Cryoelectron Tomographic Analysis of an HIV-neutralizing Protein and Its Complex with Native Viral gp120

Adam Bennett,Jun Liu,Donald Van Ryk,Donald Bliss,James Arthos,Robert M Henderson,Sriram Subramaniam

doi:10.1074/jbc.m702025200

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.

Highlights

Identifying structural determinants of human immunodeficiency virus (HIV) neutralization is an important component of rational drug and vaccine design
D1D2-IgP is a multimeric CD4-immunoglobulin fusion construct with exemplary neutralization activity against primary isolates of Human immunodeficiency virus type 1 (HIV-1) [6, 12]
The atomic force microscopy (AFM) micrographs, which are isoforce surfaces and not true three-dimensional data, do not necessarily provide a faithful description of the native state molecular morphology, because apparent structures are potentially affected by variability in the manner of absorption to the mica and by preferential absorption of certain species

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—D1D2-IgP was expressed and purified as described [12], with modifications. Solution Atomic Force Microscopy—A solution of D1D2-IgP, IgM, or MICA-IgP in 50 mM Tris-buffered saline (TBS), pH 7.4, was deposited on freshly cleaved mica coated with poly-L-lysine (Sigma), incubated for 15 min, rinsed three times with TBS, and imaged in TBS in tapping mode on a Digital Instruments MultiMode atomic force microscope (Veeco, Santa Barbara, CA) with a Veeco NP-S scanning probe. Air Atomic Force Microscopy—An IgM solution of 3.3 ␮g/ml protein in 50 ␮l of TBS was incubated on poly-L-lysine-coated mica for 2 min at room temperature, followed by rinsing with 2 ml of 0.1ϫ TBS and drying under nitrogen. Uranyl acetate pretreatment was performed by incubation of 50 ␮l of protein in TBS on freshly cleaved mica for 2 min at room temperature, followed by rinsing twice with 1 ml of 0.1ϫ TBS. Images were acquired in air tapping mode on a Digital Instruments MultiMode atomic force microscope (Veeco) with a SuperSharp silicon scanning probe (NanoSensors, Neuchatel, Switzerland)

RESULTS

Besides the presence of additional densities on the surface of

DISCUSSION