Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP

The 70 kD heat shock proteins (Hsp70s) are ubiquitous and highly conserved molecular chaperones essential for cellular protein folding and proteostasis. Each Hsp70 has two functional domains: a nucleotide-binding domain (NBD) that binds and hydrolyzes ATP, and a substrate-binding domain (SBD) that binds extended polypeptides. NBD and SBD interact little when in ADP; however, ATP binding allosterically couples the polypeptide- and ATP-binding sites. ATP binding promotes polypeptide release; polypeptide rebinding stimulates ATP hydrolysis. This allosteric coupling was poorly understood. To explore the molecular mechanism of this essential ATP-induced allosteric coupling, we solved a crystal structure of an intact Hsp70 from E. coli in complex with ATP at 1.96 A resolution. NBD-ATP adopts a unique conformation, forming extensive interfaces with a radically changed SBD that has its α-helical lid displaced and the polypeptide-binding pocket of its β-subdomain flipped open. Our biochemical analysis inspired by this structure provides a long-sought mechanistic explanation of how ATP binding allosterically opens the polypeptide-binding site.