Ronan O'Brien

Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone.

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.

ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo.

Hsp90 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.

The ATPase cycle of Hsp90 drives a molecular ‘clamp’ via transient dimerization of the N-terminal domains

How 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‐PNP‐ promoted 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 temperature‐ sensitive (ts) mutant T101I had normal ATP affinity 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.

Regulation of Hsp90 ATPase activity by tetratricopeptide repeat (TPR)- domain co-chaperones

The in vivo function of the heat shock protein 90 (Hsp90) molecular chaperone is dependent on the binding and hydrolysis of ATP, and on interactions with a variety of co‐chaperones containing tetratricopeptide repeat (TPR) domains. We have now analysed the interaction of the yeast TPR‐domain co‐chaperones Sti1 and Cpr6 with yeast Hsp90 by isothermal titration calorimetry, circular dichroism spectroscopy and analytical ultracentrifugation, and determined the effect of their binding on the inherent ATPase activity of Hsp90. Sti1 and Cpr6 both bind with sub‐micromolar affinity, with Sti1 binding accompanied by a large conformational change. Two co‐chaperone molecules bind per Hsp90 dimer, and Sti1 itself is found to be a dimer in free solution. The inherent ATPase activity of Hsp90 is completely inhibited by binding of Sti1, but is not affected by Cpr6, although Cpr6 can reactivate the ATPase activity by displacing Sti1 from Hsp90. Bound Sti1 makes direct contact with, and blocks access to the ATP‐binding site in the N‐terminal domain of Hsp90. These results reveal an important role for TPR‐domain co‐chaperones as regulators of the ATPase activity of Hsp90, showing that the ATP‐dependent step in Hsp90‐mediated protein folding occurs after the binding of the folding client protein, and suggesting that ATP hydrolysis triggers client‐protein release.

The high-resolution crystal structure of a 24-kDa gyrase B fragment from E. coli complexed with one of the most potent coumarin inhibitors, clorobiocin

Coumarin antibiotics, such as clorobiocin, novobiocin, and coumermycin A1, inhibit the supercoiling activity of gyrase by binding to the gyrase B (GyrB) subunit. Previous crystallographic studies of a 24‐kDa N‐terminal domain of GyrB from E. coli complexed with novobiocin and a cyclothialidine analogue have shown that both ligands act by binding at the ATP‐binding site. Clorobiocin is a natural antibiotic isolated from several Streptomyces strains and differs from novobiocin in that the methyl group at the 8 position in the coumarin ring of novobiocin is replaced by a chlorine atom, and the carbamoyl at the 3′ position of the noviose sugar is substituted by a 5‐methyl‐2‐pyrrolylcarbonyl group. To understand the difference in affinity, in order that this information might be exploited in rational drug design, the crystal structure of the 24‐kDa GyrB fragment in complex with clorobiocin was determined to high resolution. This structure was determined independently in two laboratories, which allowed the validation of equivalent interpretations. The clorobiocin complex structure is compared with the crystal structures of gyrase complexes with novobiocin and 5′‐adenylyl‐β,γ‐imidodiphosphate, and with information on the bound conformation of novobiocin in the p24‐novobiocin complex obtained by heteronuclear isotope‐filtered NMR experiments in solution. Moreover, to understand the differences in energetics of binding of clorobiocin and novobiocin to the protein, the results from isothermal titration calorimetry are also presented.

Metal-dependent Folding and Stability of Nuclear Hormone Receptor DNA-binding Domains

The nuclear/hormone receptors are an extensive family of ligand-activated transcription factors that recognise DNA targets through a highly conserved, structurally autonomous DNA-binding domain. The compact structure of the DNA-binding domain is supported by two zinc ions, each of which is co-ordinated by the tetrahedral arrangement of thiol groups from four cysteine residues. Metal binding is expected to be linked with deprotonation of the co-ordinating thiol groups and folding of the polypeptide. Using a variety of biophysical approaches, we characterise these linked equilibria for the isolated DNA-binding domains (DBD) of the receptors for estrogen and glucocorticoid. Mass spectrometry and equilibrium denaturation indicate that, near neutral pH, approximately four of the eight co-ordinating thiol groups release protons with zinc uptake, in agreement with the expected pK(a) change for the -SH group in the presence of the metal. Mass spectrometry reveals that the protein charge distribution changes with the uptake of zinc and that metal binding is co-operative. The co-operativity is consistent with observations from equilibrium denaturation, which indicate that the folding event is a two-state process. A crucial residue that stabilises the equilibrium structure of the DBD fold itself is a cysteine residue situated in the hydrophobic core of all known nuclear hormone receptors (but not involved in metal binding): it appears to be conserved absolutely for its unique combination of size and hydrophobicity. Stabilisation of the DBDs could be achieved by truncating the flexible, basic termini, suggesting that like-charge clusters may have deleterious effects on protein folds. While the metal-free apo protein and the chemically denatured state have little defined secondary structure, these states were expanded only partially in comparison with the native structure, according to data from small-angle X-ray scattering. The comparatively compact shapes of the denatured and apo forms may explain, in part, the marginal stability of the native fold.

Molecular design using electrostatic interactions. Part 1. Synthesis and properties of flexible tripodand tri- and hexa-cations with restricted conformations. Molecular selection of ferricyanide from ferrocyanide

Abstract 1,3,5-Tri- and hexa-cations derived from 1,3,5-tris(bromomethyl)-2,4,6-trimethylbenzene and DABCO or quinuclidine have been prepared and their interaction with polyanions in aqueous solution investigated by 1H NMR titration and by isothermal titration calorimetry. The results from the two techniques are compared and the behaviour of these comparatively simple systems in water is shown to be complex. The trication 3b forms a crystalline precipitate with ferricyanide but not with ferrocyanide and can select for the one anion in the presence of the other. The structure of the ferricyanide salt 3e has been determined by X-ray crystallography

Halophilic adaptation of protein–DNA interactions

Pyrococcus woesei ( Pw ) is an archaeal organism adapted to living in conditions of elevated salt and temperature. Thermodynamic data reveal that the interaction between the TATA-box-binding protein (TBP) from this organism and DNA has an entirely different character to the same interaction in mesophilic counterparts. In the case of the Pw TBP, the affinity of its interaction with DNA increases with increasing salt concentration. The opposite effect is observed in all known mesophilic protein-DNA interactions. The halophilic behaviour can be attributed to sequestration of cations into the protein-DNA complex. By mutating residues in the Pw TBP DNA-binding site, potential sites of cation interaction can be removed. These mutations have a significant effect on the binding characteristics, and the halophilic nature of the Pw TBP-DNA interaction can be reversed, and made to resemble that of a mesophile, in just three mutations. The genes of functionally homologous proteins in organisms existing in different environments show that adaptation is most often accompanied by mutation of an existing protein. However, the importance of any individual residue to a phenotypic characteristic is usually difficult to assess amongst the multitude of changes that occur over evolutionary time. Since the halophilic nature of this protein can be attributed to only three mutations, this reveals that the important phenotype of halophilicity could be rapidly acquired in evolutionary time.

Reversal of Halophilicity in a Protein-DNA Interaction by Limited Mutation Strategy

Comparison of the genes of functionally homologous proteins in organisms existing in different environments shows that adaptation is most often accomplished by mutation of an existing protein. However, from such comparisons, the significance of individual residues to the particular environmental adaptation is not generally discernable among the mass of changes that occur over evolutionary time. This can be exemplified by the general transcription factor found in eukaryotes and archaea, the TATA binding protein (TBP). TBP from Pyrococcus woesei is adapted for optimal binding to DNA at high salt and high temperature, with 34% of the amino acids altered in comparison to its nearest known mesophilic counterpart. We demonstrate that the halophilic nature of this protein can be attributed to only three mutations, revealing that the important phenotype of halophilicity could be rapidly acquired in evolutionary time.