Project Summary/Abstract – Project 2 Structural analysis of how HIV-1 Envelope (Env) interacts with antigen receptor on B cells (BCR) to initiate B cell signaling and activation is key to understanding antibody responses against HIV protein immunogens. Recent studies of the organization of the BCR complex on the cell membrane supports a model in which BCRs exist in different signaling states that require definition in structural terms. We hypothesize the following distinct states of the BCR complex – 1) “signaling-inhibited’, 2) signaling-competent and 3) ‘signaling-disrupted’. Advances in HIV-1 Env design including those that target germline precursors have led to the development of Env immunogens as potential candidates for HIV vaccines. How BCRs of distinct specificities and signaling-states interact with Env protein immunogens that trigger signaling and activate these cells are not clearly understood. The overall goal of the project is to define BCR-antigen structures with specificity of broadly neutralizing antibodies (bnAb), non-canonical glycan-binding bnAbs and to-be isolated autologous neutralizing antibodies (anAb). In this project, we will perform biophysical/biochemical, structural and immunological analyses to define properties of HIV Env-BCR interactions for activation of B cells expressing bnAb or germline precursor BCRs. In Aim 1, we will perform Cryo-EM and molecular dynamic simulation analyses to define structures of antigen- liganded BCR complex with specificities of autologous or broadly neutralizing HIV-1 antibodies. In addition, we will define the structures of BCRs with specificities of glycan-binding bnAbs that present non-canonical Fab configurations (I-shaped versus Y-shaped). In Aim 2, we will study specificity of bnAb precursors with disrupted proximal signaling and determine whether such BCRs show distinct cell surface interaction dynamics with Env proteins. Studies in Aim 3 will define antigen-BCR interaction parameters that enhance B cell signaling, antigen internalization and MHC class II-peptide presentation of Env protein eptiopes. The proposed studies will bridge high-resolution structures and antigen-BCR interaction dynamics to B cell signaling and activation. The long- range goals of these studies are to provide the mechanistic basis for understanding the humoral response to HIV-1 vaccines and guide development of strategies to enhance vaccine efficacy.