Computational Analysis of Residue Interaction Networks and Coevolutionary Relationships in the Hsp70 Chaperones: A Community-Hopping Model of Allosteric Regulation and Communication

Abstract

Allosteric interactions in the Hsp70 proteins are linked with their regulatory mechanisms and cellular functions. Despite significant progress in structural and functional characterization of the Hsp70 proteins fundamental questions concerning modularity of the allosteric interaction networks and hierarchy of signaling pathways in the Hsp70 chaperones remained largely unexplored and poorly understood. In this work, we proposed an integrated computational strategy that combined atomistic and coarse-grained simulations with coevolutionary analysis and network modeling of the residue interactions. A novel aspect of this work is the incorporation of dynamic residue correlations and coevolutionary residue dependencies in the construction of allosteric interaction networks and signaling pathways. We found that functional sites involved in allosteric regulation of Hsp70 may be characterized by structural stability, proximity to global hinge centers and local structural environment that is enriched by highly coevolving flexible residues. These specific characteristics may be necessary for regulation of allosteric structural transitions and could distinguish regulatory sites from nonfunctional conserved residues. The observed confluence of dynamics correlations and coevolutionary residue couplings with global networking features may determine modular organization of allosteric interactions and dictate localization of key mediating sites. Community analysis of the residue interaction networks revealed that concerted rearrangements of local interacting modules at the inter-domain interface may be responsible for global structural changes and a population shift in the DnaK chaperone. The inter-domain communities in the Hsp70 structures harbor the majority of regulatory residues involved in allosteric signaling, suggesting that these sites could be integral to the network organization and coordination of structural changes. Using a network-based formalism of allostery, we introduced a community-hopping model of allosteric communication. Atomistic reconstruction of signaling pathways in the DnaK structures captured a direction-specific mechanism and molecular details of signal transmission that are fully consistent with the mutagenesis experiments. The results of our study reconciled structural and functional experiments from a network-centric perspective by showing that global properties of the residue interaction networks and coevolutionary signatures may be linked with specificity and diversity of allosteric regulation mechanisms.

Fig 1. The Functional Cycle of the Hsp70 Chaperones.

The main steps of the biochemical chaperone cycle are schematically illustrated for the E. coli Hsp70 (DnaK) clockwise: a) a closed, ADP-bound form (pdb id 2KHO); b) a partially closed ATP-bound form (intermediate 1); c) a partially open ATP-bound form (intermediate 2); d) substrate is released and compact ATP-bound structure is formed (pdb id 4B9Q); e) substrate association is coupled with unlocking of the lid in the ATP-bound intermediate state (intermediate 3); f) ATP hydrolysis is coupled with transition to a partly undocked substrate-bound form (intermediate 4). The hypothetical intermediate forms schematically illustrate a progression of major conformational changes and reorganization of the inter-domain interfaces NBD/SBD-β, NBD/SBD-α and SBD-β/SBD-α interfaces during functional cycle. The Hsp70 structures along functional cycle are shown in ribbons. Structural subdomains are annotated and colored according to the following scheme: IA (blue), IB (red), IIA (green), IIB (cyan), the linker (black), SBD-α (magenta), and SBD-β (orange).

Fig 2. Crystal Structures and Domain Organization of the Simulated Hsp70 Proteins.

Crystal structures and domain organization of the additional simulated Hsp70 structures are annotated according to the PFAM nomenclature of the Hsp70 family (PF00012). (Left upper panel) the crystal structure of the DnaK_GEOKA in post-ATP hydrolysis state (pdb id 2V7Y); (middle upper panel) a solution structure of an ADP-bound DnaK_ECOLI (pdb id 2KHO); (right upper panel) the crystal structure of ATP-bound DnaK_COLI constructs (pdb id 4B9Q, 4JNE, 4JN4); (left middle panel) the crystal structure of the HSC bovine construct E213A/D214A mutant (pdb id 1YUW); (central middle panel) the crystal structure of the of the HSC bovine construct E213A/D214A mutant (pdb id 4FL9); (right middle panel) the crystal structure of the native ATP-bound Sse1 (HSP7F_YEAST) (pdb id 2QXL); (left lower panel) the crystal structure of ATP-Sse1 nucleotide exchange complex with the NBD of HSC70 bovine (pdb id 3C7N); (middle lower panel) the crystal structure of the selenomethionine-derivatized Sse1 construct in a complex with the NBD of hHsp70 (pdb id 3D2E); (right lower panel) the crystal structure of the native Sse1 in a complex with the NBD of hHsp70 (pdb id 3D2F).

Fig 3. Sequence Conservation and Coevolutionary Analysis of the Hsp70 Proteins.

(A) Sequence conservation profile of the Hsp70 residues. The KL conservation score was calculated using multiple sequence alignment (MSA) profile of the Hsp70 protein families obtained from PFAM. The reference sequence in the alignment corresponds to DNAK_ECOLI (residues 4–604). The residue numbering in the sequence conservation profile corresponds to the residue numbering in the DnaK crystal structures. The positions of experimentally known functional residues involved in regulation are indicated as filled brown squares. These residues include K70, R71 (subdomain IB), P143, Y145, F146, D148, R151, K155, R167, I168, N170, E171 (subdomain IA), D393 (inter-domain linker), K414, N415, I438, V440, Q442, L454, D431, R467, D481, L484 (SBD-β), M515, D540, H544 (SBD-α). The important coevolutionary residues is also highlighted: the top 5% of highest pMI residues are shown in black filled circles and the top 5% of highest cMI residues are depicted in red filled diamonds. (B) The cMI score of the Hsp70 residues measures the cumulative value of mutual information shared by a given residue with other protein residues. (C) Structure-based pMI profile of the ADP-DnaK. (D) The pMI profile of the ATP-DnaK. pMI scores for each residue position are evaluated as the sum of cMI values of all residues within 5Å proximity from a given residue. The distance between each pair of residues in the structure was calculated as the shortest distance between any two non-hydrogen atoms from respective two residues. pMI values are evaluated by averaging computations over equilibrium conformational ensembles of the DnaK structures. The sequence conservation and coevolutionary residue scores are shown in colored bars. The adopted coloring scheme is based on subdomain nomenclature as in Fig 1.

Fig 4. Conformational Dynamics and Residue Coevolution of the DnaK Structures.

Conformational dynamics of the ADP-DnaK structure (pdb id 2KHO) (A) and ATP- DnaK structure (pdb id 4B9Q) (B). Residue-based conformational mobility profiles are annotated and colored according to the subdomain nomenclature as in Fig 1. The GMM-derived essential mobility profiles averaged over the three slowest modes are shown for the ADP-DnaK (C) and ATP- DnaK structure (D). The same coloring scheme for the chaperone subdomains is applied. The top 5% of highest pMI residues (black filled circles) and top 5% of highest cMI residues (red filled diamond) are mapped onto conformational dynamics and essential mobility profiles.

Fig 5. Structural Mapping of Coevolving Residues in the DnaK Forms.

Structural mapping of high pMI residues (top 5%) onto ADP-DnaK (A) and ATP-DnaK forms (B). Mapping of high cMI residues (top 5%) onto ADP-DnaK and ATP-DnaK is shown on panels (C) and (D) respectively. The top 5% of high pMI residues (A, B) and high cMI residues (C, D) are in spheres (only C_α atoms are shown).

Fig 6. Network Analysis of the DnaK Structures: Residue Centrality Profiles.

Residue-based centrality distributions for the ADP-DnaK (A) and ATP-DnaK (B). Residue centrality profiles are also obtained by averaging computations of network parameters over MD-based equilibrium ensembles. The position of experimentally known functional residues is indicated as filled black diamonds. (C, D) Structural mapping of functional residues onto the ADP-DnaK (pdb id 2KHO) and ATP-DnaK conformations. The experimentally known functional residues involved in allosteric regulation are shown in spheres (only C_α atoms of these residues are depicted as spheres) and colored according to the respective subdomain they belong to. These residues include K70, R71 (subdomain IB), P143, Y145, F146, D148, R151, K155, R167, I168, N170, E171 (subdomain IA), D393 (inter-domain linker), K414, N415, I438, V440, Q442, L454, D431, R467, D481, L484 (SBD-β subdomain), M515, D540, H544 (SBD-α subdomain).

Fig 7. The Relationships between Coevolutionary and Residue Interaction Network Parameters in the Hsp70 Proteins.

(A) A scatter graph of cMI residue scores and residue centrality values. (B) A scatter plot of pMI residue scores and residue centrality values. The data points (shown in black filled squares) aggregated coevolutionary propensities and residue centrality profiles in all simulated Hsp70 proteins. pMI scores and residue centrality values were evaluated by averaging computations over MD-based conformational ensembles of the following Hsp70 structures: DnaK_GEOKA (pdb is 2V7Y) DNAK_ECOLI (pdb id 2KHO, 4B9Q, 4JNE, 4JN4), HS7C_BOVIN (pdb id 1YUW, 4FL9), and HSP7F_YEAST (Hsp homolog Sse1) proteins (pdb id 2QXL, 3C7N, 3D2E, 3D2F). Functional sites of allosteric regulation in the DnaK chaperone are highlighted as gold filled squares. These residues include K70, R71 (subdomain IB), P143, Y145, F146, D148, R151, K155, R167, I168, N170, E171 (subdomain IA), D393 (inter-domain linker), K414, N415, I438, V440, Q442, L454, D431, R467, D481, L484 (SBD-β subdomain), M515, D540, H544 (SBD-α subdomain). The frequency distributions of pMI scores (C) and residue centrality (D) in the Hsp70 structures are shown for all residues (in blue bars) and for functional residues (in green bars). All experimentally known functional sites of the Hsp70 proteins were included in these frequency distributions.

Fig 8. Structural Mapping of Local Interaction Communities in the DnaK Structures.

Structural characterization of local interaction communities in the ADP-DnaK (pdb id 2KHO) (A) and ATP-DnaK conformations (pdb id 4B9Q) (B). Residues that form communities are depicted in spheres (only C_α atoms are shown). The key inter-domain communities are annotated and contributing residues are shown in spheres with side chain atoms included. The annotated inter-domain communities are anchored by functional sites and involve highly coevolving residues. In the ADP-DnaK structure (A) the depicted communities are (K70-E171-P143), (I4-V139-G379-T383), (D481-K387-D385), (N415-D393-T395), (L397-E444-M515), (R445-N451-M515), (L484-V440-L45-I501), (R467-D431-H544). The annotated local communities in the ATP-DnaK structure (B) are (K70-E171-P143-D201), (I73-V142-F146-T154), (K414-N415-D326-T221), (Q442-L454-L484-D148), (D481-K155-R167-I168), (A111-Q114-L507-M515).

Fig 9. Structural Mapping of Allosteric Communication Pathways in the DnaK Structures.

Structural mapping of most probable communication pathways in the ADP-DnaK structure (pdb id 2KHO) (A) and ATP-DnaK structure (pdb id 4B9Q) (B). The forward NBD-SBD pathways (shown in green spheres) connect residue K70 (NBD) with residue D431 (SBD). The reverse SBD-NBD pathways (shown in light blue spheres) connect residue D431 (SBD) with K70 (NBD). A close-up view of the communication pathways is also shown. Residues involved in allosteric communication pathways are annotated and depicted as spheres (the C_α atoms only). The most probable pathways in each direction are shown for the DnaK structure. A complete list of residues that form two most probable forward and reverse communication pathways in the DnaK structures is presented in Table 2.