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A mechanism by which binding of the broadly neutralizing antibody b12 unfolds the inner domain ?1 helix in an engineered HIV-1 gp120.

ABSTRACT: Using all-atom simulations, we examine the role of the I109C/Q428C disulfide "stitch" in altering the conformational distribution of engineered HIV-1 gp120 core relevant for binding of the broadly neutralizing recombinant antibody b12. In particular, we propose that the I109C/Q428C stitch results in a conformational distribution favoring an unfolded inner-domain ?1-helix upon binding of b12. Using targeted molecular dynamics, we show that folded ?1 in the b12-bound conformation of gp120 is stable both with and without the stitch, but that with folded ?1, the stitch requires an orientation of the ?20/?21 sheet that is sterically incompatible with b12 binding. Forcing ?20/?21 into the orientation displayed by the b12-bound conformation after folding ?1 with the stitch intact results in partial unfolding of ?1, whereas without the stitch, ?20/?21 reorientation does not affect the conformation of ?1. These findings collectively support the hypothesis that the disulfide stitch shifts the conformational distribution of ?1 to the unfolded state, meaning an unfolded ?1 is not a strict requirement of the b12-bound conformational ensemble of gp120's lacking the I109C/Q428C stitch.

SUBMITTER: Emileh A

PROVIDER: S-EPMC3059550 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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A mechanism by which binding of the broadly neutralizing antibody b12 unfolds the inner domain α1 helix in an engineered HIV-1 gp120.

Emileh Ali A Abrams Cameron F CF

Proteins 20110201 2

Using all-atom simulations, we examine the role of the I109C/Q428C disulfide "stitch" in altering the conformational distribution of engineered HIV-1 gp120 core relevant for binding of the broadly neutralizing recombinant antibody b12. In particular, we propose that the I109C/Q428C stitch results in a conformational distribution favoring an unfolded inner-domain α1-helix upon binding of b12. Using targeted molecular dynamics, we show that folded α1 in the b12-bound conformation of gp120 is stabl ...[more]

PMID: 21117239

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Project description:The V1/V2 domain of the HIV-1 envelope protein gp120 possesses two important epitopes: a glycan-dependent epitope recognized by the prototypic broadly neutralizing monoclonal antibody (bN-mAb), PG9, as well as an epitope recognized by non-neutralizing antibodies that has been associated with protection from HIV infection in the RV144 HIV vaccine trial. Because both of these epitopes are poorly immunogenic in the context of full length envelope proteins, immunization with properly folded and glycosylated fragments (scaffolds) represents a potential way to enhance the immune response to these specific epitopes. Previous studies showed that V1/V2 domain scaffolds could be produced from a few selected isolates, but not from many of the isolates that would be advantageous in a multivalent vaccine. In this paper, we used a protein engineering approach to improve the conformational stability and antibody binding activity of V1/V2 domain scaffolds from multiple diverse isolates, including several that were initially unable to bind the prototypic PG9 bN-mAb. Significantly, this effort required replicating both the correct glycan structure as well as the ?-sheet structure required for PG9 binding. Although scaffolds incorporating the glycans required for PG9 binding (e.g., mannose-5) can be produced using glycosylation inhibitors (e.g., swainsonine), or mutant cell lines (e.g. GnTI(-) 293 HEK), these are not practical for biopharmaceutical production of proteins intended for clinical trials. In this report, we describe engineered glycopeptide scaffolds from three different clades of HIV-1 that bind PG9 with high affinity when expressed in a wildtype cell line suitable for biopharmaceutical production. The mutations that improved PG9 binding to scaffolds produced in normal cells included amino acid positions outside of the antibody contact region designed to stabilize the ?-sheet and turn structures. The scaffolds produced address three major problems in HIV vaccine development: (1) improving antibody responses to poorly immunogenic epitopes in the V1/V2 domain; (2) eliminating antibody responses to highly immunogenic (decoy) epitopes outside the V1/V2 domain; and (3) enabling the production of V1/V2 scaffolds in a cell line suitable for biopharmaceutical production.

Dataset Information

A mechanism by which binding of the broadly neutralizing antibody b12 unfolds the inner domain ?1 helix in an engineered HIV-1 gp120.

Publications

A mechanism by which binding of the broadly neutralizing antibody b12 unfolds the inner domain α1 helix in an engineered HIV-1 gp120.

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