INVESTIGATING NEUTRALIZING ANTIBODY INDUCED CONFORMATIONAL CHANGES AND MATURATIO

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Little three-dimensional structural information is available for trimeric forms of the HIV-1 Env protein, mostly now limited to cryoEM reconstructions where there is some disagreement. We have essed and purified multiple soluble, clade A and B HIV-1 glycoproteins as gp140 trimers, soluble D1D2 CD4 and a panel of neutralizing anti-HIV antibodies. While these reagents are used in quantitative binding studies and ongoing crystallization trials, SAXS can be used to obtain complementary structural information that can independently have significant impact on our studies of the biophysical basis of the process of neutralization by antibodies. After preliminary SAXS data collection, we generated good SAXS models for a highly-sensitive clade B (SF162) gp140 trimer and a highly-resistant clade A (Q461) gp140 trimer (plus a series of control structures). The comparison clearly showed a dramatic conformational difference between these two, demonstrated the viability of using SAXS to examine the overall structure of gp140 trimers ? and revealed potentially unrecognized aspects of trimer structure. Now we propose a cohesive set of experiments to map out gp140 sub-structures using a cross-clade panel of gp140 trimers, including a series of V-loop deletions and forms with reduced N-linked CHO, a set of neutralizing antibodies (b12, 447-52D, 4E10, 2F5) and soluble CD4.