Cerutti N.,Elevation Biotech |
Mendelow B.V.,University of Witwatersrand |
Napier G.B.,Elevation Biotech |
Papathanasopoulos M.A.,University of Witwatersrand |
And 4 more authors.
Journal of Biological Chemistry | Year: 2010
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 Ser60 on the CD4 domain 1 α-helix with Cys positions a thiol in proximity of the gp120 V1/V2 loop disulfide (Cys126-Cys 196), satisfying the stereochemical and geometric conditions for redox exchange between CD4 Cys60 and gp120 Cys126, 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. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Source
Joubert M.K.,South African Council for Scientific and Industrial Research |
Joubert M.K.,Amgen Inc. |
Kinsley N.,Elevation Biotech |
Capovilla A.,Elevation Biotech |
And 5 more authors.
Biochemistry | Year: 2010
The HIV-1 envelope glycoprotein, gp120, is a key target for a class of drugs called entry inhibitors. Here we used molecular modeling to construct a three-dimensional model of an anti-gp120 RNA aptamer, B40t77, alone and in complex with gp120. An initial model of B40t77 was built from the predicted secondary structure and then subjected to a combination of energy minimization and molecular dynamics. To model the B40t77-gp120 complex, we docked the B40t77 predicted structure onto the CD4-induced epitope of the gp120 crystal structure. A series of gp120 point mutations in the predicted B40t77-gp120 interface were measured for their binding affinity for B40t77 by surface plasmon resonance. According to the model, of the 10 gp120 amino acids that showed a reduction in the level of binding when mutated to alanine, all of them are modeled as making direct contact with B40t77 as part of a hydrogen bonding network. Comparison by electron microscopy of the B40t77-gp120 complex with gp120 alone revealed that only the longest dimension of the complex significantly increased in length, in a manner consistent with the predicted model. Binding assays revealed that B40t77 can weaken the binding of gp120 to the monoclonal antibodies B6, B12, and 2G12, none of which have binding sites that overlap with B40t77, as well as strengthen the binding to the antibody 19b. Thus, B40t77 may induce distant conformational changes in gp120 that disrupt its association with host cells and may suggest a mechanism for aptamer neutralization of HIV-1. © 2010 American Chemical Society. Source