduct composed of cross-linked side chains along with an associated mobile Arg is essential for catalase activity in catalase-peroxidases. In addition, acidic residues in the entrance channel, in particular an Asp and a Glu ∼7 and ∼15 Å, respectively, from the heme, significantly enhance catalase activity. The mechanism by which these channel carboxylates influence catalase activity is the focus of this work. Seventeen new variants with fewer and additional acidic residues have been constructed and characterized structurally and for enzymatic activity, revealing that their effect on activity is roughly inversely proportional to their distance from the heme and adduct, suggesting that the electrostatic potential of the heme cavity may be affected. A discrete group of protonable residues are contained within a 15 Å sphere surrounding the heme iron, and a computational analysis reveals that the pKa of the distal His112, alone, is modulated within the pH range of catalase activity by the remote acidic residues in a pattern consistent with its protonated form having a key role in the catalase reaction cycle. The electrostatic potential also impacts the catalatic reaction through its influence on the charged status of the Met-Tyr-Trp adduct.

Structural determinants for the membrane insertion of the transmembrane peptide of hemagglutinin from influenza virus.

Membrane fusion is a process involved in a high range of biological functions, going from viral infections to neurotransmitter release. Fusogenic proteins increase the slow rate of fusion by coupling energetically downhill conformational changes of the protein to the kinetically unfavorable fusion of the membrane lipid bilayers. Hemagglutinin is an example of a fusogenic protein, which promotes the fusion of the membrane of the influenza virus with the membrane of the target cell. The N-terminus of the HA2 subunit of this protein contains a fusion domain described to act as a destabilizer of the target membrane bilayers, leading eventually to a full fusion of the two membranes. On the other hand, the C-terminus of the same subunit contains a helical transmembrane domain which was initially described to act as the anchor of the protein to the membrane of the virus. However, in recent years the study of this peptide segment has been gaining more attention since it has also been described to be involved in the membrane fusion process. Yet, the structural characterization of the interaction of such a protein domain with membrane lipids is still very limited. Therefore, in this work, we present a study of this transmembrane peptide domain in the presence of DMPC membrane bilayers, and we evaluate the effect of several mutations, and the effect of peptide oligomerization in this interaction process. Our results allowed us to identify and confirm amino acid residue motifs that seem to regulate the interaction between the segment peptide and membrane bilayers. Besides these sequence requirements, we have also identified length and tilt requirements that ultimately contribute to the hydrophobic matching between the peptide and the membrane. Additionally, we looked at the association of several transmembrane peptide segments and evaluated their direct interaction and stability inside a membrane bilayer. From our results we could conclude that three independent TM peptide segments arrange themselves in a parallel arrangement, very similarly to what is observed for the C-terminal regions of the hemagglutinin crystallographic structure of the protein, to where the segments are attached.

Fusing simulation and experiment: The effect of mutations on the structure and activity of the influenza fusion peptide

During the infection process, the influenza fusion peptide (FP) inserts into the host membrane, playing a crucial role in the fusion process between the viral and host membranes. In this work we used a combination of simulation and experimental techniques to analyse the molecular details of this process, which are largely unknown. Although the FP structure has been obtained by NMR in detergent micelles, there is no atomic structure information in membranes. To answer this question, we performed bias-exchange metadynamics (BE-META) simulations, which showed that the lowest energy states of the membrane-inserted FP correspond to helical-hairpin conformations similar to that observed in micelles. BE-META simulations of the G1V, W14A, G12A/G13A and G4A/G8A/G16A/G20A mutants revealed that all the mutations affect the peptide’s free energy landscape. A FRET-based analysis showed that all the mutants had a reduced fusogenic activity relative to the WT, in particular the mutants G12A/G13A and G4A/G8A/G16A/G20A. According to our results, one of the major causes of the lower activity of these mutants is their lower membrane affinity, which results in a lower concentration of peptide in the bilayer. These findings contribute to a better understanding of the influenza fusion process and open new routes for future studies.

The impact of using single atomistic long range cutoff schemes with the GROMOS 54A7 force field

With the recent increase in computing power, the molecular modeling community is now more focused on improving the accuracy and overall quality of biomolecular simulations. For the available simulation packages, force fields, and all other associated methods used, this relates to how well they describe the conformational space and thermodynamic properties of a biomolecular system. The parameter sets of GROMOS force fields have been parametrized and validated with the reaction field (RF) method using charge groups and a twin-range cutoff scheme (0.8/1.4 nm). However, the most recent versions of GROMACS (since v.2016) discontinued the support for charge groups. To take full advantage of the newer and faster versions of this software package with GROMOS 54A7 and RF, we need to evaluate the impact of using a single cutoff scheme (vs twin-range) and of using the Verlet list update method (which is atomistic) compared to the group-based cutoff scheme. Our results show that the GROMOS 54A7 force field seems consistent with a single cutoff, since the resulting conformation and protonation ensembles were indistinguishable. The GROMOS parametrization procedure was also reproduced using an atomistic cutoff scheme, and we have observed that the hydration free energy values of small amino acid side-chain analogues were similar to the ones obtained with the group-based protocol. We do observe a small impact of the atomistic cutoff scheme in the conformational space of the model systems studied (G1-PAMAM and DMPC). However, since the structural properties of these systems are well converged for the cutoff range used (1.4-2.0 nm), unlike with the group-based cutoff schemes, we are confident that the atomistic cutoff can be adopted with RF for MD and constant-pH MD biomolecular simulations using the GROMOS 54A7 force field.