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Discovering the Structural Basis for Biological Function at the Molecular and Cellular Levels

Research Summary

David Agard's research is focused on elucidating the mechanism of Hsp90 chaperone function and its role in human disease, microtubule nucleation & centrosome structure, and the structure and cell biology of phage-encoded tubulins. Accompanying this is an effort to develop new technologies for high resolution cryoEM and fluorescence light microscopy.

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.

Our main focus is on how the energy of ATP is used to remodel client proteins and how the many cochaperones participate in this process. Using a partially folded model client protein, we showed the dominant client protein binding site was at the juncture of the middle and C-terminal domains. This novel bi-partite binding site can adjust to differing client interactions by changing the relationship of these two domains, explaining at least some of Hsp90’s ability to interact with a broad range of completely dissimilar client proteins. In the case of the model client, an a-helix normally buried in the native state is what binds directly to this interface. 

A recent crystal structure of the closed mitochondrial Hsp90 (TRAP1) has begun to reveal how ATP hydrolysis might be coupled to client remodeling. Notably, the TRAP1 closed state is remarkably asymmetric, with maximal asymmetry at the client binding interface at the juncture of the middle and C-terminal domains. Combinations of DEER, FRET and kinetic crystallography reveal that the more asymmetric side hydrolyzes ATP first, leading to a switch of asymmetry to the other protomer. Finally that protomer hydrolyzes its ATP, leading to the open apo state. Thus the first ATP hydrolyzed could alter the conformation of the bound client while the second ATP hydrolyzed would reset the system.

We are simultaneously pursuing several classes of natural clients that in vivo are absolutely dependent upon Hsp90 for function, including the glucocorticoid receptor (GR) and several kinases. Paradoxically, our efforts on GR revealed that Hsp70 inactivates GR and Hsp90 reverses this inhibition. Thus much of the Hsp90 dependence is due to relieving the Hsp70 inhibition, in a process requiring ATP hydrolysis on Hsp90. A low-resolution cryoEM structure of Hsp90, Hsp70, the cochaperone Hop, and GR shows that that the nucleotide binding domains of the two chaperones interact explaining the coupling of the two ATPases.

Perhaps most informative is the just completed 3.9Å resolution cryoEM structure of the 240KDa complex between Hsp90, the kinase-specific cochaperone Cdc37 and the Cdk4 kinase. In this remarkable structure the N-lobe of the kinase is unfolded and threaded through the interior of the closed HSp90 while Cdc37 wraps around the outside of Hsp90 and interacts with the kinase C-lobe. Together with the GR studies, this suggests how chaperones can be playing a role throughout the lifetime of a client protein by catalyzing unfolding/folding transitions as a means to assess whether ligands, interacting partners, etc. are around. Thus protein folding is used to facilitate the assembly and disassembly of functional complexes while protecting vulnerable partially folded states from aggregation or degradation.

Mechanisms of Microtubule Formation and the Role of γ-Tubulin Complexes as Nucleators
The spatial and temporal regulation of tubulin polymerization into microtubules (MTs) is a central question in cell biology. Our goal is to understand, in atomic detail, the molecular mechanisms underlying dynamic MT behavior and MT nucleation.

Of key importance is the structural and functional analysis of γ-tubulin complexes, which act in vivo to nucleate MT growth. Although central to all MT nucleation, the γ-tubulin small complex (γTuSC) is a surprisingly poor MT nucleator. We have determined the structure of the isolated yeast γTuSC by single-particle EM (electron microscopy). We can assemble γTuSCs into either ring complexes or filaments, and we have determined the cryo-EM structure of the filament at ~6.5Å resolution. These structures have 6.5 γTuSCs/turn, resulting in the display of 13 γ-tubulins explaining how pairs of g-tubulins present within gTuSCs can nucleate 13 protofilament MTs. The cryoEM structure also reveals that the g-tubulins within each gTuSC are too far apart to efficiently nucleate MTs, indicating an allosteric activation required for potent MT nucleation. We actively seek to understand this regulation.

Although yeast γTuSC assemblies can form spontaneously, they are only stable at low pH. However, Spc110p, which links γTuSC to the spindle pole body (SPB), stabilizes the assemblies to physiological conditions. Using a novel FRET assay, we have shown that Spc110p’s ability to assemble the gTuSCs depends upon its own oligomerization into structures of at least a tetramer. Organization of Spc110p oligomers at the SPB likely ensure that γTuSC assemblies are only formed at the SPB, ensuring high fidelity of MT nucleation. High-resolution cryoEM structures of isolated gTuSCs and Spc110p driven assemblies are underway.

The Structure and Cell Biology of Bacteriophage Tubulins
In collaboration with Joe Pogliano (UCSD) we have discovered a family of tubulins encoded by very large bacteriophage (~300Kb), and have determined the crystal structure of the first phage cytoskeletal element—a tubulin family member known as PhuZ. High resolution cryoEM structures sow that PhuZ forms novel three-stranded filaments whit an inverted geometry compared to microtubules. While the tubulin C-terminus is exposed and used for binding associated proteins, in PhuZ, it is extended and located at the center of the polymer where it makes interactions with 3 separate subunits. CryoEM is also revealing the orginis of metastability via structures of post-hydrolysis filaments.

In vivo observations of infected cells reveals that PhuZ forms a spindle like structure that position phage particles at the cell midline. Altering PhuZ dynamics leads to phage mis-positioning and a significantly decreased burst size. In a continued collaboration, we now focus on understanding the cell and structural biology underlying this remarkable observation.

This work has been supported by HHMI and NIH grants.

As of April 29, 2016

Scientist Profile

Investigator
University of California, San Francisco
Cell Biology, Structural Biology