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Structural Studies of Macromolecular Assemblies
Summary: Eva Nogales is interested in the structure of large macromolecular complexes and the regulation of their assembly and function.
My lab is involved in the structural characterization of complex biological assemblies, their architecture, conformational flexibility, and their interactions with ligands and cellular partners. One major area of interest is the structural basis of cytoskeletal self-assembly and regulation during cell division. We are also studying the architecture, dynamics, and complex interactions of a number of large molecular machines involved in nucleic acid transactions. We use electron microscopy (EM), image analysis, and functional biochemical and biophysical assays, with the final aim of a mechanistic understanding of the regulated function of these complex macromolecular systems.
Cytoskeleton
Microtubule structure and dynamics. The dynamic behavior of microtubules, an intrinsic property of tubulin GTPase activity, is essential to their functions. Our studies of tubulin in polymerized, straight protofilaments established the structural basis of nucleotide exchange and polymerization-coupled hydrolysis. More recently we have obtained the structures of two intermediates corresponding to the start and end points in the polymerization cycle. These structures illustrate the conformational consequences of the nucleotide state and how they relate to longitudinal and lateral assembly.
The flexibility of tubulin and the consequent versatility of its self-assembly can hardly be an accident. We propose that the polymorphism of assembly unique to tubulin reflects an exquisite tuning mechanism for the complex interaction of different microtubule intermediates with cellular factors that need to detect or make direct use of the growing or shortening state of microtubules to play functional roles at the right time and place in the cell. We are now pursuing the idea that the transient sheets at the end of growing microtubules may be the landing site for microtubule plus-end tracking proteins. The characterization of the molecular interplay between tubulin polymers and protein factors that affect tubulin assembly and/or are able to select tubulin polymerization states has only just begun. It promises to create new paradigms of microtubule cellular function, where tubulin polymers are seen not as passive platforms but as molecular machines capable of carrying out work by switching conformational and polymerization states.
Microtubule-kinetochore attachment interface. In collaboration with the labs of Georjana Barnes and David Drubin (University of California, Berkeley), we are studying the yeast Dam1 kinetochore complex. This complex self-assembles around microtubules into rings and spiral-like structures and has a higher affinity for the GTP-containing ends of growing microtubules. Dam1 ring structures interact with the microtubule via flexible elements and lack a footprint on the microtubule lattice, allowing for diffusion that becomes unidirectional when the microtubule depolymerizes and effectively pushes along the ring. At this point the Dam1 complex uses the conformational strain of GDP-tubulin as it relaxes into its curved state to move processively and without energy consumption of its own.
To understand the mechanisms by which rings couple processive movement to microtubule disassembly and thus contribute to the end-on attachment of chromosomes to the mitotic spindle, it is necessary to define the architecture of the Dam1 complex and its microtubule-driven self-assembly. This knowledge is also crucial to determine how the assembly of the ring is regulated and how the ring attaches to other components of the kinetochore. We have used EM-based single-particle and helical analyses to obtain initial structures of the Dam1 complex before and after its oligomerization around microtubules. This work has allowed us to define the architecture of the Dam1 complex and the self-assembly mechanism. Ring oligomerization seems to be facilitated by a conformational change upon binding to microtubules, suggesting that the Dam1 ring is not preformed, but self-assembles around kinetochore microtubules. The C terminus of the Dam1p protein, where most of the Aurora kinase Ipl1 phosphorylation sites reside, is in a strategic location to affect oligomerization and interactions with the microtubule. One of Ipl1's roles might be to fine-tune the coupling of the microtubule interaction with the conformational change required for oligomerization, with phosphorylation resulting in ring breakdown.
Septin filament assembly and architecture. Recently we have initiated a collaboration with the lab of Jeremy Thorner (UC Berkeley) to study septin filaments, an additional cytoskeletal element involved in cell division. Septins comprise a discrete family of GTP-binding proteins conserved from fungi to humans. Mitotic yeast cells express five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1/Sep7). Only Shs1 is nonessential. These septins form filaments to define a collar at the bud neck during cytokinesis. The collar filaments impose a barrier to diffusion of integral membrane proteins between mother and bud cells and act as a scaffold to recruit proteins required for bud-site selection and a morphogenesis checkpoint. We carried out single-particle analysis by EM and revealed that the hetero-oligomer of the four essential mitotic septins is an octameric linear rod. We identified the location of each subunit within the rod by examining complexes lacking a given septin, by antibody decoration, and by fusion to marker proteins (green fluorescent protein or maltose-binding protein). The rod has the order Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11 and, hence, lacks polarity. At low ionic strength, rods assemble end to end to form filaments, but not when Cdc11 is absent or its N terminus is altered. Filaments invariably pair into long parallel "railroad tracks." Lateral association seems to be mediated by heterotetrameric coiled coils between the paired C-terminal extensions of Cdc3 and Cdc12 projecting orthogonally from each filament. Our findings provide insights into the molecular mechanisms underlying the function and regulation of cellular septin structures. We are now interested in characterizing the interplay of septins and lipids and defining the structural bases of septin regulation by kinases.
Nucleic Acid Transactions
Basal eukaryotic transcriptional machinery. Regulated gene transcription in eukaryotes requires the assembly of a complex molecular machinery that includes general factors, activators, cofactor complexes, and chromatin-remodeling factors. Our most recent work has highlighted the highly flexible nature of these large protein assemblies and its potential relevance in their affinity for DNA, their catalytic cycles, or the integration of regulatory signals.
Using new three-dimensional variance and focused classification EM methodology, we have shown (in collaboration with Robert Tjian [HHMI, UC Berkeley]) that in TFIID, the first general transcription factor to land at the promoter, a number of discrete structural elements use hinges in the structure to move in a concerted manner. TFIID's central cavity and channels between lobes rearrange, changing their size and likely their chemical character, thus giving the complex a versatility of potential binding sites that can be cooperatively linked through the scaffold of the structure. We propose that this conformational versatility could play a distinct role in directing the formation of an active preinitiation complex at different promoters through interactions of TFIID with different sets of activators and cofactors.
TFIID is also far from being biochemically unique. As an example of the importance of compositional variation in TFIID, proper ovarian development requires the cell-type-specific TAF4b subunit. We obtained a 3D reconstruction of a cell type-specific core promoter recognition complex containing TAF4b and TAF4 (4b/4-IID), which is responsible for directing transcriptional synergy between c-Jun and Sp1 at a TAF4b target promoter. Our studies reveal that TAF4b incorporation into TFIID induces an open conformation at the lobe involved in TFIIA and putative activator interactions. This conformation correlates with differential activator-dependent transcription and promoter recognition by 4b/4-IID.
We have also used 3D variance methods to analyze the structure and conformational flexibility of human RNA polymerase II (Pol II). We have seen that the flexibility of the complex in solution parallels the conformational flexibility of the yeast structures crystallized in different states, but also illustrates a more extensive conformational flexibility with potential biological significance. The importance of this flexibility in transcription-coupled repair, a process that is essential for normal postnatal growth and development in humans, is of particular interest to our lab (in collaboration with Priscilla Cooper, Lawrence Berkeley National Laboratory).
The SWI/SNF family of chromatin-remodeling enzymes (remodelers) uses ATP hydrolysis to display or modify the structure of nucleosomes to allow access of DNA-interacting proteins to their target sequences. In our studies of the yeast RSC remodeler (in collaboration with the lab of Bradley Cairns [HHMI, University of Utah]), using our new 3D reconstruction methodology (the orthogonal tilt reconstruction method), we have visualized a deep central cavity within RSC that displays a remarkable surface complementarity for the nucleosome. We have also defined two distinct RSC states, revealing a major conformational change in a large protein "arm" that may shift to further envelop the nucleosome. Our structures both support and challenge current models for remodeling that involve the formation of small DNA loops/waves that propagate on the surface of the nucleosome, and/or the generation of a large DNA loop that accumulates on the nucleosome surface. The remarkable fit of the cavity to the nucleosome appears to set limits on the size of the DNA waves/loop that can be propagated along the entire length of the nucleosome surface. However, our structures reveal several areas where the nucleosomal DNA is accessible to solvent, suggesting that the large DNA loops observed in optical trap experiments could be generated and maintained at one or more of these positions on the nucleosome surface.
RNA processing. The eukaryotic core exosome (CE) is a conserved 9-subunit protein complex important for 3'-end trimming and degradation of RNA. In yeast, the Rrp44 protein constitutively associates with the CE and provides the sole source of processive 3'-to-5' exoribonuclease activity. In collaboration with Ailong Ke (Cornell University), we have obtained EM reconstructions of the core and Rrp44-bound exosome complexes and used bioinformatics tools and docking to generate a pseudoatomic model of most of the complex. The two-lobed Rrp44 protein binds to the RNase PH-domain side of the exosome and buttresses the bottom of the exosome-processing chamber. The Rrp44 C-terminal body part containing an RNase II–type active site is anchored to the exosome through a conserved set of interactions mainly to the Rrp45 and Rrp43 subunit, whereas the Rrp44 N-terminal head part is anchored to the Rrp41 subunit and may function as a roadblock to restrict access of RNA to the active site in the body region. The Rrp44-exosome (RE) architecture suggests an active-site sequestration mechanism for strict control of 3' exoribonuclease activity in the RE complex.
Eukaryotic translation initiation. Protein synthesis begins with recruitment of the 40S ribosomal subunit to an mRNA. In eukaryotes, at least 12 translation initiation factors and an mRNA-methylated G-cap are required, while in many viruses a structured RNA element, or internal ribosome entry site (IRES), replaces most factors and the mRNA cap. In both cases the translation factor complex eIF3 is required to coordinate formation of an initiation complex and prevent premature association of the 40S and 60S subunits. In collaboration with Jennifer Doudna's lab (HHMI, UC Berkeley), we have recently obtained the cryo-EM structure of human eIF3 and characterized its interaction with hepatitis C virus IRES and eIF4G, the major component of the cap-binding complex. eIF3 has a mutually exclusive binding site for IRES and eIF4G, implying a mechanistic overlap that helps explain why this RNA obviates the requirement for cap-binding complex in translation initiation. We have combined our own and published structural analyses of different binary complexes to produce models for the ternary complexes 40S-eIF3-IRES and 40S-eIF3-eIF4G. These models show how eIF3 positions IRES and anchors the attached mRNA near the exit site on the 40S ribosomal subunit. The eIF3 position provides a plausible explanation for how this factor inhibits premature assembly of the 40S with the 60S ribosomal subunit. This work suggests conserved interactions coordinated by eIF3 that place the mRNA in the ribosomal decoding center and prepare the 40S subunit for assembly into active ribosomes, both for the canonical initiation pathway and for the simplified, IRES-mediated pathway.
Last updated: July 3, 2008
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