Ilan E. Chemmama

Integrative structure and functional anatomy of a nuclear pore complex

Nuclear pore complexes play central roles as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm. However, their large size and dynamic nature have impeded a full structural and functional elucidation. Here we determined the structure of the entire 552-protein nuclear pore complex of the yeast Saccharomyces cerevisiae at sub-nanometre precision by satisfying a wide range of data relating to the molecular arrangement of its constituents. The nuclear pore complex incorporates sturdy diagonal columns and connector cables attached to these columns, imbuing the structure with strength and flexibility. These cables also tie together all other elements of the nuclear pore complex, including membrane-interacting regions, outer rings and RNA-processing platforms. Inwardly directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized into distinct functional units. This integrative structure enables us to rationalize the architecture, transport mechanism and evolutionary origins of the nuclear pore complex.

The Proteasome-Interacting Ecm29 Protein Disassembles the 26S Proteasome in Response to Oxidative Stress

Oxidative stress has been implicated in multiple human neurological and other disorders. Proteasomes are multi-subunit proteases critical for the removal of oxidatively damaged proteins. To understand stress-associated human pathologies, it is important to uncover the molecular events underlying the regulation of proteasomes upon oxidative stress. To this end, we investigated H2O2 stress–induced molecular changes of the human 26S proteasome and determined that stress-induced 26S proteasome disassembly is conserved from yeast to human. Moreover, we developed and employed a new proteomic approach, XAP (in vivo cross-linking–assisted affinity purification), coupled with stable isotope labeling with amino acids in cell culture (SILAC)–based quantitative MS, to capture and quantify several weakly bound proteasome-interacting proteins and examine their roles in stress-mediated proteasomal remodeling. Our results indicate that the adapter protein Ecm29 is the main proteasome-interacting protein responsible for stress-triggered remodeling of the 26S proteasome in human cells. Importantly, using a disuccinimidyl sulfoxide–based cross-linking MS platform, we mapped the interactions of Ecm29 within itself and with proteasome subunits and determined the architecture of the Ecm29–proteasome complex with integrative structure modeling. These results enabled us to propose a structural model in which Ecm29 intrudes on the interaction between the 20S core particle and the 19S regulatory particle in the 26S proteasome, disrupting the proteasome structure in response to oxidative stress.

Reconstruction of 3D structures of MET antibodies from electron microscopy 2D class averages

Dynamics of three MET antibody constructs (IgG1, IgG2, and IgG4) and the IgG4-MET antigen complex was investigated by creating their atomic models with an integrative experimental and computational approach. In particular, we used two-dimensional (2D) Electron Microscopy (EM) images, image class averaging, homology modeling, Rapidly exploring Random Tree (RRT) structure sampling, and fitting of models to images, to find the relative orientations of antibody domains that are consistent with the EM images. We revealed that the conformational preferences of the constructs depend on the extent of the hinge flexibility. We also quantified how the MET antigen impacts on the conformational dynamics of IgG4. These observations allow to create testable hypothesis to investigate MET biology. Our protocol may also help describe structural diversity of other antigen systems at approximately 5 Å precision, as quantified by Root-Mean-Square Deviation (RMSD) among good-scoring models.