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.
We can now assemble γTuSC into either ring complexes or filaments. 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. We are now pursuing high-resolution structures of γTuSC filaments by cryo-EM and crystal structures of γTuSC and its domains.
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.
MT assembly has been interpreted as a nucleation-polymerization mechanism where an unstable nucleus is formed during the lag phase. Features of our experimental kinetic data led us to develop a physically based quantitative model for MT assembly. Our modeling indicates that MT assembly is not a nucleation-polymerization mechanism, but instead resembles two-dimensional crystallization, and that polymer growth does not happen until thousands of tubulins have assembled, instead of 12–18.
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.
Recently, 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. Moreover, our EM single-particle data of bacterial, yeast, and human Hsp90s indicate that the open-closed equilibrium is species specific. SAXS studies of the yeast and human proteins confirm these results and reveal a distinct apo conformation conserved across eukaryotic Hsp90s. These remarkable conformational dynamics have important implications for the mechanism of Hsp90 action. First, they indicate that the ATP conformational cycle is stochastic and not deterministic. Second, they place limitations on the amount of energy available from ATP binding that could be utilized to drive substrate activation. Third, they suggest that release of ADP or phosphate could be the energetically most important step, much as in myosin, and could be coupled to client release. Our current efforts are directed toward understanding the mechanism of client protein remodeling by Hsp90.
The Function and Evolution of Kinetic Stability: A Study of α-Lytic Protease Folding
The central dogma in protein folding is that the native state of a protein is at the global free-energy minimum. This allows spontaneous folding to the active conformation. The family of extracellular bacterial proteases, typified by α-lytic protease (αLP), provides striking counterexamples in which the native state is substantially less stable than the fully unfolded molecule. Instead of being thermodynamically stabilized, αLP is trapped in its active conformation by a large energy barrier that effectively blocks unfolding (t1/2 = 1.2 years). Our work suggests that αLP's combination of a large and remarkably cooperative barrier to unfolding provides an optimal solution for making the native state resistant to proteolysis. Thus a protein's folding pathway can have a profound impact on the properties of the native state and not just dictate how that state is reached.
A combination of ultra-high-resolution x-ray crystallography (0.83 Å) and comparative structural and functional analysis with family members that survive extremes of pH and temperature is providing insights into the structure of the unfolding transition state. These studies indicate that a Phe in the αLP C-terminal domain is substantially bent and tie this directly to the barrier height. Other architectural features and electrostatic interactions also play key roles in determining the energetic landscape. These results have broad implications for the evolution of extremophiles.
