Biophysical Society Conference | Tahoe 2022

Molecular Biophysics of Membranes

Thursday Speaker Abstracts

CHARACTERIZATION OF THE ELUSIVE SERINC5-AP2-NEF COMPLEX IN THE CONTEXT OF A LIPID BILAYER Joana Paulino 1 ; Alisa Bowen 1 ; Phuong Nguyen 1 ; Janet Finer-Moore 1 ; Robert M Stroud 1 ; 1 University of California San Francisco, Dept Biochemistry and Biophysics, San Francisco, CA, USA The human Serine Incorporator 5 (Serinc5) is an anti-HIV restriction factor that actively inhibits the fusion of the virion with a new host cell. HIV antagonizes this intrinsic immune response by targeting Serinc5 for degradation utilizing its accessory protein, Nef. Nef hijacks the host’s clathrin adaptor protein complex 2 (AP2), directing clathrin mediated endocytosis to target many of the host cell anti-HIV lines of defense. Previous structural studies of Nef-AP2-host protein complexes have shown that although the Nef-AP2 interaction is constant, the interaction between Nef and the host protein is variable. In Serinc5 a large intracellular loop devoid of a canonical AP2 cargo motif is essential for Nef antagonism. This loop remains uncharacterized by structural efforts due to its dynamics. We have assembled the complex between Nef-AP2 and Serinc5 incorporated into lipid nanodiscs and our preliminary cryo-EM density (res.10Å) suggests that binding of AP2 to the lipid bilayer is a contributing factor to the formation of the complex. Interestingly, the interaction of AP2 with lipids alters the conformation of AP2 in comparison with other AP2-Nef complex structures without changing how Nef and AP2 interact. Currently, we are optimizing our cryo-EM structural characterization pipeline to account for the sample dynamics and sensitivity to the blotting/freezing process. Simultaneously, we are investigating the role of the native N-terminal myristoylation of Nef on complex formation and possibly on directly interacting with Serinc5, as well as measuring binding affinities of AP2-Nef to Serinc5 in the context of the lipid bilayer and its lipidic composition.

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