Stabilization of HIV-1 gp120-CD4 receptor complex through targeted interchain disulfide exchange.
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 alpha-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.
Elevation Biotech, 8 Blackwood Avenue, Parktown 2193, Johannesburg, South Africa.
Nichole
N
Cerutti
Barry V
BV
Mendelow
Grant B
GB
Napier
Maria A
MA
Papathanasopoulos
Mark
M
Killick
Makobetsa
M
Khati
Wendy
W
Stevens
Alexio
A
Capovilla
D015704:CD4 Antigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D015699:HIV Envelope Protein gp120 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008954:Models, Biological; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D015704:CD4 Antigens; D004220:Disulfides; D015699:HIV Envelope Protein gp120; D011994:Recombinant Proteins