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Summary: David Agard's research is focused on elucidating the mechanism of microtubule nucleation, Hsp90 chaperone function, and the role of dynamics in enzyme function and folding.

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 yeast γTuSC by single-particle EM (electron microscopy). A combination of in vivo FRET (fluorescence resonance energy transfer), gold labeling, and localizing YFP (yellow fluorescent protein)-tagged subunits has allowed us to assign the locations and orientations of all components within the complex. Remarkably, the two γ-tubulin heads are significantly separated, resulting in an MT-incompatible configuration and explaining the poor nucleating potential of γTuSC. Movement of a mobile arm is required to bring the γ-tubulins together, providing a template for MT growth.

	Cellular proteins: folding and function... Hsp90 and protein folding... Simulation of alpha-lytic protease... Binding mode prediction for a flexible ligand...

We can now assemble γTuSC into either ring complexes or filaments, and we have recently determined the cryo-EM structure at ~8-Å resolution. These structures have 6.5 γTuSCs/turn, resulting in the display of 13 γ-tubulins precisely as required to nucleate MTs with the 13 protofilaments observed in vivo. 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. Thus, γTuSC assemblies are only likely to form at the SPB, ensuring high fidelity of MT nucleation. Although Spc110p stabilizes γTuSC, it does not activate nucleation. We are searching for the required factor or post-translational modification. We are also pursuing high-resolution crystal structures of γTuSC and fragments.

As part of our efforts to understand the molecular basis of MT nucleation, we solved the atomic structures of human γ-tubulin complexed with GTP and GDP, providing the first eukaryotic GTP/GDP pair. These structures provided two key insights: γ-tubulin forms MT-like lateral interactions independent of nucleotide, and γ-tubulin remains in a curved conformation independent of nucleotide, contrasting sharply with the prevailing allosteric hypothesis for activation of tubulin assembly by GTP. Solution studies (conformation-specific ligand binding and small-angle x-ray scattering [SAXS]) on αβ-tubulin confirm that it too remains in a curved conformation independent of nucleotide.

As a major paradigm shift, we propose that the lattice and not the nucleotide is the allosteric effector. In this lattice model, GTP acts to tune the longitudinal affinity. Lattice metastability is determined not by GTP hydrolysis but by the mechanical spring constant for straightening. This new view has a dramatic impact on understanding MT formation.

As part of our quest to understand the basis of nucleotide regulation of tubulin assembly we also are examining a set of bacterial tubulins that enhance fidelity of plasmid segregation. We recently determined the crystal structure of the first phage cytoskeletal element—a tubulin family member known as PhuZ. Crystal packing mimics the arrangement of PhuZ that we observe by EM. The structure reveals a unique C-terminal tail that seems to directly stabilize filament formation.

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 substrate proteins.

We solved the x-ray structures of the Escherichia coli Hsp90 in the apo and ADP states. Along with a concurrent structure of the ATP state from Laurence Pearl's lab (Institute of Cancer Research, London), our work profoundly affected models of the 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.

Using small-angle x-ray scattering and our newly developed molecular modeling methods, we determined the solution structure of apo HtpG and found that it is more extended than the crystal form. In addition to this novel conformation, we discovered that under physiologically relevant conditions, multiple conformations coexist in equilibrium: there is both a pH-dependent equilibrium (pKa ~ 7.3) and an equilibrium with the ATP state. Our EM single-particle data of bacterial, yeast, and human Hsp90s indicate that all three species employ a conserved 3-state ATP conformational cycle, but that the open-closed equilibrium is species specific, reflecting optimization for the unique requirements of the ensemble of client proteins present in each species.

Our current efforts focus on elucidating the structural basis for Hsp90-client and Hsp90-cochaperone interactions and understanding how Hsp90 models its clients. We are simultaneously pursuing several classes of clients, including nuclear receptors, kinases, E3 ligases, and model systems. Because Hsp90 prefers to interact with non-native conformational states, we have been exploring protein substrates whose native states are destabilized by mutations. We have shown that several of these interact with Hsp90 and that they alter Hsp90 conformational states. One of these model systems, a permanently unfolded, but nonaggregating mutant of staphylococcal nuclease (SN), shows particular promise. By 15N-labeling SN, we not only show an interaction with Hsp90 but also show that a 25- to 30-residue segment, corresponding to the most folded segment within SN, is responsible for the interaction. Using small-angle x-ray scattering and nuclear magnetic resonance, we have been able to map where SN binds on Hsp90. Crystallographic trials with the peptide are under way. This work sets the stage for determining for the first time what Hsp90 does to its clients in atomic resolution.

While we develop client systems that can form stable complexes with Hsp90 suitable for crystallography, we have begun building human Hsp90-cochaperone complexes. Cryo-EM structures of client-loading complexes (Hop-Hsp90 and Hop-Hsp70-Hsp90) are under way, as is a structure of the immunophilin FKBP52-Hsp90 complex.

Grants from the National Institutes of Health provided partial support for the microtubule/centrosome efforts. A grant from the National Science Foundation is funding a collaborative effort (University of California, San Francisco/Lawrence Berkeley National Laboratory/GATAN) to develop a next-generation direct-detect electron camera for high-resolution cryo-EM.

Last updated September 08, 2010