Abstract
Heat shock protein 70 (Hsp70) is an important molecular chaperone of the proteostasis network, mediating protein folding and stability. Malfunction of proteostasis contributes to diseases as diverse as cancer and Alzheimer’s. Hsp70 consists of the substratebinding and nucleotide-binding domains connected by an interdomain linker, and cycles between two distinct nucleotide states: the ADP-bound and ATP-bound conformations. The ATP-bound structure of Hsp70 was not characterised at the onset of this thesis; understanding this form will give crucial insight into the mechanism of ATP catalysis and interdomain communication. We have purified, produced crystals of and characterised a modified Hsp70 bound to ATP. The modified Hsp70 was designed to capture an ATP-bound state similar to that of the distant homologue Hsp110. It lacks the last 33 amino acids of the disordered carboxy-terminal, and carries a T199A mutation to block ATP hydrolysis. Two additional cysteines (E47C and F529C) were introduced to form a cross-link designed to lock the two domains of Hsp70 together in the ATP-bound state. Proteinase K assays confirmed modified Hsp70 can adopt the wild type ATP-bound conformation. A X-ray crystallography 2.8 structure of our modified Hsp70 was characterised bound to ATP by molecular replacement methods and de novo model building with phase extension techniques. The final model displays refinement statistics of Rwork at 0.2599 and Rfree at 0.2874 (Rwork at 0.3639 and Rfree at 0.3885 in the highest resolution shell). In the ATP-bound conformation the NBD makes intimate contacts with the SBD, and a highly specific NBD: SBDα interface is formed upon conformational rearrangement of the individual domains. The interdomain linker is buried in a hydrophobic groove at the base of the NBD, and is essential in orienting the SBDβ for formation of the NBD: SBDβ interface. ATP is bound in the nucleotide binding cavity, adjacent to this linker binding site. We propose that the proximity of this linker binding site to the active site is important for signalling interdomain communication. The NBD: SBDβ interface and linker binding site also define potential targets for discovery of drugs to control proteostasis diseases.