Mechanism of HSP90 Function

In eukaryotes, the ubiquitous Hsp90 molecular chaperone facilitates the folding and activation of a broad array of proteins important in cell signaling, proliferation, and survival. Unlike other molecular chaperones, Hsp90 preferentially stabilizes near-native-state structures, aiding the dynamic assembly and disassembly of signaling complexes. Hsp90 is thus an important therapeutic target. Our goal is to understand Hsp90 action and the structural basis for its requirement by its substrate "client" proteins, and to use this information in the discovery of novel small molecule modulators.

We solved the x-ray structures of the Escherichia coli Hsp90 in the apo and ADP states; a structure of the yeast ATP state was determined concurrently from Laurence Pearl's lab (Institute of Cancer Research, London). Through small angle X-ray scattering and single particle EM, we demonstrated that bacterial, yeast, and human Hsp90s have a conserved 3-state ATP conformational cycle, and that the open-closed equilibrium is species specific, reflecting optimization for the unique requirements of each species. These results profoundly affected models of the Hsp90 conformational cycle, suggesting where client proteins may bind and how nucleotide binding and hydrolysis propel the chaperone through conformational changes that lead to the release of client proteins.

Our current efforts focus on elucidating the structural basis for Hsp90-client and Hsp90-cochaperone interactions and understanding how Hsp90 remodels its clients. This is challenging as Hsp90 has a strong preference for partially folded states which are normally only sparingly populated. To remedy this, we developed a model system based on a largely unfolded, but nonaggregating mutant of staphylococcal nuclease (SN). By 15N-NMR we defined the binding site for SN at the interface between Middle and C-terminal Hsp90 domains, showed that a 25- to 30-residue SN segment, corresponding to the most folded region within SN, is responsible for the interaction and remains structured as an α-helix when bound. Kinetic experiments show an unexpected coupling between conformational transition states in both client and Hsp90. Notably model and natural clients accelerate the rate-limiting conformation transitions in Hsp90, thereby speeding ATP hydrolysis. 

We are simultaneously pursuing several classes of natural clients, including the glucocorticoid receptor (GR), kinases, and E3 ligases and have obtained cryoEM structures and mechanistic information on Hsp90:Cdc37:Kinase at ~12Å resolution, Hsp90:Hop at 16Å and are making progress on Hsp90:Hop:Hsp70:GR complex. The human mitochondrial Hsp90 (TRAP1) is linked to Parkinson's disease and cancer. We have solved its atomic structure revealing an unexpected asymmetric ATP state that likely has significant functional importance and are using this system to discover small molecule allosteric modulators.

Relevant Publications

"The conserved arginine 380 of Hsp90 is not a catalytic residue, but stabilizes the closed conformation required for ATP hydrolysis." Cunningham CN, Southworth DR, Krukenberg KA, Agard DA. Protein Sci. 2012 May 31. (htmlpdf)

"Cross-Monomer Substrate Contacts Reposition the Hsp90 N-Terminal Domain and Prime the Chaperone Activity." Street TO, Lavery LA, Verba KA, Lee CT, Mayer MP, Agard DA. J Mol Biol. (2012), 415(1), 3-15. (pdf)

"A small molecule that preferentially binds the closed conformation of Hsp90," Alexander LD, Partridge JR, Agard DA, McAlpine SR. Bioorg Med Chem Lett. (2011), 21(23), 7068-71. (pdf)

"Client-Loading Conformation of the Hsp90 Molecular Chaperone Revealed in the Cryo-EM Structure of the Human Hsp90:Hop Complex," Southworth, Daniel R.; Agard, David A., Molecular Cell (2011), 42(6), 771-781. (pdf)

"Conformational dynamics of the molecular chaperone Hsp90," Krukenberg, Kristin A.; Street, Timothy O.; Lavery, Laura A.; Agard, David A., Quarterly Reviews of Biophysics (2011), 44(2), 229-255. (htmlpdf)

"Substrate Binding Drives Large-Scale Conformational Changes in the Hsp90 Molecular Chaperone," Street, Timothy O.; Lavery, Laura A.; Agard, David A., Molecular Cell (2011), 42(1), 96-105. (pdf)

Vasko RC, Rodriguez RA, Cunningham CN, Ardi VC, Agard DA, and McAlpine, SR, "Mechanistic Studies of Sansalvamide A-Amide: An Allosteric Modulator of Hsp90," ACS Med. Chem. Lett, 2010, 1(1), 4-8. (htmlpdf)

Street TO, Krukenberg KA, Rosgen J, Bolen DW, Agard DA, "Osmolyte-induced conformational changes in the Hsp90 molecular chaperone, " Protein Sci. 2010, 19, 57-65. (htmlpdf)

Krukenberg KA, Böttcher UM, Southworth DR, Agard DA, "Grp94, the endoplasmic reticulum Hsp90, has a similar solution conformation to cytosolic Hsp90 in the absence of nucleotide," Protein Sci. 2009 Jun 24, 18(9), 1815-27. (htmlpdf)

Krukenberg KA, Southworth DR, Street TO, Agard DA, "pH-Dependent Conformational Changes in Bacterial Hsp90 Reveal a Grp94-Like Conformation at pH 6 That Is Highly Active in Suppression of Citrate Synthase Aggregation," J Mol Biol. (2009) May 7. (htmlpdf)

Southworth DR, Agard DA, "Species-dependent ensembles of conserved conformational states define the Hsp90 chaperone ATPase cycle," Mol Cell. (2008) Dec 5, 32(5):631-40 (htmlpdf)

Förster F, Webb B, Krukenberg KA, Tsuruta H, Agard DA, Sali A., "Integration of Small-Angle X-Ray Scattering Data into Structural Modeling of Proteins and Their Assemblies." J Mol Biol. (2008), 382(4), 1089-106 (htmlpdf)

Cunningham CN, Krukenberg KA, Agard DA., " Intra- and inter-monomer interactions are required to synergistically facilitate ATP hydrolysis in Hsp90.", J Biol Chem. (2008) 283(30), 21170-8. (htmlpdfsupplemental data)

Krukenberg KA, Förster F, Rice LM, Sali A, Agard DA., "Multiple Conformations of E. coli Hsp90 in Solution: Insights into the Conformational Dynamics of Hsp90." Structure. (2008), 16(5), 755-65. (pdf, supplemental data)

Shiau AK, Harris SF, Southworth DR, Agard DA, "Structural Analysis of E. coli hsp90 Reveals Dramatic Nucleotide-Dependent Conformational Rearrangements," Cell. 2006 Oct 20;127(2):329-340(html or pdf).

Seth F. Harris, Andrew K. Shiau and David A. Agard, "The Crystal Structure of the Carboxy-Terminal Dimerization Domain of htpG, the Escherichia coli Hsp90, Reveals a Potential Substrate Binding Site," Structure, (2004) 12(6):1087-1097 (pdf).