Multiscale Simulations Give Insight into the Hydrogen In and Out Pathways of [NiFe]-Hydrogenases from Aquifex aeolicus and Desulfovibrio fructosovorans

[NiFe]-hydrogenases catalyze the cleavage of molecular hydrogen into protons and electrons and represent promising tools for H₂-based technologies such as biofuel cells. However, many aspects of these enzymes remain to be understood, in particular how the catalytic center can be protected from irreversible inactivation by O₂. In this work, we combined homology modeling, all-atom molecular dynamics, and coarse-grain Brownian dynamics simulations to investigate and compare the dynamic and mechanical properties of two [NiFe]-hydrogenases: the soluble O₂-sensitive enzyme from <i>Desulfovibrio fructosovorans</i>, and the O₂-tolerant membrane-bound hydrogenase from <i>Aquifex aeolicus</i>. We investigated the diffusion pathways of H₂ from the enzyme surface to the central [NiFe] active site, and the possible proton pathways that are used to evacuate hydrogen after the oxidation reaction. Our results highlight common features of the two enzymes, such as a Val/Leu/Arg triad of key residues that controls ligand migration and substrate access in the vicinity of the active site, or the key role played by a Glu residue for proton transfer after hydrogen oxidation. We show specificities of each hydrogenase regarding the enzymes internal tunnel network or the proton transport pathways

Partner prediction based on a restricted conformational space.

<p> because of no common residue between the small predicted interface and the docked one); hence, we cleaned the original Mintseris dataset of these three complexes and marked the affected subsets with the * symbol. Performance of protein prediction is evaluated through AUC values computed on the Mintseris dataset and its different subsets. Sensitivity () and specificity () are also given at a threshold cutoff of 0.5 for predictions based on experimental interfaces, and at a threshold cutoff of 0.25 for predicted interfaces. Calculations based on JET predicted interfaces use weights , (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003369#s4" target="_blank">Methods</a>), with the exception of the analysis run for Antibody-Antigen and Antigen-Bound Antibody where , . The analysis is realized by assuming knowledge of either the experimental interfaces or the predicted interfaces. In both cases, we report the results obtained on the restricted (by evolutionary information) conformational space. On three complexes (1ML0, 1GCQ, 1DFJ), JET provided too small interaction sites (leading to a </p

Species represented in the Mintseris Benchmark 2.0.

<p>Right: matrix reporting whether (orange entries) or not (cyan entries) any two protein structures of the Mintseris Benchmark 2.0 are represented by a common species at sequence identity. Each line in the matrix represents a protein and the matrix is not symmetric (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003369#s4" target="_blank">Methods</a>). The proteins are ordered by functional classes: Others (O), Antibody (Ab), Bound Antibody (AbB), Antigens (Ag), Inhibitors (I) and Enzymes (E). The -axis follows the same order as the -axis, from bottom to top. Compare with the matrices of Figures S74 and S75 in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003369#pcbi.1003369.s001" target="_blank">Text S1</a>, based on homology computed for and sequence identity respectively. The matrix labelled with protein names is reported in Figure S76 in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003369#pcbi.1003369.s001" target="_blank">Text S1</a>. Left: an example of IRs analysis where the species information reported in the matrix on the right is plotted. Colors in the two lines of the matrix corresponding to the Enzyme-Inhibitor complex 1MAH are mapped on the dots of the plots for the receptor 1MAH_r and the ligand 1MAH_l (see legend of <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003369#pcbi-1003369-g005" target="_blank">Figure 5</a> for the plots description). The black contour line on some of the proteins identifies bottom black dots in the IR analysis of Figures S38–S51 in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003369#pcbi.1003369.s001" target="_blank">Text S1</a>. The red contour identifies the true interacting partner. 1MAH_r is a <i>Mus musculus</i> protein structure and 1MAH_l a <i>Dendroaspis angusticeps'</i> one, a highly venomous snake. The analysis of all proteins in the dataset is reported in Figures S60–S73 in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003369#pcbi.1003369.s001" target="_blank">Text S1</a>.</p

Interaction ranks distribution for the Mintseris Benchmark 2.0.

<p> of complexes obtained by docking the proteins with all 168 proteins in the environment (this means that the NII score of the native complex falls in the top scores). Native complexes identification is realized either by knowing the experimental interface (exp) or by predicting it (pred). Cumulative counts and percentages are displayed. The selected set of 56 monomers considered in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003369#pcbi.1003369-Wass1" target="_blank">[33]</a> is also evaluated against the 168 proteins (fifth and sixth columns). The number of top proteins corresponding to the of the total number of proteins in the specified environment is given (second column). Over the 168 proteins in the Mintseris dataset, we report the number of proteins (third and fourth columns) whose native complex is identified within the top </p

Robustness of the native complex predictions with respect to the environment composition.

<p>Partner predictions are based on predicted interfaces. Average Interaction Rank (IR) of the true partners is computed over 100 random sets made of 40 proteins each (with error bars in red). The 84 complexes are ordered with respect to their increasing average IR value. For three of the 84 complexes (1BVN, 1BUH, 1N2C), detailed plots show the IR of the complex within each of the 100 random sets and the corresponding AUC value (black dots); green dots correspond to the IR of the complex computed over the Mintseris dataset; orange dots correspond to the IR of the complex computed over complexes in the same functional class. Note that the absence of the green dot on the 1N2C plot corresponds to a too large IR () of the complex. See Table S2 in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003369#pcbi.1003369.s001" target="_blank">Text S1</a> for the names of complexes ranked on the -axis. See Figure S55 in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003369#pcbi.1003369.s001" target="_blank">Text S1</a> for robustness of predictions based on experimental interfaces.</p

Normalized Interaction Index (NII) matrix for the complete dataset of 168 proteins.

<p>The matrix is ordered with the experimental complexes lying on the trailing diagonal. Protein structures corresponding to columns and rows are grouped in functional classes: Enzymes (E), Inhibitors (I), Antibody (Ab), Antigen (Ag), Bound Antibody (AbB), Others (O). Each entry of the matrix corresponds to the NII value computed for the corresponding pair of proteins (receptor on the y-axis and ligand on the x-axis). High interaction scores (between 0.7 and 1, blue and black in the color scale) indicate a high interaction probability. Interaction scores are computed using knowledge of the experimental interfaces. The plot corresponds to an . In the color bar the intervals correspond to values, where the upper bound is included in each interval. Rows and columns are labeled with protein names in Figure S1 in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003369#pcbi.1003369.s001" target="_blank">Text S1</a>.</p

NII matrices for functional classes of proteins.

<p>Enzyme-Inhibitors (EI; top left), Antibody-Antigen (AbAg; top right), Antigen-Bound Antibody (AgAbB; bottom left), Others (O; bottom right). See legend of <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003369#pcbi-1003369-g001" target="_blank">Figure 1</a> for matrix description and color scale. Protein structures are grouped in functional classes. (See Figure S2 in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003369#pcbi.1003369.s001" target="_blank">Text S1</a> for the version of the figure reporting protein names on matrices columns and rows.)</p

Average IR for true Enzyme-Inhibitor complexes and number of false positives.

<p>For each protein, we plot as false positives (FPs, black curve) the number of partners (excepted the true one) showing an average IR , where the IR is computed over 100 random sets of 20 complexes selected from the set of 46 Enzyme-Inhibitor proteins. The cyan dots indicate the average IR of the true partner. A dot corresponds to a complex. For five complexes, conformations associated to the best FIR are represented with different colors: 1AVX (green), 1BVN (blue), 2PCC (orange), 1EWY (cyan), 1KKL (yellow). All residues with a JET+NIP score display interaction propensity and are colored in a color range going from light pink (weak signal) to deep purple (strong signal). 2PCC: the JET+NIP signal is distributed all around the receptor surface enabling different possibilities for the ligand to bind. The predicted interacting site covers only the 5% of the true binding site of the receptor. 1AVX: the predicted receptor binding site shares no residue with the real interaction site, leading to a bad prediction. 1BVN and 1KKL: despite the important size of the receptors 1BVN (496 residues) and 1KKL (3 chains of 205 residues each), corresponding binding sites are well predicted and true partners are identified with and respectively.</p

The protocol.

<p>The protocol is based on docking calculations and JET predictions and produces an interaction matrix for the proteins in a database. Here, two protein structures, the receptor 1AY7 and the ligand 2MTA, are analyzed. The first step consists in cross-docking 1AY7 and 2MTA respectively, against all structures in the database (see cyan box for 1AY7 and blue box for 2MTA). A structure will be crossed dock against another in several conformations (from 100000 up to 450000, depending on the size of the proteins). In the schema, 1AY7 and 2MTA are also docked one against the other (see intersection between blue and cyan boxes). As a result of the cross-docking, a NIP score is associated to each protein leading to the prediction of an interaction site (color range from light blue to dark blue, corresponding to weak and strong signals respectively). In parallel, each protein is analyzed with JET, a JET score is associated to it and leads to the prediction of an interaction site based on evolutionary information (color range from yellow to red, corresponding to weak and strong signals respectively). JET and NIP scores are finally combined to obtain a JET+NIP score for each protein structure (color range from light pink to deep purple, corresponding to weak and strong signals respectively). Then, for each docked conformation, the JET+NIP score is combined to the corresponding energy value (to compute the FIR) to discriminate the best conformation of 1AY7 and 2MTA among all possible conformations computed by cross-docking (grey box, corresponding to the intersection of cyan and blue boxes - notice that the orientation of 1AY7 is the same in all conformations represented in the box). For the full dataset, the FIR values of the best conformation computed for each pair of proteins are recorded in the matrix. Notice that the schema describing the computation for 1AY7 and 2MTA leads to one entry of the matrix. Finally, a normalization step produces the NII matrix used to discriminate potential partners.</p

Robustness of the IR for the true partners and the false ones for the 1BUH complex.

<p>A. Each partner of the 1BUH complex is coupled with one of the 168 proteins (including the monomer itself) of the dataset forming either a false (167 cases) or the native complex. For each complex, we computed the corresponding average IR and average AUC over 100 random sets of 40 proteins, obtained by using the experimental interfaces and the full exploration of the conformational space. These values are reported as a point in a plot. Each plot contains 168 points. The red circle in each plot corresponds to the values of the native complex. Dots are colored in a scale from black, blue, cyan to yellow. A color corresponds to the value of the standard deviation of the distribution of 100 IRs computed for a complex: black if , blue if , cyan if and yellow otherwise (i.e. ). B. The analysis in A is realized here with 1BUH coupled with 166 proteins (here we have not considered the complex 1ML0 of the Mintseris Benchmark 2.0 because JET made no predictions and this turned out to provide no value), with predicted interfaces and the full exploration of the conformational space. Dots are colored in a scale from black, cyan, green to yellow. A color corresponds to the value of the standard deviation of the distribution of 100 IRs computed for a complex: black if , cyan if , green if and yellow otherwise (i.e. ). The structures of the native complex (red circle) and of four selected false complexes (orange circles) are shown to illustrate the conformations corresponding to the best II value. Notice that the II value is always the same for the 100 random runs while the NII varies with respect to the dataset of proteins used in a run. The receptor 1BUH_r is colored in light blue while the ligand 1BUH_l is colored in dark blue. The four other proteins are colored in grey. All residues with a JET+NIP score display interaction propensity and are colored in a color range going from light pink (weak signal) to deep purple (strong signal) for the 6 structures. See Figures S24–S37 and S38–S51 in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003369#pcbi.1003369.s001" target="_blank">Text S1</a> for the same analysis on all complexes in the Mintseris Benchmark 2.0.</p