The stoichiometry of trimeric SIV glycoprotein interaction with CD4 differs from that of anti-envelope antibody Fab fragments.

Mikyung Kim,David Montefiori,Bing Chen,Stephen C Harrison,Yasmin Chishti,Olwyn Byron,Rebecca E Hussey,Gordon Campbell,Ellis L Reinherz,James A Hoxie

doi:10.1074/jbc.m104166200

Abstract

Human and simian immunodeficiency viruses infect host lymphoid cells by binding CD4 molecules via their gp160 envelope glycoproteins. Biochemical studies on recombinant SIVmac32H (pJ5) envelope ectodomain gp140 precursor protein show that the envelope is a trimer. Using size exclusion chromatography, quantitative amino acid analysis, analytical ultracentrifugation, and CD4-based competition assay, we demonstrate that the stoichiometry of CD4 receptor-oligomeric envelope interaction is 1:1. By contrast, Fab fragments of both neutralizing and non-neutralizing monoclonal antibodies bind at a 3:1 ratio. Thus, despite displaying equivalent CD4 binding sites on each of the three gp140 protomers within an uncleaved trimer, only one site binds the soluble 4-domain human CD4 extracellular segment. The anti-cooperativity and the faster k(off) of gp140 trimer:CD4 versus gp120 monomer:CD4 interaction suggest that CD4-induced conformational change is impeded in the intact envelope. The implications of these findings for immunity against human immunodeficiency virus and simian immunodeficiency virus are discussed.

Highlights

The acquired immunodeficiency syndrome (AIDS)1 in humans and monkeys is caused, respectively, by the human immunodeficiency viruses (HIV1 and HIV2) and the related primate lentiviruses, designated simian immunodeficiency viruses (SIVs) (1–7)
Quantitative amino acid analysis, analytical ultracentrifugation, and CD4-based competition assay, we demonstrate that the stoichiometry of CD4 receptor-oligomeric envelope interaction is 1:1
Structures have been determined for the gp41 ectodomain, in a state that probably corresponds to the final conformational rearrangement (23–25) and for a truncated, monomeric HIV1 gp120 complexed with CD4 and a monoclonal Fab that covers the co-receptor site (26)

Summary

EXPERIMENTAL PROCEDURES

Expression and Purification of SIV gp140, gp140 Variants, gp120, and Human CD4 —SIV gp140 and variants from SIVmac32H (pJ5) were expressed in Lec3.2.8.1 cells using the pEE14 expression system as previously described and referred to as gp160e (28, 29). Each mixture was incubated overnight at 4 °C and subsequently analyzed by gel filtration using a Superdex 200 column equilibrated with 20 mM Tris-HCl, and 200 mM NaCl, pH 8.0, at a flow rate of 0.7 ml/min controlled by the AKTA FPLC (Amersham Pharmacia Biotech). Where ␦c is proportional to the baseline absorbance; n is the total number of species present in the self-association equilibrium; K1,i is the equilibrium constant for the association of monomer to q(i)-mer; C1,0 is the monomer concentration at the radial position of the first data point; ␴ is the reduced molecular weight ((M␻2(1-␯៮␳)/RT) where M is the monomer mass; ␻ is the rotor speed in radian/s; ␯៮ is the partial specific volume of the protein (ml/g); ␳ is the solvent density (g/ml); R is the gas constant (8.31432 ϫ 107 erg/mol K); T is the absolute temperature (K)); ␰ and ␰0 are the values of r2/2 at any point r in the sample column and at the radial position of the first data point respectively; B is the second virial coefficient (here with units of inverse concentration); Ci(r) is the concentration of the ith species at radial position r; q(i) is the degree of association for the ith species. Equilibrium solute distribution data were analyzed with the Beckman XL-A data analysis software, which uses a modified form of Equation 1

RESULTS

DISCUSSION

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