The Hsp70 family of molecular chaperones provides a well defined and experimentally powerful model system for understanding allosteric coupling between different protein domains.New extensions to the statistical coupling analysis (SCA) method permit identification of a group of co-evolving amino-acid positions—a sector—in the Hsp70 that is associated with allosteric function.Literature-based and new experimental studies support the notion that the protein sector identified through SCA underlies the allosteric mechanism of Hsp70.This work extends the concept of protein sectors by showing that two non-homologous protein domains can share a single sector when the underlying biological function is defined by the coupled activity of the two domains.
Allostery is a biologically critical property by which distantly positioned functional surfaces on proteins functionally interact. This property remains difficult to elucidate at a mechanistic level (Smock and Gierasch, 2009) because long-range coupling within proteins arises from the cooperative action of groups of amino acids. As a case study, consider the Hsp70 molecular chaperones, a large and diverse family of two-domain allosteric proteins required for cellular viability in nearly every organism (Figure 1) (Mayer and Bukau, 2005). In the ADP-bound state, the two domains act independently, the C-terminal substrate-binding domain displays a stable configuration in which the so-called ‘lid' region is docked against the β-sandwich subdomain, and substrates bind with relatively high affinity (Figure 1A) (Moro et al, 2003; Swain et al, 2007; Bertelsen et al, 2009). Exchange of ADP for ATP in the N-terminal nucleotide-binding domain causes significant local and propagated conformational change, formation of an interface with the substrate-binding domain, opening of the lid subdomain, and a decrease in the binding affinity for substrates (Figure 1B) (Rist et al, 2006; Swain et al, 2007). Upon ATP hydrolysis by the nucleotide-binding domain, Hsp70 is returned to the ADP-bound configuration suitable for another round of substrate binding and release. This process of cyclical substrate binding and release underlies all biological functions of Hsp70 proteins.
What is the structural basis for the long-range functional coupling within Hsp70? When allostery is a conserved property of a protein family, one approach to this problem is to analyze the correlated evolution of amino acids in the family—the expected statistical signature of cooperative action of protein residues (Lockless and Ranganathan, 1999; Kass and Horovitz, 2002; Suel et al, 2003). Previous work using an implementation of this concept (the statistical coupling analysis or SCA) showed that proteins contain sparse networks of co-evolving amino acids termed ‘sectors' that link protein active sites with distinct functional surfaces through the protein core (Halabi et al, 2009). This architecture is consistent with known allosteric mechanisms in protein domains (Suel et al, 2003; Halabi et al, 2009).
However, the principle of co-evolution of protein residues need not be limited to the study of individual protein domains. Indeed, conserved allosteric coupling between two (or more) non-homologous domains implies the existence of shared sectors that span functional sites on different domains. Here, we test this concept by extending the SCA method to consider the allosteric mechanism acting between the two domains of the Hsp70 proteins. Hsp70-like proteins include not only the allosteric Hsp70s, but also the Hsp110s—homologs that contain both domains and are regarded as structural models for Hsp70s, but that do not exhibit allosteric coupling. In this study, we take advantage of the functional divergence between the Hsp70s and Hsp110s to reveal patterns of co-evolution between amino acids that are specifically associated with the allosteric mechanism.
To identify the allosteric sector in Hsp70, we used SCA to compute a weighted correlation matrix, C̃, that describes the co-evolution of every pair of amino-acids positions in a sequence alignment of 926 members of the Hsp70/110 family. We then applied a mathematical method known as singular value decomposition to simultaneously evaluate the pattern of divergence between sequences and the pattern of co-evolution between amino-acid positions. The basic idea is that if the pattern of sequence divergence is able to classify members of a protein family into distinct functional subgroups, then we can rigorously identify the group of co-evolving residues that correspond to the underlying mechanism. Figure 2A shows the principal axis of sequence variation in the Hsp70/110 family, showing a clear separation of the allosteric (Hsp70) and non-allosteric (Hsp110) members of this family. The corresponding axis of co-evolution between amino-acid positions reveals a subset of Hsp70/110 positions (∼20%, 115 residues out of 605 total) that underlie the divergence of Hsp70 and Hsp110 proteins (Figure 2B). These positions derive roughly equally from the nucleotide-binding domain (in blue, 56 positions) and the substrate-binding domain (in green, 59 positions) and are more conserved within the Hsp70 sub-family. These results define a protein sector that is predicted to underlie the allosteric mechanism of Hsp70.
What is the structural arrangement of the putative allosteric sector within the Hsp70 protein? Consistent with a function in allosteric coupling, the 115 sector residues form a physically contiguous network of atoms, linking the ATP-binding site on the nucleotide-binding domain to the substrate recognition site on the substrate-binding domain through the interdomain interface (Figure 2C). The physical connectivity is remarkable given that only ∼20% of overall Hsp70 residues is involved (Figure 2B). Thus, functionally coupled but non-homologous protein domains can share a single sector of co-evolving residues that connects their respective functional sites.
We compared the Hsp70 sector mapping with the large body of biochemical studies that have been carried out in this family. We find strong experimental support for the involvement of sector positions in the Hsp70 allosteric mechanism in several regions: (1) within the ATP-binding site, (2) at the interface linking the two domains, and (3) within the β-sandwich core of the substrate-binding domain. The sector analysis also makes predictions about the involvement of some previously untested residues; we show that mutations at two such sites in fact reduce the allosteric coupling within Hsp70 in vitro and fail to complement a DnaK knockout strain of E. coli in a stress-response assay. Taken together, we conclude that sector positions are associated with the allosteric mechanism of Hsp70.
This work also adds a new finding with regard to the concept of protein sectors. Previous work showed that multiple quasi-independent sectors, each of which contributes a different aspect of function, are possible within a single protein domain (Halabi et al, 2009). This work shows that a single sector can also span two different protein domains when biological function (here, nucleotide-dependent substrate binding) arises from their coupled action. This result emphasizes the point that sectors are units of functional selection and are not obviously related to traditional hierarchies of structural organization in proteins. An interesting possibility is that evolution of allostery between proteins might evolve through the joining of protein sectors, a conjecture that can be tested in future work.
Allosteric coupling between protein domains is fundamental to many cellular processes. For example, Hsp70 molecular chaperones use ATP binding by their actin-like N-terminal ATPase domain to control substrate interactions in their C-terminal substrate-binding domain, a reaction that is critical for protein folding in cells. Here, we generalize the statistical coupling analysis to simultaneously evaluate co-evolution between protein residues and functional divergence between sequences in protein sub-families. Applying this method in the Hsp70/110 protein family, we identify a sparse but structurally contiguous group of co-evolving residues called a ‘sector', which is an attribute of the allosteric Hsp70 sub-family that links the functional sites of the two domains across a specific interdomain interface. Mutagenesis of Escherichia coli DnaK supports the conclusion that this interdomain sector underlies the allosteric coupling in this protein family. The identification of the Hsp70 sector provides a basis for further experiments to understand the mechanism of allostery and introduces the idea that cooperativity between interacting proteins or protein domains can be mediated by shared sectors.