NMR studies of the heat shock protein 90 N-terminal domain

The Hsp90 based chaperone is a ubiquitous protein-folding system in the cytoplasm of eukaryotes. Several signal transduction systems and cell cycling pathways utilise an interaction with Hsp90 as an essential component. The Hsp90 chaperone is an ATP dependent chaperone, which is active as a dimer. The N- terminal domain of Hsp90 itself has very weak ATPase activity and plays an essential role in the mechanism of dimerisation. This study attempts to elucidate the nucleotide binding effects on the Hsp90 N-terminal domain by NMR. Accomplishing backbone assignments of the apo- and AMP-PNP bound forms provided a system for which individual residues could be investigated. About 200 backbone amide peaks in the HSQC were observed out of a reported 207 residues for the Hsp90 N-terminal domain. Out of these, 192 for the apo- and 182 for the AMP-PNP bound forms were assigned. Assignments were also obtained for the HN, N, C, and CO nuclei. Comparison of apo and AMP-PNP HSQC spectra showed large shift differences in areas where the nucleotide binds and in a conserved loop, which has been proposed to act as a lid to the active site. The chemical shift pattern of the AMP-PNP bound form compared to that of the ADP bound form showed a different local environment for at least 30 residues, suggesting that different nucleotide bound conformations are more than just nucleotide structural differences. Analysing the NMR results suggests that binding of AMP-PNP to the Hsp90 N-terminal domain is not sufficient to cause lid closure as previously thought. Relaxation studies highlighted regions that had different local motions in the apo- and nucleotide bound form based on individual residues within the Hsp90 N- terminal domain. The protein rotational correlation time was measured at 12.5 ns in the apo and 14 ns in the AMP-PNP bound form. No interaction between the labelled N-terminal domain and non-labelled middle domain of Hsp90 was observed. The antibiotic, novobiocin, which inhibits other members of the GHKL superfamily, was also shown not to bind to the N-terminal domain, consistent with previous studies. The study of two mutants, A107N and T101I, showed the effects of mutation in the lid region of the N-terminal domain causing chemical shifts of between 20-30 residues. Neither of these two mutants were able to bind AMP-PNP. In all nucleotide bound X-ray crystal structures solved to date, no difference between the ADP and ATP bound forms has been observed and only one conformation was found. The results presented here suggest that the structure in solution is much more variable than previously envisaged