Chaperone Mediated Protein Activation

Section: Section of Structural Biology

Structural and Function of Hsp90 and Co-chaperones

C Prodromou, (P Meyer), C Vaughan, MMU Ali, M Zhang, SM Roe, LH Pearl; in collaboration with P Workman CR-UK Centre for Cancer Therapeutics; PW Piper, J Ladbury, Dept of Biochemistry and Molecular Biology, UCL; B Panaretou, Life Sciences KCL; G Siligardi, Pharmacy, KCL; H Saibil, U. Gohlke, Birkbeck College.

Source of external funding: The Wellcome Trust

Structure of complex between the N-terminal nucleotide-binding domain of HSP90 (gold) and the C-terminal segment of p50cdc37 (blue/green)

The molecular chaperone, Hsp90, is responsible for the activation of an important set of client proteins involved in signal transduction pathways, and frequently implicated in cancer. The mechanism by which these proteins are activated is fundamental to understanding the activation of signal transduction pathways. Previously we discovered that ATP binding and hydrolysis are essential for the in vivo activity of Hsp90, and have determined the crystal structure of the N-terminal nucleotide-binding domain, which is also the site of action of anti-tumour drugs such as geldanamycin and radicicol. We have shown that Hsp90 operates via an ATPase-coupled molecular clamp mechanism involving transient association of the N-terminal domains in the Hsp90 dimer, which is the conformational state in which client protein activation occurs, and that co-chaperones associated with early stages of client protein loading (Hop/p60/Sti1 and p50cdc37) arrest the ATPase activity, while late associated TPR-domain co-chaperones such as Cpr6 relieve this inhibition.

In the last year we have determined the crystal structure of the middle segment of Hsp90, which contains the major binding surfaces for client proteins, and provides a catalytic arginine residue required for ATP hydrolysis. We have also identified and characterised a new stress-regulated co-chaperone, Aha1, which activates Hsp90s ATPase activity and localised its binding site on the chaperone. We have now determined the crystal structure of the core complex between the N-terminal conserved domain of Aha1 and the middle segment of Hsp90, revealing an activation mechanism in which Aha1 promotes a conformational change in the catalytic loop from a retracted conformation. We have also determined the crystal Following our observation that the protein kinase-specific co-chaperone p50^cdc37 arrests Hsp90s ATPase activity, we have localised the binding site for p50^cdc37 and determined the crystal structure of the complex between the C-terminal part of p50^cdc37 and the N-terminal nucleotide binding domain of Hsp90. This structure shows how binding of p50^cdc37 arrests Hsp90s ATPase by locking the "lid" of the nucleotide-binding domain in an open comformation and preventing the trans-activating association of the N-terminal domains within the Hsp90 dimer. Ongoing work is focussed on structural analysis of Hsp90-p50^cdc37-kinase complexes, and structural and biochemical analysis of interactions between Hsp90 and other co-chaperones. In parallel, we are actively engaged in a drug development programme for inhibitors of Hsp90s ATPase as anti-cancer drugs, as part of a collaboration with the Cancer Research UK Centre for Cancer Therapeutics and Vernalis Cambridge Ltd.

Cytosolic Chaperonin-Containing T-complex Polypeptide 1 (CCT)

SM Roe, LH Pearl; in collaboration with KR Willison, C Dekker, Cancer Research UK Centre for Cell and Molecular Biology

Source of external funding: Cancer Research UK

Cytosolic chaperonin-containing T-complex polypeptides 1 (CCT) is the only known chaperonin in the cytosol of eukaryotes. CCT functions to assist in the folding and assembly of newly synthesised and denatured tubulins and actins, Von Hippel-Lindau protein, and a range of proteins contaning WD40 β-propellor domains, including several key cell-cycle regulators. CCT is composed of a double torus consisting of eight different but related (sharing 30% identity), gene products. The eight constitutively expressed subunit species are; CCTα, -β, -δ, -ε, -γ, -ξ, -η, -θ. A close specific functional relationship exists between different CCT subunits and the domains of its main client proteins, tubulins and actins.

We are using biochemical and structural techniques to define the structural basis of the specific interactions between CCT and its client protens. Previously we established E. coli expression systems for the apical domains of all eight subunits and determined the structure of the CCTγ apical domain, revealing an overwhelmingly polar client-protein binding site. We have now established a large-scale purification system for the full ~1 MDa CCT complex and have succeeded in identifying crystallisation conditions, opening the way to a full crystallographic structure determination.