Fig 1.
Ribbon representation of the crystal structure of the ATP-bound nitrogenase complex (PDB ID: 2AFK).
Fe1, red; Fe2, yellow; α1β1 unit of MoFe, blue; α2β2 unit of MoFe, green. The ADP-bound complex (PDB ID: 2AFI [I]) shown in white, is aligned to the ATP-bound complex on the MoFe protein. In the ATP-bound complex, the P-loop (cyan), switch I region (orange) and switch II region (magenta) are highlighted in Fe2, and the ATP analogues (AMPPCP), [4Fe-4S] clusters, P-clusters and FeMo-co are shown in spheres.
Fig 2.
Mechanical coupling matrix M for (A) the ATP-bound nitrogenase complex and (B) the ADP-bound nitrogenase complex. The mechanical couplings between Fe1 and α1β1, Fe2 and α2β2, Fe1 and Fe2 are highlighted in dashed blocks.
Fig 3.
Maximum mechanical coupling M of (A) residues in α1β1 to any residue in Fe1 and (B) residues in Fe2 to any residue in Fe1 in ATP-bound complex. The ATP analogues, [4Fe-4S] clusters, P-clusters, and FeMo-co are shown in mesh surface. Residue 186–190 in β1 are highlighted in spheres in (A); one α-helix and three β-sheets in each monomer of Fe2 appear red in (B) (following the numbering in the 2AFK PDB file).
Fig 4.
Change in RMSF of the MoFe protein upon ATP hydrolysis.
The change is shown as deviation of the RMSF of the MoFe protein in the ADP-bound complex relative to the ATP-bound complex reported as a color scale and is projected on the ribbon representation of the protein. The positive changes, whereby the ADP-bound complex shows larger flexibility than the ATP-bound complex, are shown in red, and the negative values are shown in blue. For clarity of representation, the RMSF change for the Fe proteins is not color-coded. The ATP analogues, [4Fe-4S] clusters, P-clusters, and FeMo-co are shown in mesh surface. Residues 186–190 in β1 are highlighted as space-filling structures (spheres). Residues at Fe protein/MoFe protein interface with large RMSF change upon ATP hydrolysis appear in orange or red.
Fig 5.
Change in M between residues in α1β1 to any residue in Fe1 upon ATP hydrolysis.
The change is shown as largest deviation of M in the ADP-bound complex relative to the ATP-bound complex, either positive (red) or negative (blue) following the same projection scheme discussed in Fig 4. The Fe1, Fe2, α2β2 proteins are shown in white. The ATP analogues, [4Fe-4S] clusters, P-clusters, and FeMo-co are shown in mesh surface. Residues 186–190 in β1 are highlighted in spheres and residues 119–120 in α1 are indicated by arrow. Residues at Fe protein/MoFe protein interface with large deviation upon ATP hydrolysis appear in dark red or dark blue. For clarity of representation only the change in one half of the MoFe protein is shown.
Fig 6.
Important residues for mechanical coupling between (A) Fe1 and α1β1, (B) Fe1 and Fe2 and (C) P-loops in Fe1 and P-loops in Fe2 as identified by cost-weighted betweenness centrality, χ. For clarity, only residues with χ > 40% of maximum value are shown as hot pink space-filling structures, whose size is proportional to the magnitude of the corresponding χ value. The ribbon presentation of the ATP-bound nitrogenase complex is colored as follows: Fe1, red; Fe2, yellow; α1β1 unit of MoFe, blue; α2β2 unit of MoFe, green. ATP, [4Fe-4S] clusters, P-clusters and FeMo-co are shown in spheres. Residues among top 10% of χ or residues in/near the sites of interest are labeled.
Fig 7.
Top 9 normal modes of the ATP-bound nitrogenase complex (top panels) and their influences on the mechanical coupling between (A) Fe1 and α1β1, (B) Fe1 and Fe2, (C) P-loops in Fe1 and P-loops in Fe2; (D) overlap between the normal modes of the ATP-bound nitrogenase and the vector describing the displacement of the Fe protein that takes place upon ATP hydrolysis as inferred from the ATP- and ADP-bound crystal structures.
Fig 8.
HDX Mass Spectrometry measured deuterium exchange in the MoFe protein alone and in the presence of the ATP-bound Fe protein.
The percent deuterium uptake at 1 hour in selected regions of MoFe alone and in complex with Fe protein is shown as a histogram. Complete exchange was based on a 24-hour time point.
Fig 9.
Correlation patterns of protein dynamics in (A) free MoFe protein and (B) MoFe protein in presence of ATP-bound Fe protein based on HDX measurements recorded after 1h of the deuterium exchange. Red and blue circles correspond to negative and positive correlations, respectively. An empty spot in the matrix (or white circle) signifies lack of any correlation. Size of the circle represents significance of the results: the larger the circle the more significant the correlation. Squares are drawn to highlight clusters with similar correlation patterns.