Structural Basis of Interdomain Communication in the Hsc70 Chaperone

Abstract

Hsp70 family proteins are highly conserved chaperones involved in protein folding, degradation, targeting and translocation, and protein complex remodeling. They are comprised of an N-terminal nucleotide binding domain (NBD) and a C-terminal protein substrate binding domain (SBD). ATP binding to the NBD alters SBD conformation and substrate binding kinetics, but an understanding of the mechanism of interdomain communication has been hampered by the lack of a crystal structure of an intact chaperone. We report here the 2.6 angstroms structure of a functionally intact bovine Hsc70 (bHsc70) and a mutational analysis of the observed interdomain interface and the immediately adjacent interdomain linker. This analysis identifies interdomain interactions critical for chaperone function and supports an allosteric mechanism in which the interdomain linker invades and disrupts the interdomain interface when ATP binds.

Molecular Structure, Function, and Dynamics of Clathrin-Mediated Membrane Traffic

“…The agent of clathrin uncoating is the cytosolic heat-shock cognate protein Hsc70 (Rothman and Schmid 1986). Like all members of the Hsp70 family, Hsc70 has an actin-like ATPase domain linked to a characteristic "molecular clamp" domain, which can capture hydrophobic peptides, exposed on an unfolded polypeptide chain or projecting from an assembly destined for dissociation (Jiang et al 2005). When the enzyme is in the ATP-bound state, the clamp-domain/peptide association is relatively weak, ATP hydrolysis (to ADP and Pi) tightens the clamp, and nucleotide exchange (dissociation of ADP and binding of ATP) opens the clamp, releases the peptide, and completes the cycle.…”

A sequential mechanism for clathrin cage disassembly by 70-kDa heat-shock cognate protein (Hsc70) and auxilin

“…It was recently demonstrated that dynamic light scattering can be used effectively to monitor clathrin cage disassembly (21,22), thus providing better time resolution than previous studies that were predominantly based on centrifugation and densitometry of SDS-PAGE (19,20). We have further increased the time resolution by which disassembly kinetics can be measured, by monitoring simple perpendicular light scattering using stopped-flow methods to capture events on the milliseconds-to-seconds timescale.…”

Heat shock cognate protein 70 isoform D is required for clathrin-dependent endocytosis of Japanese encephalitis virus in C6/36 cells

“…In past decades, the entry of JEV into host cells, either mammalian or mosquito, was demonstrated to be significantly influenced by treatment with bafilomycin A1 (Andoh et al, 1998;Nawa, 1998). In fact, a number of flaviviruses like West Nile virus (Jiang et al, 2005), DENV (Acosta et al, 2008) and JEV (Chai et al, 2013), have been known to infect host cells via this pathway. Furthermore, the cationic amphiphilic drug chlorpromazine can interfere with the entry process (Chai et al, 2013;Nawa et al, 2003), revealing that the mode of infection is receptormediated endocytosis and is clathrin-dependent.…”

“…Hsc70s are highly conserved in structure from prokaryotes to eukaryotes (Boorstein et al, 1994). In general, Hsc70 can be involved in folding/ unfolding and assembly/disassembly of macromolecular structures (Elefant & Palter, 1999;Jiang et al, 2005) as well as anti-apoptotic activity (Powers et al, 2008). Thus, Hsc70 expression is usually beneficial for cells by resisting injury under conditions of minimal stress (Sens et al, 1997).…”