Hsp90 molecular chaperones in eukaryotic cells play essential roles in the folding and activation of a range of client proteins involved in cell cycle regulation, steroid hormone responsiveness, and signal transduction. The biochemical mechanism of Hsp90 is poorly understood, and the involvement of ATP in particular is controversial. Crystal structures of complexes between the N-terminal domain of the yeast Hsp90 chaperone and ADP/ATP unambiguously identify a specific adenine nucleotide binding site homologous to the ATP-binding site of DNA gyrase B. This site is the same as that identified for the antitumor agent geldanamycin, suggesting that geldanamycin acts by blocking the binding of nucleotides to Hsp90 and not the binding of incompletely folded client polypeptides as previously suggested. These results finally resolve the question of the direct involvement of ATP in Hsp90 function.

The cellular activity of several regulatory and signal transduction proteins, which depend on the Hsp90 molecular chaperone for folding, is markedly decreased by geldanamycin and by radicicol (monorden). We now show that these unrelated compounds both bind to the N-terminal ATP/ADP-binding domain of Hsp90, with radicicol displaying nanomolar affinity, and both inhibit the inherent ATPase activity of Hsp90 which is essential for its function in vivo. Crystal structure determinations of Hsp90 N-terminal domain complexes with geldanamycin and radicicol identify key aspects of their nucleotide mimicry and suggest a rational basis for the design of novel antichaperone drugs.

B.Panaretou and C.Prodromou contributed equally to this workHsp90 is an abundant molecular chaperone essential to the establishment of many cellular regulation and signal transduction systems, but remains one of the least well described chaperones. The biochemical mechanism of protein folding by Hsp90 is poorly understood, and the direct involvement of ATP has been particularly contentious. Here we demonstrate in vitro an inherent ATPase activity in both yeast Hsp90 and the Escherichia coli homologue HtpG, which is sensitive to inhibition by the Hsp90-specific antibiotic geldanamycin. Mutations of residues implicated in ATP binding and hydrolysis by structural studies abolish this ATPase activity in vitro and disrupt Hsp90 function in vivo. These results show that Hsp90 is directly ATP dependent in vivo, and suggest an ATP-coupled chaperone cycle for Hsp90-mediated protein folding.

Pleckstrin homology (PH) domains are found in many signaling molecules and are thought to be involved in specific intermolecular interactions. Their binding to several proteins and to membranes containing 1-Caphosphatidylinositol 4,5-bisphosphate [PtdIns(4,5) The importance of modular binding domains in regulating interactions between signaling modules, as well as their activity, is well established (1, 2). The pleckstrin homology (PH) domain is thought to represent such a module. It contains '120 aa and was identified.as a region of sequence homology with pleckstrin that appears in a large variety of proteins involved in intracellular signaling (3)(4)(5)(6)(7)(8). Recent x-ray crystallography (9, 10) and NMR (11-14) studies have demonstrated that PH domains have a distinct characteristic structure. Their fold is best described as a seven-stranded ,-sandwich of two orthogonal 1-sheets, closed off at one corner by a C-terminal a-helix. The domain is electrostatically polarized, with the three most variable loops falling in the most positively charged surface (9).Reported ligands for PH domains include 13y subunits (Gg,) of heterotrimeric G proteins (15,16) We report here that the isolated PH domain of PLC-81 interacts specifically, and with high affinity, with both Ptdlns(4,5)P2 and Ins(1,4,5)P3. The PH domain is, therefore, likely to represent the portion of PLC-&1 responsible for high-affinity binding to PtdIns(4,5)P2 and for the observed product inhibition by Ins(1,4,5)P3. These results are a demonstration of a stereo-specific high-affinity ligand for a PH domain and support a proposed role for PH domains in regulation of PLC isoforms. MATERIALS AND METHODSGeneration of Recombinant PH Domains. Dynamin PH (DynPH) was produced in Escherichia coli as described (9). Constructs expressing residues 11-140 of rat PLC-81 (28) (PLCS-PH) and residues 1-105 of pleckstrin (29) (PlecN-PH) were produced and purified as described (9,30

C.Prodromou and B.Panaretou contributed equally to this workHow the ATPase activity of Heat shock protein 90 (Hsp90) is coupled to client protein activation remains obscure. Using truncation and missense mutants of Hsp90, we analysed the structural implications of its ATPase cycle. C-terminal truncation mutants lacking inherent dimerization displayed reduced ATPase activity, but dimerized in the presence of 5¢-adenylamido-diphosphate (AMP-PNP), and AMP-PNPpromoted association of N-termini in intact Hsp90 dimers was demonstrated. Recruitment of p23/Sba1 to C-terminal truncation mutants also required AMP-PNP-dependent dimerization. The temperaturesensitive (ts) mutant T101I had normal ATP af®nity but reduced ATPase activity and AMP-PNP-dependent N-terminal association, whereas the ts mutant T22I displayed enhanced ATPase activity and AMP-PNP-dependent N-terminal dimerization, indicating a close correlation between these properties. The locations of these residues suggest that the conformation of the`lid' segment (residues 100±121) couples ATP binding to N-terminal association. Consistent with this, a mutation designed to favour`lid' closure (A107N) substantially enhanced ATPase activity and N-terminal dimerization. These data show that Hsp90 has a molecular`clamp' mechanism, similar to DNA gyrase and MutL, whose opening and closing by transient N-terminal dimerization are directly coupled to the ATPase cycle.

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Our data provide an explanation for the specificity and high affinity of the interaction with phosphatidylinositol 3,4,5-trisphosphate and lead to a classification of the XLA mutations that reside in the Btk PH domain. Mis-sense mutations that do not simply destabilize the PH fold either directly affect the interaction with the phosphates of the lipid head group or change electrostatic properties of the lipid-binding site. One point mutation (Q127H) cannot be explained by these facts, suggesting that the PH domain of Btk carries an additional function such as interaction with a Galpha protein.

Here we describe how the systematic redesign of a protein's hydrophobic core alters its structure and stability. We have repacked the hydrophobic core of the four-helix-bundle protein, Rop, with altered packing patterns and various side chain shapes and sizes. Several designs reproduce the structure and native-like properties of the wildtype, while increasing the thermal stability. Other designs, either with similar sizes but different shapes, or with decreased sizes of the packing residues, destabilize the protein. Finally, overpacking the core with larger side chains causes a loss of native-like structure. These results allow us to further define the roles of tight residue packing and the burial of hydrophobic surface area in the construction of native-like proteins.Keywords: hydrophobic core; molecular packing; molten globule; protein design What makes a protein a protein and distinguishes it from a collapsed polymer? A pressing goal of protein engineering and design is to understand the features that give rise to the physical properties associated with a stable, "native-like" protein structure. A well-developed secondary structure is essential and the intrinsic requirements for a-helix and P-sheet formation are becoming increasingly clear (Regan, 1994;Bryson et al., 1995). Although important for stability, the turn and loop connections between elements of secondary structure often do not play a principal role in specifying the structure and properties of a protein (Brunet et al., 1993;Castagnoli et al., 1994;Predki et al., 1996) Regan, in prep). By contrast, the packing of residues in the hydrophobic core appears to be extremely important for the structure, stability, and native-like properties of natural proteins (Kellis et al