HYBRID STRUCTURE DETERMINATION OF TRAFFICKING COMPLEXES BY SAXS, PROTEIN CRYSTAL

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Membrane trafficking pathways are essential for normal physiological processes such as signal transduction, antigen presentation, organelle biogenesis, and many others, and for pathophysiological processes such as HIV budding. Membrane trafficking via endosomes is carried out by a series of multi-protein complexes, including the ESCRT complexes and the retromer complex. We have obtained a series of crystal structures of the cores of these assemblies, and in combination with the structures of individual domains and homology modeling, models for the complete structures can be generated. The models have been assessed by comparison to hydrodynamic studies of multiple truncation constructs, providing constraints on the solution structures of the intact complexes. We have applied coarse-grained Monte Carlo simulations using residue-based potentials with rigid domains and core structures to model the assemblies. To complete the structural analysis of the intact complexes, we urgently wish to obtain SAXS data, which will provide more powerful constraints on the solution structural models than is possible using hydrodynamic data alone. Two structural publications are currently in preparative for top tier journals, thus the experiments, if successful, would be publishable almost immediately in high profile venues. These papers deal with highly competitive topics, with publications to be submitted in 1-2 months. If there is a delay in access, we will probably not be able to include the SAXS analysis in these studies.