1 Hsp90 nucleotide-dependent conformations Using glutaraldehyde to - PDF document

Hsp90: A molecular chaperone that activates specific substrate client proteins Client Proteins >150 Trapping Dynamic Conformational States and Cochaperone General Protein Apoptosis Interactions of the Hsp90 Molecular Chaperone Cochaperones p53 Folding >20 IP6K2 ASK1 Aha1 Hop Hsp90 Daniel Southworth Steroid Hormone Polymerases Receptors Telomerase Progesterone p23 Viral Polymerases David Agard Laboratory Estrogen Rev. Transcriptase Hsp70 Glucocorticoid UCSF kinases Sse1 src akt NOS raf eNOS p50 (Cdc37) Immunophilins nNOS centrosome Polo kinase Mitochondrial Import Three Nucleotide-Stabilized Conformational Full-Length Crystal Structures of 180 kDa Hsp90 Dimer Identify States in E. coli Hsp90 Different Conformational States Apo ATP ADP p23 E. coli Hsp90 •Are these conformations conserved between species? NTD •How are the conformational states coupled to client activation? MD •How does the human Hsp90 chaperone cycle function? 150 Å CTD Apo Extended Closed, ATP conformation Compact ADP Dimer E. coli Hsp90 Yeast Hsp90:AMPPNP:p23 Modeled from E. coli Hsp90:ADP Negative stain w/ Shiau, et al 2006 -Ali MM, et al . 2006 tetramer Uranyl Formate Shiau, et al 2006 Defocus: 1.5 nM 120 keV 20 Å resolution 1

Hsp90 nucleotide-dependent conformations Using glutaraldehyde to crosslink and trap appear to vary between species transiently sampled states Apo ATP ADP E. coli Apo human Hsp90 + AMPPNP Hsp90 Glutaraldehyde Yeast (0.005%) Hsp90 Human Hsp90 Are the mechanisms fundamentally different? Glut. (0.005%) + protein (200 nM), 37° for 15 minutes. Stopped w/ 20 mM Tris. Crosslinked States are Nucleotide Dependent Hsp90 Chaperone Cycle Involves Three and Match EM Structures Universally Conserved Conformational States Human Hsp90 Human Hsp90 Yeast Hsp90 AMPPNP ADP ADP E.coli E.coli Single RF Hsp90:ATP Single RF Single RF ADP ADP Particles Avgs. model Particles Avgs. Particles Avgs. model model •Hsp90 chaperone cycle involves a unique conformational equilibrium that is different between species. 150 Å 2

The Human Hsp90 Chaperone Cycle Involves Coordinated Quantitation of the open and closed state Interactions with Specific Co-Chaperones conformational equilibrium Client Loading Fraction of Hsp90 Match single particle data to a set of unique 2D projections of ATP the open and closed structures of Hsp90 Client Maturation Yeast Human Human E. coli E. coli Yeast ATP apo ATP apo ATP apo •Conformational states are isoenergetic. Complexes Dissociate: Low affinity and transient •Nucleotide binding shifts inherent conformational equilibrium. Stable 300 kDa Hsp90:Hop tetramer complex Negative-stain EM of 300 kDa Hsp90:Hop formed following disulfide crosslinking HOP HOP HOP HOP + Cys Cys Cys light scattering/chromatography Normalized Refractive Index Crosslinked Molar Mass (Daltons) Hsp90:Hop 300 kDa Hsp90:Hop 250 kDa Hsp90:Hop 0.005% Glutaraldehyde Hsp90 alone Hsp90 alone 180 kDa 180 kDa Hop 60 kDa Volume (ml) 100 Å 3

Cryo-EM of Hsp90:Hop 18.5 Å Cryo-EM Reconstruction of Hsp90:Hop •Data collected at Scripps (Leginon). •15,000 particles (10,000 in final model). •Defocus: 1.5 to 3.5 µ M. 8k x 8k Image 4k x 4k Image •CTF correction (CTFFIND). 1.19 Å/pixel 1.19 Å/pixel •Refinement using SPIDER, C2 symmetry. Technai TF20 120 Kev Tietz 8k Camera 18.5 Å resolution FSC 30 nM Apo Middle-C ATP N-Middle + Conformation Conformation 150 Å 30 nM 1/Å 0.054 5.0 µ M defocus Conclusions Disulfide Crosslinking Stabilizes Multiple Hsp90 Complexes •Significant information can be gained from low-resolution structures Hsp90:Hop:Hsp70 •Glutaraldehyde crosslinking at low concentrations for short times improves Hsp70 homogeneity and traps transient states. 360 kDa Client •Disulfide crosslinking in known binding sites improves stability. E E V D •Information about biological function gained from sorting heterogeneos data. Client Loading Hsp90:AMPPNP:FKBP52 FKBP52 300 kDa Hsp90:Hop Hsp90:Hop:Hsp70 ATP TPR Client Maturation 20 Å negative-stain reconstructions. Hsp90:AMPPNP:FKBP52 4

Acknowledgements David Agard Yifan Cheng Shawn Zheng Justin Kollman AMI, Scripps San Diego Bridget Carragher Clint Potter Joel Quispe Funding American Cancer Society HHMI 5