NefCD4AP2 Complex Crosslinking with DSSO for Integrative Structure Determination

Objective: HIV-1 Nef suppresses immune surveillance mechanisms to promote viral pathogenesis. Cellular receptor CD4 is essential for HIV entry into host cells, though becomes problematic at later stages in the virus replication cycle. CD4 at the plasma membrane is targeted by Nef for endocytosis and lysosomal degradation via recruitment of clathrin adaptor complex 2 (AP2 complex). Our goal is to use cross-linking mass spectrometry and integrative modeling to aid in structure determination of flexible portions of the Nef-CD4-AP2 complex. Methods: We use disuccinimidyl sulfoxide (DSSO), a MS-cleavable, bifunctional amine-reactive small molecule, to cross-link proximal Lys residues or N-termini of a purified reconstituted Nef-CD4 C-terminal domain fusion protein bound to the AP2Δμ2-CTD complex. Cross-linked proteins were separated by SDS-PAGE, excised from the gel and digested with trypsin. The resulting peptides were analyzed by specialized LC-MS3 experiments for identification of cross-linked residues using MSconvert (to obtain individual MS2 and MS3 level mgf files), ProteinProspector (to obtain peptide identification results files), and XLTools (to identify cross-linked peptides from results, MS2, and MS3 files). Additional scripts were used to summarize cross-linked results. Results: We find that an intricate combination of conformational changes occurs in both Nef and AP2 to enable CD4 binding and downregulation. A pocket on Nef previously identified as crucial for recruiting class I MHC is also responsible for recruiting CD4, revealing a potential approach to inhibit two of Nef’s activities and sensitize the virus to immune clearance.