Giuliano Siligardi

Activation of the ATPase activity of Hsp90 by the stress-regulated cochaperone Aha1

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.

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.

Regulation of Hsp90 ATPase activity by the co-chaperone Cdc37p/p50cdc37

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.

Reassessment of the electronic circular dichroism criteria for random coil conformations of poly(l-lysine) and the implications for protein folding and denaturation studies

The circular dichroism (CD) spectra of poly(L-lysine) in water and ethanediol/water (2:1) solutions in the temperature range -110 to 85 degrees C are presented. The results combined with vibrational CD data are interpreted in terms of a two-state conformational equilibrium with a left-handed trans polyproline II conformation being preferred at low temperatures. The relevance of these studies to the CD criteria for random-coil conformations, the study of helix-coil transitions and protein/peptide folding is pointed out.

Oligomerization of the chromatin-structuring protein H-NS

H‐NS is a major component of the bacterial nucleoid, involved in condensing and packaging DNA and modulating gene expression. The mechanism by which this is achieved remains unclear. Genetic data show that the biological properties of H‐NS are influenced by its oligomerization properties. We have applied a variety of biophysical techniques to study the structural basis of oligomerization of the H‐NS protein from Salmonella typhimurium. The N‐terminal 89 amino acids are responsible for oligomerization. The first 64 residues form a trimer dominated by an α‐helix, likely to be in coiled–coil conformation. Extending this polypeptide to 89 amino acids generated higher order, heterodisperse oligomers. Similarly, in the full‐length protein no single, defined oligomeric state is adopted. The C‐terminal 48 residues do not participate in oligomerization and form a monomeric, DNA‐binding domain. These N‐ and C‐terminal domains are joined via a flexible linker which enables them to function independently within the context of the full‐length protein. This novel mode of oligomerization may account for the unusual binding properties of H‐NS.

Measuring Protein Structure and Stability of Protein–Nanoparticle Systems with Synchrotron Radiation Circular Dichroism

We measure the structural and stability changes of proteins at nanomolar concentration upon interaction with nanoparticles. Using synchrotron radiation circular dichroism (SRCD), we measure a decrease of 6 °C in the thermal unfolding of human serum albumin upon interaction with silver nanoparticles while this does not happen with gold. The use of SRCD allows measuring critical parameters on protein-nanoparticle interactions, and it will provide experimental data on the relative stability of key biological proteins for nanotoxicology.

The Role of the Phospho-CDK2/Cyclin A Recruitment Site in Substrate Recognition

Phospho-CDK2/cyclin A, a kinase that is active in cell cycle S phase, contains an RXL substrate recognition site that is over 40 Å from the catalytic site. The role of this recruitment site, which enhances substrate affinity and catalytic efficiency, has been investigated using peptides derived from the natural substrates, namely CDC6 and p107, and a bispeptide inhibitor in which the γ-phosphate of ATP is covalently attached by a linker to the CDC6 substrate peptide. X-ray studies with a 30-residue CDC6 peptide in complex with pCDK2/cyclin A showed binding of a dodecamer peptide at the recruitment site and a heptapeptide at the catalytic site, but no density for the linking 11 residues. Kinetic studies established that the CDC6 peptide had an 18-fold lower Km compared with heptapeptide substrate and that this effect required the recruitment peptide to be covalently linked to the substrate peptide. X-ray studies with the CDC6 bispeptide showed binding of the dodecamer at the recruitment site and the modified ATP in two alternative conformations at the catalytic site. The CDC6 bispeptide was a potent inhibitor competitive with both ATP and peptide substrate of pCDK2/cyclin A activity against a heptapeptide substrate (Ki = 0.83 nm) but less effective against RXL-containing substrates. We discuss how localization at the recruitment site (KD 0.4 μm) leads to increased catalytic efficiency and the design of a potent inhibitor. The notion of a flexible linker between the sites, which must have more than a minimal number of residues, provides an explanation for recognition and discrimination against different substrates.

Analysis of mutants of tetanus toxin HC fragment: ganglioside binding, cell binding and retrograde axonal transport properties

Tetanus toxin binds neuronal tissue prior to internalization and trafficking to the central nervous system. Binding of the carboxy‐terminal 50 kDa HC fragment of tetanus toxin to polysialogangliosides is important for this initial cell binding step. Using the three‐dimensional structure of HC, mutants were designed to investigate the role of individual residues in ganglioside binding. Mutant proteins were tested for binding to GT1b gangliosides, to primary motoneurons and for their ability to undergo retrograde transport in mice. Two classes of mutant were obtained: (i) those containing deletions in loop regions within the C‐terminal β‐trefoil domain which showed greatly reduced ganglioside and cell binding and did not undergo retrograde transport and (ii) those that showed reduced ganglioside binding, but retained primary neuronal cell binding and retrograde transport. The second class included point mutants of Histidine‐1293, previously implicated in GT1b binding. Our deletion analysis is entirely consistent with recent structural studies which have identified sugar‐binding sites in the immediate vicinity of the residues identified by mutagenesis. These results demonstrate that ganglioside binding can be severely impaired without abolishing cell binding and intracellular trafficking of tetanus toxin.

Expression and characterisation of a highly repetitive peptide derived from a wheat seed storage protein

The high molecular weight (HMW) subunit group of wheat seed storage proteins impart elasticity to wheat doughs and glutens. They consist of three domains: non-repetitive N- and C-terminal domains, which contain cysteine residues for covalent cross-linking, and a central domain consisting of repeated sequences. The circular dichroism and infrared (IR) spectra of an intact HMW subunit were compared with those of a peptide corresponding to the central repetitive domain expressed in Escherichia coli. This allowed the structure of the central domain to be studied in the absence of the N- and C-terminal domains and the contributions of these domains to the structure of the whole protein to be determined. In solution the peptide showed the presence of beta-turns and polyproline II-like structure. Variable temperature studies indicated an equilibrium between these two structures, the polyproline II conformation predominating at low temperatures and the beta-turn conformation at higher temperatures. IR in the hydrated solid state also indicated the presence of beta-turns and intermolecular beta-sheet structures. In contrast, spectroscopy of the whole subunit showed the presence of alpha-helix in the N- and C-terminal domains. The content of beta-sheet was also higher in the whole subunit, indicating that the N- and C-terminal domains may promote the formation of intermolecular beta-sheet structures between the repetitive sequences, perhaps by aligning the molecules to promote interaction.