Figure 1.
Structure of Hsp70 ATPase domain and its complexes with different nucleotide exchange factors (NEFs).
(a) ATPase domain structure colored by subdomains: IA (red; residues 1–39 and 116–188), IB (blue; residues 40–115), IIA (green; residues 189–228 and 307-C-terminus) and IIB (orange; residues 229–306). Several subdomain IIB residues are involved in NEF recognition and binding, including residues at the C-terminal part of helix 8 (G230-H249), the helix 9 (K257-S275), and the β-sheet E (strands Q279-I284 and F293-T298 connected by a long exposed loop). Residue identifications and secondary structure nomenclature are based on the PDB entry 1HPM. In yellow stick representation is a bound ADP. (b–e) Interactions with four different NEFs. (b) DnaK ATPase fragment from E. coli complexed with GrpE, (c) bovine Hsc70 complexed with BAG-1, (d) human Hsc70 with Sse1, and (e) human Hsc70 with HspBP1. In each case the NEF is colored cyan, ATPase fragment white, and interface residues, shown in space-filling representation, are colored according to their subdomain locations. See Table S1 and Table S2, and the Materials and Methods for more information on the examined complexes, and the identity of NEF-recognition residues in each case. All ribbon diagrams are created using PyMol (http://www.pymol.org).
Figure 2.
Intrinsic dynamics of the Hsp70 ATPase domain: high mobility of NEF-recognition sites in contrast to restricted mobility of nucleotide-binding residues.
(a) Distribution of residue mobilities Mi(1) in the global mode of motion calculated for the unbound Hsp70 ATPase domain. The horizontal bars on the upper abscissa indicate the ranges of the four subdomains IA, IB, IIA and IIB, colored as in Figure 1a. Subdomain IIB is distinguished by its enhanced mobility, with peaks at two regions: the C-terminal part of helix 8, and the β-hairpin loop. NEF-binding residues are indicated by the blue open circles (based on atom-atom distances) and red filled circles (based on ΔSASA). The diagram in the inset is color-coded to illustrate the global mobility profile (red: most mobile, blue: most rigid). (b) Weighted-average mobility profiles based on top-ranking ten GNM modes of motion, calculated using Equation 1 for the unbound ATPase domain (blue) and for the NEF-bound structures (red), averaged over three mammalian complexes (Table S1). Nucleotide-binding residues (G12-Y15, G201-G203, G230, E231, E268, K271, R272, S275, G338- S340, R342, I343, D366) are indicated by filled squares. (c) Change in mobility between bound and unbound ATPase domain, obtained by taking the difference of two curves shown in panel b. The dashed line corresponds to the zero level. NEF-binding residues are marked by filled squares.
Figure 3.
Schematic description of ET calculations for Hsp70 family.
(a) The phylogenetic tree is constructed using the ET server [30] and the set of 1627 ATPase domain sequences retrieved from PFAM database for the Hsp70 family. Each vertical line corresponds to a given distance threshold. The boxes in different colors refer to the partitions obtained at the 12th distance threshold (also called level). Each box yields a different consensus sequence. The class consensus sequence for each partitioning level is then identified, as illustrated. Dots therein refer to positions that are sequentially variable between the members of the class. The ET sequence for the particular level is determined by assigning letter code X to all positions that are conserved within classes, but not conserved across classes. Those amino acids conserved across classes are indicated by their single letter code (e.g., glycine G in the illustrated ET sequence). (b) Results are shown for a 20-level partitioning of the phylogenetic tree. Peaks indicate the most conserved residues (among the 380 amino acids represented in each sequence), with their conservation level (or ET rank) indicated by the row numbers on the left. The columns highlighted in gray correspond to nucleotide binding residues. Those corresponding to the NEF binding residues are colored by the subdomains to which they belong (see Figure 1a). A high-resolution version of this figure can be seen in the Figure S3 of the SM.
Figure 4.
Comparison of experimentally observed and computationally predicted structural changes in the Hsp70 ATPase domain.
Experimental changes are illustrated for BAG-1-bound and free forms of the bovine Hsp70 ATPase domain (respective PDB Ids: 1HX1 and 1HPM). Computational results are obtained by the ANM applied to the respective two structures. (a) Structural alignment of NEF-bound and unbound ATPase fragments. The unbound ATPase fragment (1HPM) is colored gray. The NEF-bound ATPase fragment (1HX1) is color-coded according to its extent of deformation with respect to the unbound ATPase, the regions showing the largest deformation being colored red, and those unchanged, blue. The distance between Ala60 and Arg258 Cα-atoms is 5.0 Å in the closed form and 10.9Å in the open form. Panel b displays the results for the unbound (black) and BAG-1-bound (red) ATPase domain. The solid curve represents the correlation cosine between the experimentally observed deformation vector d and the ANM modes 1–20 accessible to the ATPase domain (either NEF-bound or -free). The curve with circles describes the cumulative overlap (Equation 2). A subset of 6 slow modes accessible to the unbound form ensures the passage to the NEF-bound conformer with an overlap of 0.86. The NEF-bound form exhibits an even stronger potential to be reconfigured back to its closed form, consistent with the preferred conformation of the ATPase domain in the absence of NEF binding: top ranking two modes yield a cumulative overlap of 0.88 with the experimental deformation d.
Figure 5.
Correlation between residue mobility and its sequential variability.
(a) Ribbon diagram colored by the ET rank of residues, from red (most conserved) to blue (most variable). (b) The average mobility of residues corresponding to different ET ranks. The mobilities are evaluated using Equation 1. The bars display the standard error for each ET rank. Best fitting second order polynomial (red curve) is shown to guide the eye (correlation coefficient of 0.92). See also SM Figure S7 panel b for a similar plot, based on ConSurf score (instead of ET rank).
Figure 6.
Co-evolution of NEF-binding residues revealed by mutual information (MI) maps constructed for the Hsp70 ATPase domain.
(a) Amino acids distinguished by their high co-evolutionary patterns in the maps c and d (residues with average MI value greater than 0.32), shown in stick representation and colored by subdomains. Among them, the NEF contacting residues (Table S1) are labeled black, and others are colored by subdomain. Note the large proportion of charged or polar residues. (b) MI map for the ATPase domain sequence included in the MSA (residues 6–385). The color-coded bar on the right indicates the level of correlation between the evolution of residue pairs. Two regions containing a large number of NEF-binding residues are enlarged in panels c and d. The bar plots under the map display the contribution of each residue to the most correlated residue pairs (top 1%, 720 pairs) in the MI matrix (upper plot), and the frequency of NEF-ATPase domain contacts in three mammalian complexes (lower plot). (c) and (d) Close-up views of the MI map portions between residues 246–305 (containing the helix 9 and β-sheet E of subdomain IIB) and residues 16–75 (containing NEF-contacting segments in subdomains IA and IB) (c), and within residues 246–305 (d). The corresponding secondary structural elements are indicated along the abscissa by cylinders (α-helices) and arrows (β-strands). The bar plots display the average MI per residue, NEF binding residues being colored by their subdomain.
Figure 7.
Average MI values calculated for different structural elements (helices/strands) and for different subdomains.
The panels demonstrate the mean MI value between and within (a) pairs of secondary structure elements (the names of helices and sheets are based on the PDB entry 1HPM, H: α-helix, S: β-sheet) and (b) pairs of subdomains.