title: Cryoelectron tomographic analysis of an HIV-neutralizing protein and its complex with native viral gp120.
abstract: Identifying structural determinants of human immunodeficiency virus (HIV) neutralization is an important component of rational drug and vaccine design. We used cryoelectron tomography and atomic force microscopy to characterize the structure of an extremely potent HIV-neutralizing protein, D1D2-Ig alpha tp (abbreviated as D1D2-IgP), a polyvalent antibody construct that presents dodecameric CD4 in place of the Fab regions. We show that D1D2-IgP has a novel structure, displaying greater flexibility of its antibody arms than the closely related IgM. Using simian immunodeficiency virus in complex with D1D2-IgP, we present unequivocal evidence that D1D2-IgP can cross-link surface spikes on the same virus and on neighboring viruses. The observed binding to the viral envelope spikes is the result of specific CD4-gp120 interaction, because binding was not observed with MICA-IgP, a construct that is identical to D1D2-IgP except that major histocompatibility complex Class I-related Chain A (MICA) replaces the CD4 moiety. CD4-mediated binding was also associated with a significantly elevated proportion of ruptured viruses. The ratio of inactivated to CD4-liganded gp120-gp41 spikes can be much greater than 1:1, because all gp120-gp41 spikes on the closely apposed surfaces of cross-linked viruses should be incapable of accessing the target cell surface and mediating entry, as a result of inter-virus spike cross-linking. These results implicate flexibility rather than steric bulk or polyvalence per se as a structural explanation for the extreme potency of D1D2-IgP and thus suggest polyvalence presented on a flexible scaffold as a key design criterion for small molecule HIV entry inhibitors.
authors.affiliation: Laboratory of Cell Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Bennett
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.forename: Donald
authors.initials: D
authors.lastname: Van Ryk
authors.forename: Donald
authors.initials: D
authors.lastname: Bliss
authors.forename: James
authors.initials: J
authors.lastname: Arthos
authors.forename: Robert M
authors.initials: RM
authors.lastname: Henderson
authors.forename: Sriram
authors.initials: S
authors.lastname: Subramaniam
mesh_terms: D000818:Animals; D019380:Anti-HIV Agents / Q000737:chemistry; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D020285:Cryoelectron Microscopy / Q000379:methods*; D015699:HIV Envelope Protein gp120 / Q000737:chemistry*; D015658:HIV Infections / Q000378:metabolism; D006801:Humans; D021621:Imaging, Three-Dimensional; D007136:Immunoglobulins / Q000737:chemistry; D018625:Microscopy, Atomic Force; D008954:Models, Biological; D008968:Molecular Conformation; D015302:Simian Immunodeficiency Virus / Q000378:metabolism; D014054:Tomography / Q000379:methods; D014764:Viral Proteins / Q000737:chemistry*
chemical_list: D019380:Anti-HIV Agents; D015699:HIV Envelope Protein gp120; D007136:Immunoglobulins; D014764:Viral Proteins