Home Faculty and Research Faculty by Name Eva Nogales

Eva Nogales

Howard Hughes Investigator and Professor of Biochemistry and Molecular Biology*
*And Affiliate, Division of Cell and Developmental Biology

Lab Homepage: http://cryoem.berkeley.edu/

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Research Interests

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, 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.

Current Projects

Cytoskeleton and Cell Division

Microtubule Strucure 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. We later obtained the structure of two structural intermediates corresponding to the start and end points in the polymerization cycle that illustrate the conformational consequences of the nucleotide state, and how they relate to longitudinal and lateral assembly. 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 aim to characterize these interactions and their functional implications.

Microtubule-Kinetochore Interface - The yeast Dam1 kinetochore complex self-assembles around microtubules into rings that interact with the microtubule via flexible elements and lack a footprint on the microtubule lattice. This property allows 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. We have obtained initial structures of the Dam1 complex before and after its oligomerization around microtubules. 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. We are now extending our studies to other kinetochore microtubules. In particular we have been studying the structure and conformational flexibility of the Ndc80 complex, a  highly conserved, essential kinetochore component.

Septin Filament Assembly and Architecture - We have recently started work on 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 non-essential. These septins form filaments to define a collar at the bud neck during cytokinesis. We have shown that the hetero-oligamer of the four essential mitotic septins is an octameric linear rod and identified the location of each subunit. 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 altered. Filaments invariably pair into long parallel "railroad tracks." Lateral association seems to be mediated by hetero-tetrameric 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. Some of our major interests are now to characterize the interplay of septins and lipids and to define the structural basis of septin regulation by kinases. Additionally, and as an extension of our traditional area of technical expertise, we have started the in vivo study of septins, using electron tomography of yeast cells. With this work we want to link our reconstitution studies of septin assembly and lipid interaction, to the ultrastructure of septin filaments and membrane interactions in the cell.

Nucleic Acid Transactions

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. We have shown that in TFIID, the first general transcription factor to land at the promoter, a number of discreet structural elements move in a concerted manner using hinges in the structure. We propose that this conformational versatility could play a distinct role in directing the formation of an active PIC 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, it is known that proper ovarian development requires the cell type-specific TAF4b subunit. Interestingly, TAF4b incorporation into TFIID induces an open conformation at the lobe involved in TFIIA and putative activator interactions. Importantly, this conformation correlates with differential activator-dependent transcription and promoter recognition by 4b/4-IID. One of our major interests now is the interaction of TFIID with TFIIA and TFIIB, as well as with Mediator and activators, in the context of different promoters.

Chromatin Remodeling - The SWI/SNF family of chromatin remodeling enzymes (remodelers) uses ATP hydrolysis to display or modify the structure of nucleosomes in order to allow access of DNA-interacting proteins to their target sequences. In our studies of the yeast RSC remodeler we have visualized a deep central cavity within RSC that displays a remarkable surface complementarity for the nucleosome. We have also been able to define two distinct RSC states, revealing a major conformational change in a large protein 'arm' which may shift to further envelop the nucleosome. Our structures provide support for current models for remodeling that involve the formation of small DNA loops/waves that propagate on the surface of the nucleosome. Our present efforts to dissect the mechanism of function of this family of remodelers center on the study of the human complex PBAF. We are localizing the position of different subunits by immunolabeling and using the specificity for a pattern of histone modifications to obtain PBAF-nucleosome complexes to study by cryo-EM analysis.

Additional studies include the structural characterization of macromolecular complexes involved in replication initiation, DNA repair, and RNA processing.

Methods Development

Electron microscopy and image analysis have become the methodology of choice in the structural characterization of large, low-abundance complexes. A major challenge, as well as a most exciting promise, is that these methods will be able to detect and characterize conformational and biochemical variability in the sample. Such capabilities are going to be essential for our molecular understanding of complexes involved, for example, in gene regulation, where the combinatorial nature of the process involves many different interactions and alternative conformations. We are presently involved in a collaborative effort of a multidisciplinary team including mathematicians and computer scientists to face this exciting challenge.

Selected Publications

Selected Recent Publications

Liu, W-L., Coleman, R.A., Grob, P., Geles, K.G., King, D.S., Ramey, V.H., Nogales, E. and Tjian, R. (2008) Structural changes in TAF4b-TFIID correlated with promoter selectivity. Mol. Cell 29, 81-91.

Bertin, A., McMurray, M.A., Grob, P., Park, S-S., Garcia, G. III, Patanwala, I., Ng, H-L., Alber, T.C., Thorner, J. and Nogales, E. (2008) Saccharomyces cerevisiae septins: Supramolecular organization of hetero-oligomers and the mechanism of filament assembly. PNAS 105, 8274-8279. 

Wang, H-W, Wang, J., Ding, F., Callahan, K., Bulter, J.S., Nogales, E., Ke, A. (2007) Architecture of the yeast Rrp44-exosome complex suggests routes of RNA recruitment for 3'-end processing. PNAS 104, 16844-9.

Wang, H-W., Ramey, V.H., Westermann, S., Leschziner, A.E., Welburn, J.P.I., Nakajima, Y., Drubin, D.G., Barnes, G. and Nogales, E. (2007) Architecture of the Dam1 kinetochore ring complex: implications for microtubule-driven assembly and force-coupling mechanisms. Nat Struct Mol Biol., 14, 721-726

Leschziner, A.E., Saha, A., Wittmeyer, J., Zhang, Y., Bustamante, C., Cairns, B.R. and Nogales, E. (2007) Conformational flexibility in the chromatin remodeler RSC observed by electron microscopy and the orthogonal tilt reconstruction method. PNAS, 104, 4913-4918.

Clarey, M.G., Erzberger, J.P., Grob, P., Leschziner, A.E., Berger, J.M., Nogales, E. and Botchan, M. (2006) Nucleotide-dependent conformational changes in the DnaA-like core of the origin recognition complex. Nat Struct Mol Biol., 13, 684-690.

Wang, H-W. and Nogales, E. (2006) Structural intermediates in microtubule assembly and disassembly: how and why? Curr Opin Cell Biol., 18(2), 179-284.

Westermann, S., Wang, H-W., Avila-Sakar, A., Drubin, D.G., Nogales, E. and Barnes, G. (2006) The Dam1 kinetochore ring complex moves processively on depolymerizing microtubule ends. Nature, 440, 565-569.

Siridechadilok, B., Fraser, C.S., Hall, R.J., Doudna, J.A. and Nogales, E. (2005) Structural roles for human translation factor eIF3 in the initiation of protein synthesis. Science, 310, 1513-1515.

Sarker, A.H., Tsutakawa, S.E., Kostek, S., Ng, C., Shin, D.S., Peris, M., Campeau, E., Tainer, J.A., Nogales, E. and Cooper, P.K. (2005) Recognition of RNA PolII and transcription bubbles by XPG, CSB and TFIIH: Insights for transcription-coupled repair and Cockayne Syndrome. Mol Cell., 20, 1-12. 

Wang, H-W. and Nogales, E. (2005) The nucleotide-dependent bending flexibility of tubulin regulates microtubule assembly. Nature, 435, 911-915

For a full publication listing please visit http://cryoem.berkeley.edu/pubs.shtml

Last Updated 2008-08-19