Sunday, May 11, 2025
5/11/2025
The Structural basis of Protein Biogenesis - Protein translocation and biogenesis machines Abstract Cells are compartmentalized by membranes and macromolecules must cross these barriers. The SecY/Sec61 complex and the Nuclear Pore Complex (NPC) mediate transport across single and double membranes, respectively. Secretory and membrane proteins are translocated by the bacterial ribosome- SecY complex and by the 80S-Sec61 complex. The NPC is a gateway for proteins and RNPs to cross the nuclear envelope and is comprised of a spoke ring complex and a central transporter. Aim 1- We will elucidate the mechanism of co-translational translocation by determining structures of ribosome-SecYEG complexes with a defined nascent chain, to give novel views of early translocation intermediates. To this end, stalled translocation intermediates are trapped by cross-linking in E. coli, purified and reconstituted in nanodisks with native E. coli lipids. Frozen-hydrated particles are then imaged with a Volta Phase Plate to give informative maps after multi-body refinement. Active complexes will be compared to the ground state channel to provide a detailed view of this stepwise process. This work may also provide insights into the positive inside rule for membrane protein topology. An N-terminal, 2-helix bundle in SecE of medically-relevant enterobacteria, such as E. coli (O157:H7), may provide a novel antibiotic target, once we understand its role in translocation. Our pipeline will provide a path forward to study Signal Sequence Peptidase interactions with SecYEG, membrane protein integration and the role of non-canonical signal sequences in translocation. Finally, a structure of the TRAP complex will help us to understand how nascent chains with weaker signal sequences may be translocated more efficiently by the ribosome-Sec61 complex. Aim 2- We recently determined a first-pass, domain structure of the yeast NPC, which revealed the functional anatomy of this translocation machine. A composite 3D map will now be determined at higher resolution with single particle cryo-EM and/or state-of-the-art tomographic sub-volume averaging. The new 3D map will be combined with a large database of nucleoporin (Nup) crosslinks, ~25 published Nup domain structures, and a structure of the Nup84 complex, to create a multi-scale model of the NPC with Integrative Modeling. This in turn, will provide insights into Nup roles in assembly, pore stabilization, transport, and evolution of the NPC. Three-dimensional classification will reveal the density distribution of FG Nups in the central transporter, which will guide Brownian Dynamics simulations to dissect the transport mechanism.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).