Stabilization of HIV-1 gp120-CD4 Receptor Complex through Targeted Interchain Disulfide Exchange

Abstract

HIV-1 enters cells via interaction between the trimeric envelope (Env) glycoprotein gp120/gp41 and the host cell surface receptor molecule CD4. The requirement of CD4 for viral entry has rationalized the development of recombinant CD4-based proteins as competitive viral attachment inhibitors and immunotherapeutic agents. In this study, we describe a novel recombinant CD4 protein designed to bind gp120 through a targeted disulfide-exchange mechanism. According to structural models of the gp120-CD4 receptor complex, substitution of Ser 60 on the CD4 domain 1 ␣-helix with Cys positions a thiol in proximity of the gp120 V1/V2 loop disulfide (Cys 126 -Cys 196 ), satisfying the stereochemical and geometric conditions for redox exchange between CD4 Cys 60 and gp120 Cys 126 , and the consequent formation of an interchain disulfide bond. In this study, we provide experimental evidence for this effect by describing the expression, purification, refolding, receptor binding and antiviral activity analysis of a recombinant two-domain CD4 variant containing the S60C mutation (2dCD4-S60C). We show that 2dCD4-S60C binds HIV-1 gp120 with a significantly higher affinity than wild-type protein under conditions that facilitate disulfide exchange and that this translates into a corresponding increase in the efficacy of CD4-mediated viral entry inhibition. We propose that targeted redox exchange between conserved gp120 disulfides and nucleophilic moieties positioned strategically on CD4 (or CD4-like scaffolds) conceptualizes a new strategy in the development of high affinity HIV-1 Env ligands, with important implications for therapy and vaccine development. More generally, this chalcogen substitution approach provides a general means of stabilizing receptor-ligand complexes where the structural and biophysical conditions for disulfide exchange are satisfied.The first step of HIV-1 2 entry into host cells involves the interaction between the viral envelope protein (Env) and the host cell receptor CD4. Functionally active (fusogenic) Env exists on the surface of virions as a trimer composed of three heterodimeric gp120-gp41 complexes (1). The latter are established through noncovalent association of monomeric gp120 with membrane-embedded gp41 (2-4), such that gp120 is presented on the exterior of the viral lipid membrane for interaction with CD4. The development of both Env-directed therapeutic compounds (5-10) and Env-based immunogens (11)(12)(13)(14)(15)(16)(17)(18)(19)(20) has been frustrated by the significant sequence heterogeneity known to exist among Envs, even from viruses that are phylogenetically closely related, which limits the cross-reactive breadth of anti-Env antibodies and therapies. Furthermore, Env is extensively glycosylated and conformationally flexible, which compounds the problem of Env ligand accessibility and reactivity. In the background of this extraordinary array of strategies evolved to enable viral escape from effective immune-mediated neutralization, and apart from a very small number of studies that have ...

Bifunctional CD4–DC-SIGN Fusion Proteins Demonstrate Enhanced Avidity to gp120 and Inhibit HIV-1 Infection and Dissemination

“…In nonreducing SDS-PAGE, C35NDs60c showed mainly as monomers (60.5 kDa) (Fig. 1b, lane 3), although oligomers were detected, some of which likely formed in nonreducing conditions (8). The 214.8-kDa complex in the AUC indicated that C35NDs60c existed mainly as tetramers.…”

Disulfide Reduction in CD4 Domain 1 or 2 Is Essential for Interaction with HIV Glycoprotein 120 (gp120), which Impairs Thioredoxin-driven CD4 Dimerization

“…5B). In contrast, 2dCD4-WT preparations (30 -40% of the content of which is oxidized in D1 and D2) show far more regular and ordered ␤-pleating patterns (27,(41)(42)(43). This suggests that a degree of structural flexibility in CD4, potentially conferred through CD4 disulfide reduction, may be required for the initial engagement with gp120.…”

“…2dCD4 Redox Isomer Analysis-We previously reported the production of a recombinant protein containing the first two N-terminal domains of human CD4 (2dCD4-WT) using an E. coli-based expression system (27). Following extraction from inclusion bodies, purification by nickel-nitrilotriacetic acid affinity chromatography and refolding under conditions that promote disulfide bond formation, functional 2dCD4-WT can be prepared to apparent homogeneity, resolving as a single band of ϳ28 kDa when analyzed by denaturing, reducing SDS-PAGE (Fig.…”

“…The recombinant 2dCD4 variants were expressed and purified by standard denaturing metal chelate affinity chromatography and refolded using oxidative refolding protocols according to methods described previously (27). Recombinant HIV-1 gp120 (BaL) was expressed in a stably transfected HEK293 cell line previously generated in our laboratory and purified by two-step affinity and size-exclusion chromatography.…”

Stabilization of HIV-1 Envelope in the CD4-bound Conformation through Specific Cross-linking of a CD4 Mimetic

“…Cerutti et al (53) showed that a recombinant two-domain CD4 variant containing an S60C mutation could be covalently linked to gp120, probably through the Cys 126 -Cys 196 disulfide bond at the stem of the V1/V2 variable loop, as this cystine is well positioned to react with Cys 60 . The authors demonstrated that this cross-linking increased the efficacy of CD4-mediated viral entry inhibition.…”