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Rationally designed integrin beta3 mutants stabilized in the high affinity conformation.

ABSTRACT: Integrins are important cell surface receptors that transmit bidirectional signals across the membrane. It has been shown that a conformational change of the integrin beta-subunit headpiece (i.e. the beta I domain and the hybrid domain) plays a critical role in regulating integrin ligand binding affinity and function. Previous studies have used coarse methods (a glycan wedge, mutations in transmembrane contacts) to force the beta-subunit into either the open or closed conformation. Here, we demonstrate a detailed understanding of this conformational change by applying computational design techniques to select five amino acid side chains that play an important role in the energetic balance between the open and closed conformations of alphaIIbbeta3. Eight single-point mutants were designed at these sites, of which five bound ligands much better than wild type. Further, these mutants were found to be in a more extended conformation than wild type, suggesting that the conformational change at the ligand binding headpiece was propagated to the legs of the integrin. This detailed understanding of the conformational change will assist in the development of allosteric drugs that either stabilize or destabilize specific integrin conformations without occluding the ligand-binding site.

SUBMITTER: Luo BH

PROVIDER: S-EPMC2635054 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Rationally designed integrin beta3 mutants stabilized in the high affinity conformation.

Luo Bing-Hao BH Karanicolas John J Harmacek Laura D LD Baker David D Springer Timothy A TA

The Journal of biological chemistry 20081119 6

Integrins are important cell surface receptors that transmit bidirectional signals across the membrane. It has been shown that a conformational change of the integrin beta-subunit headpiece (i.e. the beta I domain and the hybrid domain) plays a critical role in regulating integrin ligand binding affinity and function. Previous studies have used coarse methods (a glycan wedge, mutations in transmembrane contacts) to force the beta-subunit into either the open or closed conformation. Here, we demo ...[more]

PMID: 19019827

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Project description:The V1V2 region of HIV-1 gp120 harbors a major vulnerable site targeted by a group of broadly neutralizing monoclonal antibodies (MAbs) such as PG9 through strand-strand recognition. However, this epitope region is structurally polymorphic as it can also form a helical conformation recognized by RV144 vaccine-induced MAb CH58. This structural polymorphism is a potential mechanism for masking the V1V2 vulnerable site. Designing immunogens that can induce conformation-specific antibody (Ab) responses may lead to vaccines targeting this vulnerable site. We designed a panel of immunogens engrafting the V1V2 domain into trimeric and pentameric scaffolds in structurally constrained conformations. We also fused V1V2 to an Fc fragment to mimic the unconstrained V1V2 conformation. We tested these V1V2-scaffold proteins for immunogenicity in rabbits and assessed the responses by enzyme-linked immunosorbent assay (ELISA) and competition assays. Our V1V2 immunogens induced distinct conformation-specific Ab responses. Abs induced by structurally unconstrained immunogens reacted preferentially with unconstrained V1V2 antigens, suggesting recognition of the helical configuration, while Abs induced by the structurally constrained immunogens reacted preferentially with constrained V1V2 antigens, suggesting recognition of the ?-strand conformation. The Ab responses induced by the structurally constrained immunogens were more broadly reactive and had higher titers than those induced by the structurally unconstrained immunogens. Our results demonstrate that immunogens presenting the different structural conformations of the gp120 V1V2 vulnerable site can be designed and that these immunogens induce distinct Ab responses with epitope conformation specificity. Therefore, these structurally constrained V1V2 immunogens are vaccine prototypes targeting the V1V2 domain of the HIV-1 envelope.The correlates analysis of the RV144 HIV-1 vaccine trial suggested that the presence of antibodies to the V1V2 region of HIV-1 gp120 was responsible for the modest protection observed in the trial. In addition, V1V2 harbors one of the key vulnerable sites of HIV-1 Env recognized by a family of broadly neutralizing MAbs such as PG9. Thus, V1V2 is a key target for vaccine development. However, this vulnerable site is structurally polymorphic, and designing immunogens that present different conformations is crucial for targeting this site. We show here that such immunogens can be designed and that they induced conformation-specific antibody responses in rabbits. Our immunogens are therefore prototypes of vaccine candidates targeting the V1V2 region of HIV-1 Env.

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Rationally designed integrin beta3 mutants stabilized in the high affinity conformation.

Publications

Rationally designed integrin beta3 mutants stabilized in the high affinity conformation.

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