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 implications of the binding of AMP-PNP (adenylyl-imidodiphosphate-a non-hydrolysable ATP analogue), ADP and the inhibitors radicicol and geldanamycin. Analysis of (1)H,(15)N correlation spectra showed a specific pattern of chemical shift perturbations 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 Hsp90. For AMP-PNP and ADP, additional shift perturbations of residues outside the binding pocket were observed and can be regarded as a result of conformational rearrangement upon binding. According to the crystal structures, these regions are the first alpha-helix and the "ATP-lid" ranging from amino acids 85 to 110. The N-terminal domain is therefore not a passive nucleotide-binding site, as 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.
Dehner
Alexander
A
Institut für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany.
Furrer
Julien
J
Richter
Klaus
K
Schuster
Ioana
I
Buchner
Johannes
J
Kessler
Horst
H
D000266:Adenylyl Imidodiphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D016227:Benzoquinones; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D047029:Lactams, Macrocyclic; D007783:Lactones / Q000378:metabolism*; D018942:Macrolides; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011809:Quinones / Q000378:metabolism*
D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D007783:Lactones; D018942:Macrolides; D011809:Quinones; D000266:Adenylyl Imidodiphosphate; C035359:monorden; C001277:geldanamycin