Our goal is to probe the interactive surfaces of E. coli molecular chaperones DnaJ and DnaK using NMR and to test hypotheses of how DnaJ stimulates the ATPase activity of DnaK, for example, that DnaJ stimulates the hydrolysis of ATP by interacting with DnaK at the ATP-binding site. Preliminary experiments indicate that that certain residues of a truncated DnaJ, DnaJ-2-75, approach within a few angstroms of manganese ions in a Mn-ATP-DnaK complex. Mn2+ is a paramagnetic counter ion which selectively broadens NMR signals of atoms that approach within a few angstroms, and no differences are observed in the rats of Mg-ATP and Mn-ATP hydrolysis in single turnover or steady-state assays. A Mn-ATP-DnaK complex was formed and added to 15JV - labeled DnaJ2-75. The Mn-ATP-DnaK complex specifically interacts with DnaJ2-75 as shown by the selective broadening of DnaJ2-75 peaks in an HSQC experiment. The structure of the first 75 residues of DnaJ has been determined by NMR (Szyperski et al.) 1994). Our results suggest that residues D5, E17, E42,A43 and D59 of DnaJ2-75 approach the ATP-binding site on DnaK and, surprisingly, these residues are not close in space to the conserved HPD tripeptide.
|Original language||English (US)|
|State||Published - Dec 1 1996|
ASJC Scopus subject areas
- Molecular Biology