Structural basis of client recognition by co-chaperones on the Hsp70 network

Molecular chaperones play a vital role in protein homeostasis by aiding in the folding of newly synthesized proteins, refolding damaged or misfolded proteins, and sometimes preventing or reversing protein aggregation. In the chaperone cycle of Hsp70s, ATP-independent Hsp40s (known as J-domain proteins, JDPs) are responsible for recognizing and delivering clients to their Hsp70 partner. Although all JDPs have a conserved J-domain, which binds Hsp70s, each holds multiple client bindings. While in the non-canonical JDPs, the binding region is defined as a single C-terminal-domain (CTD), in canonical class A and B JDPs, this is divided into two CTDs (I and II), where the CTDI of class A is implicated in the binding with unfolded peptides. It is yet unknown how different JDPs recognize, select, and remodel different clients.
In this work, we used a combination of solution Nuclear Magnetic Resonance (NMR) methods to explore the interactions at an atomic level between different JDPs and two distinctive client proteins in terms of primary/ secondary structure (β-sheet to α-helical) and stabilities (marginally to misfolded or partially folded). Among the studied JDPs, we found that class A recognizes these client proteins by binding its CTDI to the most exposed hydrophobic segment. This leads to a dynamic and heterogeneous bound complex, which we characterized by NMR experiments to gain kinetic and thermodynamic information. Interestingly, the chaperone preferentially binds to the unfolded ensemble in a millisecond’s timescale suggesting a conformational selection mechanism. This work presents a conserved mode of client recognition by class A JDPs co-chaperones.