Catalysis of ADP-ATP exchange by nucleotide exchange factors (NEFs) is central to the activity of Hsp70 molecular chaperones. Yet, the mechanism of interaction of this family of chaperones with NEFs is not well understood in the context of the sequence evolution and structural dynamics of Hsp70 ATPase domains. We studied the interactions of Hsp70 ATPase domains with four different NEFs on the basis of the evolutionary trace and co-evolution of the ATPase domain sequence, combined with elastic network modeling of the collective dynamics of the complexes. Our study reveals a subtle balance between the intrinsic (to the ATPase domain) and specific (to interactions with NEFs) mechanisms shared by the four complexes. Two classes of key residues are distinguished in the Hsp70 ATPase domain: (i) highly conserved residues, involved in nucleotide binding, which mediate, via a global hinge-bending, the ATPase domain opening irrespective of NEF binding, and (ii) not-conserved but co-evolved and highly mobile residues, engaged in specific interactions with NEFs (e. g. , N57, R258, R262, E283, D285). The observed interplay between these respective intrinsic (pre-existing, structure-encoded) and specific (co-evolved, sequence-dependent) interactions provides us with insights into the allosteric dynamics and functional evolution of the modular Hsp70 ATPase domain.
Many proteins are molecular machines. They function because their three-dimensional structure allows them to undergo cooperative changes in conformation that maintain the native fold while enabling their biological functions. The changes have been pointed out to be structure-encoded, intrinsically accessible to proteins, as can be deduced from simple physics-based approaches [1]. Yet, amino acid specificity is another important property that selectively mediates the interactions with specific partners and ligands [2]. Overall, a subtle balance exists between structure-encoded mechanical properties and sequence-encoded specific properties, and this balance must be evolutionarily optimized to achieve precise functioning. The interplay between these two effects becomes particularly important in the case of a number of proteins or domains that play a modular role in a variety of biomolecular interactions. The ATPase domain (also called nucleotide-binding domain) of the Hsp70 family of proteins is a typical example. This domain plays a critical role in regulating the activities of these molecular chaperones, which, in turn, promote accurate folding, and prevent unwanted aggregation by either unfolding and refolding misfolded proteins or regulating their intracellular trafficking to the protein degradation machinery [3]–[5]. Chaperones of the Hsp70 family contain two domains: the N-terminal ATPase domain and the C-terminal substrate-binding domain (SBD), which regulate each other' s activity via allosteric effects. ATP hydrolysis at the ATPase domain increases the substrate-binding affinity of the SBD, thus lowering the substrate exchange rate; on the other hand, the dissociation of the ADP produced upon ATP hydrolysis and its replacement by a new ATP trigger the release of substrate by the SBD, and therefore enhance the substrate exchange rate [3]. Regulation of substrate-binding affinity by the ATPase domain forms the basis of the chaperone activity of Hsp70s [6], [7]. The precise functioning of the Hsp70 ATPase domain involves an interaction with two families of co-factors, also called co-chaperones: the J-domain proteins that catalyze ATP hydrolysis [8], and the nucleotide exchange factors (NEFs) that assist in the replacement of ADP with ATP, by significantly increasing the ADP dissociation rate [9]. A molecular understanding of Hsp70 function requires a systemic analysis of the structural basis and mechanism of interaction with these co-chaperones. Here we focus on the interaction of their ATPase domain with NEFs. The Hsp70 ATPase domain is composed of four subdomains: IA and IB in lobe I, and, IIA and IIB in lobe II (Figure 1a). ATP binds the central cleft between the two lobes at the interface between subdomains IIA and IIB such that the geometric and energetic effects of its binding and hydrolysis are efficiently transmitted throughout the ATPase domain. To date, four classes of NEFs have been identified: GrpE in prokaryotes [10], and BAG-1 [11], HspBP1 [12] and Hsp110 [13] in eukaryotes. Their diverse three-dimensional structures (Figure 1b–e) exhibit a variety of binding geometries and interfacial interactions with the Hsp70 ATPase domain. In the present study, we examine these interactions, using sequence-, structure- and dynamics-based computations and identify their shared features. Our analysis provides insights into the generic and specific aspects of ATPase domain-NEF interactions, as well as the molecular machinery and sequence design principles of this highly versatile module, the Hsp70 ATPase domain, thus reconciling robust structure-encoded cooperative dynamics properties and highly correlated amino acid changes that enable specific recognition.
We began with 4839 sequences retrieved from the Pfam database 22. 0 [14] for the Hsp70 family of molecular chaperones (Pfam id: PF00012). We refined the generated multiple sequence alignment (MSA) by using the consensus sequence of the ATPase domain (380 residues) in the bovine cytosolic homolog of Hsp70 [15]. The refinement consists of three steps: (i) iterative implementation of Smith-Waterman algorithm (SW) for pairwise alignment [16] using our consensus sequence, and elimination of those sequences below a threshold SW score (or less than 40% sequence identity; see details in Supplementary Material (SM) Figure S1 and Text S1) to retrieve the closest orthologs to human (Hsc70) and bacterial (DnaK) chaperones; (ii) deletion of MSA columns that correspond to insertions with respect to the consensus sequence, and (iii) removal of the sequences containing more than 10 gaps. These three steps resulted in a MSA of 1627 sequences with N = 380 columns (corresponding to residues 6 to 385 in Hsc70 ATPase domain), which has been subjected to evolutionary trace (ET) and mutual information (MI) analyses for detecting residue conservation and co-evolution patterns, respectively. We retrieved from the Protein Data Bank (PDB) [17] structural data for HSP70 ATPase domains complexed with GrpE (PDB id: 1DKG [10]), BAG-1 (PDB id: 1HX1 [18]), HspBP1 (PDB id: 1XQS [19]), and Sse1 (Hsp110, PDB id: 3D2E [20]), shown in Figure 1b–e. Additionally, the structure of the above mentioned bovine Hsc70 ATPase domain resolved at 1. 7 Å resolution (PDB id: 1HPM [15]) was used for the unbound form, and the PDB structure 1S3X [21] of the human Hsp70 served as a template to reconstruct the lobe I missing in the complex with HspBP1 using the method described in the SM Figure S2 and Text S2. We performed Gaussian Network Model (GNM) [22], [23] and anisotropic network model (ANM) [24], [25] analyses for elucidating the equilibrium dynamics of Hsp70 ATPase domain both in the unbound form and in the complexes with different NEFs, including the reconstructed complex with HspBP1. Details on the methods can be found in our previous work [22]–[26]. Mainly, knowledge of the distribution of inter-residue contacts in the native structure permits us to construct the Kirchhoff (GNM) and Hessian (ANM) matrices, which, upon eigenvalue decomposition, yield information on the collective modes spectra. We focused on the low frequency modes, also called global modes, as the major determinant of functional movements. In the GNM, each mode k is represented by an N-dimensional eigenvector, u (k), and eigenvalue λk, describing the mode shape and frequency (squared), respectively. The ith element, [u (k) ]i, of u (k) describes the displacement of residue i along the kth mode axis; the plot of [u (k) ]i2 as a function of residue index i defines the mobility profile Mi (k) in mode k. See for example, the mobility profile Mi (1) for the first (lowest frequency) mode accessible to Hsp70 ATPase domain in Figure 2a. By definition, eigenvectors are normalized, i. e. , the mobility profile also represents the normalized distribution of square displacements in mode k. The reciprocal λk−1 serves as the weight of mode k, such that the slower modes, also called softer modes, make larger contributions to observed dynamics. The mobility of residue i driven by a subset of m soft modes is found from the weighted average (Figure 2b) (1) The modes predicted by the ANM for the ATPase domain both in NEF-bound and –free forms were compared to the experimentally measured changes in structure (designated by a 3N-dimensional deformation vector d) using two metrics: (i) the correlation cosine (|v (k) ·d|/|d|) between the kth ANM eigenvector v (k) (k = 1, …, 3N-6) and d, and (ii) the cumulative overlap achieved by the m softest modes [26], (2) The deformation d, is obtained by superposing the known NEF-bound and -free structures of ATPase domain and evaluating the differences in the Cα-coordinates. Kabsch' s algorithm [27] is used for optimal superposition that eliminates rigid-body translations and rotations. The ET method [28] identifies conserved residues using the MSA-derived phylogenetic tree for a given family. The application of the procedure to the Hsp70 family of chaperones is outlined in Figure 3, and details can be found in previous work [28], [29]. In summary, the method consists of three steps: (i) the phylogenetic tree is partitioned into multiple levels [30] as indicated by the vertical bars in Figure 3a; (ii) at each level, sequences are grouped into classes, each being characterized by a “class consensus sequence”. The consensus sequences are cross-examined to identify fully conserved (across classes) and class-specific or trace residues (conserved within classes but not across classes). The ET sequence for the particular level lists the fully conserved residues by their single-letter code, the trace residues by the symbol ‘X’, and the remaining residues as blank; and (iii) The ET sequences generated at each level are organized in rows (Figure 3b). An ET rank (leftmost column) is assigned to each residue. A fully conserved residue is assigned the highest rank (rank of 1). In the present case, Gly201 is the only residue with ET rank 1, i. e. , it is fully conserved among the set of 1627 sequences (see SM Figure S3 for a larger version of this panel). The conservation of a given residue in all subfamilies is a very strict condition when large sets of aligned sequences are considered. This limitation restricted the previous applications of the ET method to MSA of 100 and 200 sequences [31]. To adapt the ET method and its variations [32]–[34] to our dataset of >1,600 sequences, we relaxed the condition for defining an ET residue from conservation across “all” members in a given level to “90%”of members, and we allowed for gaps [34]. The ET method identifies conserved residues, but does not provide information on co-evolutionary relations between residues. Co-evolving residues are usually indicative of structural or functional constraints [35]–[38]. We adopted the MI content as a measure of the degree of intra-molecular co-evolution between residues in the Hsp70 ATPase domain [37], [39]–[41]. In this method, each of the N columns of the MSA is considered as a discrete random variable that takes on one of the 20 amino-acid types, or an insertion (gap, as the 21st type), with some probability. The MI associated with the ith and jth sequence positions is defined as an N×N matrix (for a MSA of N columns) of the form (3) where P (xi, yj) is the joint probability of observing amino acid types x and y at the respective sequence positions i, and j; P (xi) is the marginal/singlet probability of amino acid of type x at the ith position. I (i, j) varies in the range [0, Imax], where the lower and upper limits correspond to fully uncorrelated and most correlated pairs of residues.
Organisms comply with the evolutionary pressure to maintain their phenotype by genotypic variations that are compensated or correlated as needed, conserving certain sequence fragments vital to preserving their functions [57]. Understanding the co-evolving and conserved sequence patterns in modular domains is an interesting problem in its own right [58], [59]. Understanding these patterns in the light of structural data, if available, provides us with further insights into shared mechanisms of interactions that form the molecular basis of the biological function of such modular domains. The Hsp70 ATPase domain is such a modular protein common to functionally diverse actin, hexokinase, and Hsp70 protein families [60]. The present combined analysis of structure-encoded dynamics and sequence evolution for Hsp70 ATPase domain discloses a subtle interplay between conserved interactions and those involving co-evolved residues. Conserved interactions define generic properties of the Hsp70 ATPase domain: these include the concerted dynamics of its four subdomains, which allow for sampling functional conformations (e. g. , that stabilized upon NEF binding, allowing for ADP release; shown in Figure 3), and the physicochemical events (ATP hydrolysis) at the nucleotide-binding site. Those residues involved in NEF recognition, on the other hand, show low-to-moderate conservation, but exhibit a remarkably high tendency to co-evolve, or undergo correlated mutations, again to achieve specific NEF-dependent recognition and binding activities. Interestingly, NEF residues that interact with the Hsp70 ATPase domain appear to be rather conserved (Figure S11) to maintain this specificity. An observation of interest is the similarity between the interactions of the Hsp70 ATPase domain with different NEFs, in terms of structural dynamics. While Hsp70 ATPase domains are highly conserved both sequentially and structurally, the four NEFs examined have distinct structures and consequently different dynamics. The key point is that their binding to the ATPase domain involves in all cases the subdomain IIB of the ATPase domain, although not in exactly the same arrangement. Their binding to a common interfacial region on the ATPase domain point to a shared mechanism of interaction: The ATPase subdomain IIB is originally distinguished by its high mobility in the slowest mode, especially at the β-sheet E and the exposed loop connecting the two strands of this sheet; and after NEF binding, there is a significant suppression in its mobility. The conserved dynamics of the complexes suggests a role of subdomain IIB as an “adjustable handle”, which regulates the Hsp70 chaperone machine, to facilitate other proteins making use of its SBD. Many applications using the ANM have shown that the substrate recognition involves a region distinguished by its enhanced mobility in the most cooperative (or softest) modes, which enables the molecule to optimize its interactions with the substrate. Here we can see that the C-terminal part of helix 8 and the loop of β-hairpin E enjoy this type of high mobility/adaptability. On the other hand, substrate ‘binding’ may also involve more constrained residues in the close neighborhood, which may play a role in transmitting allosteric effects. In the opposite case of a binding site composed exclusively of floppy residues, the structural changes induced upon substrate binding could dissipate locally and not efficiently transmitted. In this respect, we propose that the involvement of residues such as Arg258, Arg261 and Arg262 in subdomain IIB, or N57, A60 and M61 in subdomains IB is critically important in establishing the communication between subdomains and transmitting allosteric signals between NEF-binding and nucleotide binding sites. A putative communication pathway that couples distant residues in different subdomains of the Hsp70 ATPase domain is suggested here by the structural mapping of correlated and conserved residues, which needs to be further established. Figure 6a displays those residues identified to be co-evolving. Notably, we observe several pairs making interdomain contacts, in addition to spatially distant residue pairs (e. g. H23 in subdomain IA and N57, A60 and M61 in subdomain IB correlated with R258, R261, E283 and D292 in subdomain IIB). In a recent study, R272, R261, Y15 and Y41 have been identified to play a central role in establishing the allosteric communication in the unbound Hsp70 ATPase domain, along with highly conserved residues K71, R72, E175 and H227 [45]. It remains to be seen if these central residues play a key role in mediating between these co-evolving, spatially distant residues. We also note that Smock et al. recently identified a sparse but structurally contiguous group of co-evolving residues at the interface between the ATPase domain and the SBD in Hsp70/110 protein family, which has been proposed to underlie the inter-domain allosteric coupling [61], in support of the role of co-evolved residues in mediating allosteric signaling. Many recent studies have pointed out the validity of “pre-existing equilibrium” concept where a substrate or ligand simply selects from amongst an ensemble of conformations already accessible to the protein prior to binding [49], [62]–[67]. The present results, and recent applications of ENMs, suggest that more important than the pre-existence of these ‘states’, is the existence of energetically accessible ‘paths’ that provide access to those states, or the intrinsic tendency of the native structure to reconfigure towards such functional states. In terms of energy landscape description, what is needed is not the existence of multiple minima, the depths of which change upon ligand or substrate binding, but the existence of one or more directions of reconfigurations, or paths along the energy landscape, that are easily accessible to the protein and lead to the targeted (functional) conformer. The softest modes provide such paths. They define directions of motion in the space of collective coordinates, which incur a minimal energy ascent as the molecule moves away from its original energy minimum. They also present the best mechanisms of dissipating energy, if the system is perturbed. These are the modes that are being exploited when proteins bind ligands or substrates. Notably these functional conformations accessible near the native state can be observed by NMR residual dipolar coupling, as shown for Hsp70 ATPase domain by Zuiderweg and coworkers [52]. Figure 4 clearly shows that movements along a handful of modes satisfactorily ensures the passage to the alternative (functional) open form, and that the open form itself has a strong tendency to restore its conformation back to the closed form, in the absence of NEF. Protein-ligand binding interfaces and protein-protein contact interfaces are characterized by different sequence variation patterns. The protein-protein contact interfaces usually expose larger contact areas [68] and exhibit high mutation rates. Moreover, if the contact interface is a common recognition site for multiple targets (possibly in different organisms), co-evolution is likely to occur among the binding residues to preserve specific interactions and conformations at the sequence motif. On the other hand, the protein-ligand interface is usually buried in the folded core of the protein; in contrast to protein-protein interaction, the protein-ligand interaction is usually characterized by higher specificity, requiring sequence conservation [28], [29]. The Hsp70 ATPase domain exhibits patterns in close agreement with these general features: Its ligand (nucleotide) binding site essentially consists of highly conserved residues, which not only precisely coordinate the ligand, but also take part in a global hinge-bending region so that they are both chemically and mechanically required to be highly conserved. NEF recognition sites, on the other hand, exhibit much lower conservation properties; and in addition to their sequence variability, the subdomain IIB, which is observed to be most often involved in NEF binding, enjoys enhanced mobility. Briefly, global dynamics requirements entail residue conservation, and specific recognition entails sequence variation along with enhanced mobility. However, neither the sequence variability, nor the conformational mobility at NEF recognition sites, is random. The sequence variability takes place under unique restrictions, compensating mutations, as unraveled by the MI map. Conformational variability, on the other hand, is uniquely defined by the ATPase architecture, and precisely adept to accommodate the passage to the functional open state that is stabilized upon NEF binding. The ATPase domain uniquely juxtaposes such structure-encoded dynamics and sequence-specific interactions, which underlie its ubiquitous activities. In general, subdomains IA and IIA are more conserved and more rigid than subdomains IB and IIB [69], as also indicated by the ET in Figure 3b; notably, they also serve as binding site to a number of proteins. For example, subdomain IA accounts for the binding of J-domain proteins [70]; subdomain IIA is reported to contain a putative binding site near its interface with subdomain IA (V189-V195) to the chaperonin-containing TCP-1 [71], and it is connected to the SBD by an inter-domain linker, which is considered important for the allosteric interactions between the two domains [72], [73]. It remains to be seen if the correlated sites on Hsp70 ATPase domain emerging from the MI analysis play a role in the functional communication with other co-chaperones or the SBD. Extensive experimental studies have been performed to date with the E. coli Hsp70, DnaK, to understand the molecular mechanism of activity of the molecular chaperones in the Hsp70 family. The analysis in the present paper will guide our interpretation of the NMR, FRET, and EPR data on different states accessible to DnaK. Each of these methods gives us a different window into the ensemble of conformational states populated in response to ATP, ADP and NEFs. Excitingly, a detailed chemical shift analysis of six different ligand bound states for the nucleotide-binding domain of DnaK, with and without the linker that connects it to the substrate-binding domain (i. e. , 12 NMR samples compared pairwise and as a group) has pointed to the same subdomain interface rearrangements indicated in the present study (Zhuravleva & Gierasch, in preparation). Moreover, the NMR results point to the fundamental feature that subdomain IIB can undergo a hinge-like movement to enable nucleotide entry and release. It is this fundamental movement, intrinsic to Hsp70 ATPase domains, that different NEFs have exploited. They bind in different, sequence-specific ways, but modulate the same fundamental movement. Further detailed analysis of the ensemble distributions and rates of interconversion between states can be achieved using a synergistic battery of computational and experimental tools.
The heat shock protein 70 (Hsp70) serves as a housekeeper in the cell, assisting in the correct folding, trafficking, and degradation of many proteins. The ATPase domain is the control unit of this molecular machine and its efficient functioning requires interactions with co-chaperones, including, in particular, the nucleotide exchange factors (NEFs). We examined the molecular motions of the ATPase domain in both NEF-bound and -unbound forms. We found that the NEF-binding surface enjoys large global movements prior to NEF binding, which presumably facilitates NEF recognition and binding. NEF binding stabilizes the ATPase domain in an open form and thereby facilitates the nucleotide exchange step of the chaperone cycle. A series of highly correlated amino acids were distinguished at the NEF-binding sites of the Hsp70 ATPase domain, which highlights the adaptability of the ATPase domain, both structurally and sequentially, to recognize NEFs. In contrast, the nucleotide-binding residues are tightly held near a global hinge center and are highly conserved. The contrasting properties of these two groups of residues point to an evolutionarily optimized balance between conserved/constrained and co-evolved/mobile amino acids, which enables the functional interactions of the modular Hps70 ATPase domains with NEFs.
computational biology/macromolecular structure analysis
computational biology/macromolecular sequence analysis
evolutionary biology/bioinformatics
computational biology
Role of Hsp70 ATPase Domain Intrinsic Dynamics and Sequence Evolution in Enabling its Functional Interactions with NEFs