ASSESSMENT OF THE ATP BINDING-PROPERTIES OF HSP90

Hsp90, one of the most prominent proteins in eucaryotic cells under ph ysiological and stress conditions, chaperones protein folding reaction s in an ATP-independent way. Surprisingly, ATP binding and ATPase acti vity of Hsp90 has been reported by several groups. To clarify this imp ortant issue, we have reinvestigated the potential ATP binding propert ies and ATPase activity of highly purified Hsp90 using a number of dif ferent techniques. Hsp90 was compared to the well characterized ATP-bi nding chaperone Hsc70 and to two control proteins, immunoglobulin G an d bovine serum albumin, that are known to not bind ATP. Hsp90 behaved very similarly to the non-ATP-binding proteins and very differently fr om the ATP-binding protein Hsc70. Like bovine serum albumin and immuno globulin G, Hsp90 (i) did not bind to immobilized ATP, (ii) could not be specifically photocross-linked with azido-ATP, (iii) failed to exhi bit significant changes in intrinsic protein fluorescence upon ATP add ition, and (iv) did not bind to three fluorescent ADP analogues. In co ntrast, Hsc70 strongly bound ATP and ADP, specifically cross-linked wi th azido-ATP, and exhibited major shifts in fluorescence upon addition of ATP. Finally, reexamination of the amino acid sequence of Hsp90 fa iled 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.