High dimensionality NMR experiments with improved spectral resolution applied to the complete structure determination of the molecular chaperone HSC-70 (heat shock cognate, 70 KDA) substrate binding domain.

Abstract

The first 3-dimensional structure of the eukaryotic molecular chaperone Hsc-70 substrate binding domain (SBD) has been determined using multidimensional Nuclear Magnetic Resonance Spectroscopy. The structure determination was performed using novel strategies and experiments in order to both improve the quality of the final structure and to compensate for the difficulties associated with the size of the protein. The strategy employed an arsenal of 7 Nuclear Overhauser Effect Spectroscopy (NOESY) experiments including novel high resolution 4-D NOESY's to unambiguously assign the Nuclear Overhauser Effect (NOE) cross-peaks leading to distance constraints, thus obviating the premature use of potentially erroneous iterative based assignment procedures. The effectiveness of this strategy was demonstrated by obtaining 4150 NOE distance constraints, the highest number of any published structure to date. The ensemble of 20 independently calculated structures had an average backbone Root Mean Square Deviation for structured regions of 0.45 Angstroms. The Hsc-70 SBD consists of two four stranded beta sheets forming a beta sandwich domain followed by an alpha helix. The structure strongly resembles the homologous prokaryotic DnaK substrate binding domain recently determined by X-ray crystallography. A major difference is the position of the helix. The Hsc-70 structure displays a unique helix position stabilized by two salt bridges and a hydrophobic cluster. Mutational and degradation studies in the literature revealed several residues that are involved in the affinity changes important for activity of the protein. The same residues were found to be involved in intramolecular interactions that determine the positions of the helix in Hsc-70 and DnaK structures. This correlation is evidence that the positions in the helix is involved in the modulation of substrate affinity. The different helix positions in DnaK and in Hsc-70 may therefore represent different stages of the chaperone-peptide binding and release mechanism.