Assessment of the ATP binding properties of Hsp90.
Hsp90, one of the most prominent proteins in eucaryotic cells under physiological and stress conditions, chaperones protein folding reactions in an ATP-independent way. Surprisingly, ATP binding and ATPase activity of Hsp90 has been reported by several groups. To clarify this important issue, we have reinvestigated the potential ATP binding properties and ATPase activity of highly purified Hsp90 using a number of different techniques. Hsp90 was compared to the well characterized ATP-binding chaperone Hsc70 and to two control proteins, immunoglobulin G and bovine serum albumin, that are known to not bind ATP. Hsp90 behaved very similarly to the non-ATP-binding proteins and very differently from the ATP-binding protein Hsc70. Like bovine serum albumin and immunoglobulin G, Hsp90 (i) did not bind to immobilized ATP, (ii) could not be specifically photocross-linked with azido-ATP, (iii) failed to exhibit significant changes in intrinsic protein fluorescence upon ATP addition, and (iv) did not bind to three fluorescent ADP analogues. In contrast, Hsc70 strongly bound ATP and ADP, specifically cross-linked with azido-ATP, and exhibited major shifts in fluorescence upon addition of ATP. Finally, reexamination of the amino acid sequence of Hsp90 failed to reveal any significant homologies to known ATP-binding motifs. Taken together, we conclude that highly purified Hsp90 does not bind ATP. Weak ATPase activities associated with Hsp90 preparations may be due to minor impurities or kinases copurifying with Hsp90.
Jakob
U
U
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, 93040 Regensburg, Federal Republic of Germany.
Scheibel
T
T
Bose
S
S
Reinstein
J
J
Buchner
J
J
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002417:Cattle; D016384:Consensus Sequence; D004926:Escherichia coli; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D018832:Molecular Chaperones / Q000737:chemistry*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
D002352:Carrier Proteins; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D018832:Molecular Chaperones; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate