NMR chemical shift perturbation study of the N-terminal domain of Hsp90 upon binding of ADP, AMP-PNP, geldanamycin, and radicicol

Hsp90 is one of the most abundant chaperone proteins in the cytosol. In an ATP-dependent manner it plays an essential role in the folding and activation of a range of client proteins involved in signal transduction and cell cycle regulation. We used NMR shift perturbation experiments to obtain information on the structural implication of the binding of AMP-PNP (adenylyl-imidodiphosphate - a non hydrolysable ATP analogue), ADP and inhibitors radicicol and geldanamycin. Analysis of ¹H,¹⁵N correlation spectra showed a specific pattern of chemical shift perturbation at N210 (ATP binding domain of Hsp90, residues 1-210)upon ligand binding. This can be interpreted qualitatively either as a consequence of direct ligand interactions or of ligand induced conformational changes within the protein. All ligands show specific interactions in the binding site, which is known from the crystal structure of the N-terminal domain of the Hsp90. For AMP-PNP and ADP, additional shift perturbation of residues outside the binding pocket were observed and can be regarded as a result of confornmational rearangement upon binding. According to the crystal structures, these regions are the first α-helix and the "ATP-lid" ranging from amino acids 85-110. The N-terminal domain is therefore not a passive nucleotide binding site, as a suggested by X-ray crystallography, but responds to the binding of ATP in a dynamic way with specific structural changes required for the progression of the ATPase cycle.