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.

Client protein activation by Hsp90 involves a plethora of cochaperones whose roles are poorly defined. A ubiquitous family of stress-regulated proteins have been identified (Aha1, activator of Hsp90 ATPase) that bind directly to Hsp90 and are required for the in vivo Hsp90-dependent activation of clients such as v-Src, implicating them as cochaperones of the Hsp90 system. In vitro, Aha1 and its shorter homolog, Hch1, stimulate the inherent ATPase activity of yeast and human Hsp90. The identification of these Hsp90 cochaperone activators adds to the complex roles of cochaperones in regulating the ATPase-coupled conformational changes of the Hsp90 chaperone cycle.

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In vivo activation of client proteins by Hsp90 depends on its ATPase-coupled conformational cycle and on interaction with a variety of co-chaperone proteins. For some client proteins the co-chaperone Sti1/Hop/p60 acts as a "scaffold," recruiting Hsp70 and the bound client to Hsp90 early in the cycle and suppressing ATP turnover by Hsp90 during the loading phase. Recruitment of protein kinase clients to the Hsp90 complex appears to involve a specialized co-chaperone, Cdc37p/p50 cdc37 , whose binding to Hsp90 is mutually exclusive of Sti1/ Hop/p60. We now show that Cdc37p/p50 cdc37 , like Sti1/ Hop/p60, also suppresses ATP turnover by Hsp90 supporting the idea that client protein loading to Hsp90 requires a "relaxed" ADP-bound conformation. Like Sti1/Hop/p60, Cdc37p/p50 cdc37 binds to Hsp90 as a dimer, and the suppressed ATPase activity of Hsp90 is restored when Cdc37p/p50 cdc37 is displaced by the immunophilin co-chaperone Cpr6/Cyp40. However, unlike Sti1/Hop/ p60, which can displace geldanamycin upon binding to Hsp90, Cdc37p/p50 cdc37 forms a stable complex with geldanamycin-bound Hsp90 and may be sequestered in geldanamycin-inhibited Hsp90 complexes in vivo.

ATP hydrolysis by the Hsp90 molecular chaperone requires a connected set of conformational switches triggered by ATP binding to the N-terminal domain in the Hsp90 dimer. Central to this is a segment of the structure, which closes like a "lid" over bound ATP, promoting N-terminal dimerization and assembly of a competent active site. Hsp90 mutants that influence these conformational switches have strong effects on ATPase activity. ATPase activity is specifically regulated by Hsp90 co-chaperones, which directly influence the conformational switches. Here we have analyzed the effect of Hsp90 mutations on binding (using isothermal titration calorimetry and difference circular dichroism) and ATPase regulation by the co-chaperones Aha1, Sti1 (Hop), and Sba1 (p23). The ability of Sti1 to bind Hsp90 and arrest its ATPase activity was not affected by any of the mutants screened. Sba1 bound in the presence of AMPPNP to wild-type and ATPase hyperactive mutants with similar affinity but only very weakly to hypoactive mutants despite their wild-type ATP affinity. Unexpectedly, in all cases Sba1 bound to Hsp90 with a 1:2 molar stoichiometry. Aha1 binding to mutants was similar to wild-type, but the -fold activation of their ATPase varied substantially between mutants. Analysis of complex formation with co-chaperone mixtures showed Aha1 and p50 cdc37 able to bind Hsp90 simultaneously but without direct interaction. Sba1 and p50 cdc37 bound independently to Hsp90-AMPPNP but not together. These data indicated that Sba1 and Aha1 regulate Hsp90 by influencing the conformational state of the "ATP lid" and consequent N-terminal dimerization, whereas Sti1 does not.

Crystallographic, isotopic labeling nmr and transferred nuclear Overhauser effect studies have highlighted the extended conformation as a very important element of secondary structure at the binding site of many peptide/protein complexes including peptide inhibitors-enzymes, B-cell epitopes-antibodies, and T-cell epitopes-major histocompatibility complex (MHC) of class I and II complexes. This paper discusses the peptide ligand conformation consequences of these findings particularly in view of the identification of the PII conformation (left-handed extended polyproline II) in free solution.

The syntheses and properties of a series of lanthanide complexes (Ln Eu, Tb, Dy, Yb) of C 4 symmetric chiral tetraamide ligands based on 1,4,7,10-tetraazacyclododecane are reported. The configuration of the chiral centre at carbon in the amide substituent (CH 2 NHCO-CH(Me)Ar) determines the helicity of the derived complex and the configuration of the macrocyclic ring. The enantiopure lanthanide complexes do not undergo D/L interconversion in the temperature range 220 to 320 K and three complexes have been characterised by X-ray crystallography, revealing nine-coordination about the lanthanide ion (Ln Eu, Dy) with a monocapped square-antiprismatic coordination geometry. The terbium complexes are highly emissive in aqueous solution following excitation into the aryl chromophore (e.g. for [Tb´(R)- 3 ) shows a strong near-IR CD and circularly polarised luminescence (CPL) associated with the 2 F 5/2 ± 2 F 7/2 transition. Overall, these emissive complexes allow control in modulating both the frequency and the polarisation of emitted light in aqueous solution.

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