Structure and mechanism of cooperation by Escherichia coli hsp40 DnaJ and hsp70 DnaK
Description
The Escherichia coli hsp40 DnaJ and hsp70 DnaK cooperate in the binding of proteins at intermediate stages of folding, assembly and translocation across membranes. Binding of protein substrates to the DnaK C-terminal domain is controlled by ATP binding and hydrolysis in the N-terminal ATPase domain. The interaction of DnaJ with DnaK is mediated at least in part by the highly conserved N-terminal J-domain of DnaJ that includes residues 2 to 75 The goal of this research is to understand at the atomic level how hsp40 and hsp70 cooperate as molecular chaperones in the management of protein-protein interactions. This dissertation consists of two parts: (I) The interaction of the J-domain, DnaJ2-75, with full-length DnaK and with the ATPase domain of DnaK, DnaK2-388, is characterized; (II) the folding and stability of 5-hydroxytryptophan-containing DnaJ variant proteins are described. In part I, residues in the J-domain involved in the interaction with DnaK were identified by heteronuclear NMR experiments. The J-domain interaction is localized to the ATPase domain of DnaK and is likely to be dominated by electrostatic interactions. Dissociation constants were calculated for the interaction of DnaJ2-75 for DnaK(MgADP) and DnaK2-388(MgADP). NMR and paramagnetic relaxation experiments examined the proximity of DnaJ2-75 to Mn2+ in DnaK-bound MnADP, and the results suggest that DnaJ2-75 does not approach within five angstroms of Mn2+ in a DnaK(MnADP) complex. In part II, DnaJ variant proteins containing 5-hydroxytryptophan (5HW) were characterized by unfolding and fluorescence, circular dichroism and transverse urea gradient gel electrophoresis. Unfolding monitored by fluorescence of 5HW residues located in the J-domain is cooperative, whereas unfolding monitored by a 5HW residue in the gly/phe-rich region is not cooperative. Unfolding of one of the DnaJ 5HW variants is cooperative and at least partially reversible. Fluorescence quenching with iodide of 5HW in variant proteins suggests that the gly/phe-rich region is more accessible to solvent than the J-domain. This is consistent with the proposal that the gly/phe-rich region is unstructured Structural data obtained in these studies contribute toward a detailed mechanism of hsp40/hsp70 cooperation. DnaJ can interact with certain polypeptide substrates prior to DnaK. Therefore, we propose that DnaJ enhances the ability of DnaK to bind polypeptide substrates by targeting DnaK to the complex, stimulating ATP hydrolysis of DnaK and thereby stabilizing the ternary DnaJ-DnaK(MgADP)-polypeptide complex