Structure information on HIV glycoproteins (gp120 and gp41) can facilitate the design of envelope-based antigens, and better understanding of HIV fusion process and host immune response. The major objective of Project 2 is to structurally characterize HIV-1 envelope glycoproteins to assist the design of HIV-1 envelope antigens that can elicit broadly cross-reactive neutralizing antibodies, with a long-term goal of developing an envelope-based protective vaccine against HIV infection. All currently known gp41 structures are very similar to each other and represent a post-fusion conformation. Structure of gp41 pre-fusion intermediate is not known. We propose to solve structure for carefully designed gp41 fragments without the N-terminal heptad region. Our approach includes a suite of carefully selected and state-of-the art structural and computational biology approaches, including crystallography, hydroxyl radical footprinting and ab initio modeling. Our current model of the gp41-64 antigen explains its antigenic reactivity and is driving ideas for its engineering.
Aim 1 provides a seamless gp41 antigen design-high resolution structure determination-redesign pipeline within the program as a whole.
Aim 2 intends to structurally characterize gp120 outer domain (gp120OD) and OD-gp41 constructs using structural mass spectrometry methods. Glycosylation signature of gp120OD would affect the immunogenicity of gp120OD-based antigens. Thus, a design-structural characterization-redesign approach where Project 2 feeds back to the Project 1 team is critical for developing improved gp120-based antigens. Present crystal structures of gp120 are mostly from protein cores, with variable loop regions and N- and Ctermini truncated. Solving a crystal structure of a full-length gp120 with intact variable loops has been extremely difficult due to the flexibility of these loop segments and extensive glycosylation of the protein.
In Aim 3, we propose to use, hydroxyl radical footprinting, homology modeling, and docking to characterize the structure of the V1/V2 variable loop regions in the presence and in the absence of the primary receptor CD4. By probing the outer surfaces of proteins accessible to solvents, we will be able to determine the conformational changes in the variable loop regions upon binding of CD4.
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