Multivalent protein-protein interactions are pivotal regulators of eukaryotic Hsp70 complexes

Abstract

Heat shock protein 70 (Hsp70) is a molecular chaperone and central regulator of protein homeostasis (proteostasis). Paramount to this role is Hsp70's binding to client proteins and co-chaperones to produce distinct complexes, such that understanding the protein-protein interactions (PPIs) of Hsp70 is foundational to describing its function and dysfunction in disease. Mounting evidence suggests that these PPIs include both "canonical" interactions, which are universally conserved, and "non-canonical" (or "secondary") contacts that seem to have emerged in eukaryotes. These two categories of interactions involve discrete binding surfaces, such that some clients and co-chaperones engage Hsp70 with at least two points of contact. While the contributions of canonical interactions to chaperone function are becoming increasingly clear, it can be challenging to deconvolute the roles of secondary interactions. Here, we review what is known about non-canonical contacts and highlight examples where their contributions have been parsed, giving rise to a model in which Hsp70's secondary contacts are not simply sites of additional avidity but are necessary and sufficient to impart unique functions. From this perspective, we propose that further exploration of non-canonical contacts will generate important insights into the evolution of Hsp70 systems and inspire new approaches for developing small molecules that tune Hsp70-mediated proteostasis.

Keywords: Bag domain; Hsp110; J-domain protein; Nucleotide exchange factor; Protein aggregation; Protein folding.

Fig. 1

Members of the Hsp70 family have a conserved architecture and ATPase cycle. A General structure of Hsp70 family members, including a nucleotide-binding domain (NBD), substrate-binding domain (SBD). The SBD is sub-divided into SBDβ, SBDα (lid) and C-terminal extension. The various Hsp70 orthologs vary in the length and composition of the C-terminal region and cytoplasmic isoforms of eukaryotic Hsp70s also have an EEVD motif. The structure of the prokaryotic Hsp70, DnaK (PDB 2KHO), is shown, along with a cartoon representation. B Schematic of the ATPase cycle of Hsp70s (PDB 2KHO and 5NRO), highlighting the conformational changes that accompany hydrolysis and the roles of the co-chaperones: J-domain proteins (JDPs) and nucleotide exchange factors (NEFs)

Fig. 2

Canonical clients bind a conserved, hydrophobic groove in Hsp70’s SBDβ. The SBDβ and lid of DnaK is pictured bound to the model client peptide NRLLLTG (PDB 1DKZ), highlighting the key residues involved. The conservation of those residues across Hsp70 orthologs in E. coli (DnaK), yeast (Ssa1), and humans (mtHsp70, BiP, Hsp72, and Hsc70) is shown from a CLUSTALW multiple sequence alignment

Fig. 3

Certain clients engage Hsp70 both canonically and non-canonically. A The domain architecture of three client proteins is shown, with known Hsp70-binding sequences displayed. A subset of these sequences is experimentally shown to bind in a nucleotide-independent, non-canonical manner and they are not competitive with canonical, model substrates for binding SBDβ. B Schematic to illustrate that some clients, including α-syn and tau, have both canonical and non-canonical binding motifs, and are able to interact with both the SBDβ and SBDα domains of Hsp70s. Still, the exact binding site of non-canonical peptides is unknown. See text for citations and details

Fig. 4

Canonical Interactions of Hsp70 NEFs are highly conserved. A NEF interaction sites are mapped onto the Hsc70 NBD (PDB 1HX1) and color coded by NEF. Binding sites shared by 2 or more NEFs are colored purple. B Domain architecture of the 5 Bag family NEFs shows significant variance outside of the C-terminal Bag domain. C The co-crystal structure of the human Bag1 Bag domain (green) and Hsc70 (gray; PDB 1HX1) highlight an important network of electrostatics stabilizes the PPI interface. Conservation of these residues across Bag family members is shown below (C-terminal Bag domain was used for Bag 5) from a CLUSTALW multiple sequence alignment

Fig. 5

Bag family NEFs contain sequences predicted to bind Hsp70’s SBD and displace clients. A Sequences predicted to bind Hsp70 SBDβ within the Bag proteins. Briefly, the ChaperISM python script was used to search human Bag protein sequences in both quantitative mode (cutoff = 2.7) and qualitative mode (cutoff = 0.2). Sequences were only included if they met cutoff thresholds in both quantitative and qualitative modes, and were found outside of the Bag domain. B Model for how a representative BAG protein, Bag3, might use both its canonical Bag domain and its non-canonical, pseudo-substrate motif to interact with two separate sites on Hsp70. In this model, the pseudo-substrate acts as a “release domain” to promote client release from the Hsp70 complex

Fig. 6

The canonical interaction of Hsp70s with JDPs is mediated by three major contacts within the J-domain, as shown in a co-crystal structure of the E. coli DnaK/DnaJ system (PDB 5NRO). The conservation of those residues across Hsp70 orthologs in E. coli (DnaJ), yeast (Ydj1 and Sis1), and humans (DnaJA1 and DnaJB1) is shown from a CLUSTALW multiple sequence alignment

Fig. 7

JDPs and Hsp70 interact using multiple sites. (A) The conserved J-domain binds to the NBD and linker of Hsp70s (see Fig. 6). In addition, Class B JDPs also bind to the EEVD motif present in cytoplasmic Hsp70s. (B) Co-crystal structure of an Hsp70 derived EEVD motif peptide (gray) bound to CTDI of human DnaJB1 (blue; PDB 3AGY), highlighting the key residues responsible for complex formation