Structural and Molecular Mechanisms of B Cell Co-Receptor Function

Abstract

B cells are central to the adaptive immune response, serving as the basis for generation of humoral immunity to foreign agents. To initiate an immune response, an antigen binds to a cell surface B cell receptor (BCR), leading to a coordinated cellular response that culminates in the secretion of neutralizing antibodies. To enhance the sensitivity of B cell signaling, the BCR functions in association with a multicomponent co-receptor complex that contains the complement receptor CD21, the signaling protein CD19, and CD81. CD81 belongs to the tetraspanin family of proteins and is critical for proper B cell function by acting both as a chaperone to enable cell surface expression of properly folded CD19 and as a signaling scaffold to regulate the localization of CD19 in the membrane during B cell signaling.

The work presented in this dissertation makes key advances toward understanding the functional partnership between CD19 and CD81 through structural and mechanistic studies. In Chapter 2, I show that CD81 relies on its ectodomain to traffic CD19 to the cell surface, and then use cell-based assays and crystallography to illuminate the binding mechanism of the anti-CD81 antibody 5A6. I show that the 5A6 antibody binds selectively to activated B cells and recognizes a conformational epitope on CD81 that is masked when CD81 is bound to CD19 on resting B cells, revealing that the CD19-CD81 interaction is dynamically regulated upon B cell activation. In Chapter 3, I engineer a stable CD19-CD81 complex and report the cryo-EM structure of this complex bound to a therapeutic antigen binding fragment (Fab). This structure, the first high-resolution structure of any tetraspanin bound to a partner protein, shows a tetraspanin captured in the “open” conformation, highlighted by a major reorganization of the transmembrane helices to occlude the cholesterol binding pocket present in apo-CD81. This structure reveals that tetraspanins are a conformationally regulated family of membrane proteins and provides a structural explanation for how changes in tetraspanin structure facilitate shuttling of protein partners in and out of cholesterol-enriched lipid rafts to regulate signal transduction. In Chapter 4, I engineer a cell-based system that enables parallel quantification of B cell co-receptor interactions with local phosphosite information in the seconds to hours after BCR stimulation. Using this system, I determine the kinetics of the response of proteins previously implicated in signaling events downstream of BCR engagement, including the BCR itself, Akt, PKC, PI3K, and Raf isoforms. I also identity many proteins with no previous connection to B cell signaling that are responsive to BCR stimulation and verify SWAP70 and the glutamate transporter SLC1A1 as novel regulators of the early B cell response. Collectively, the work described here provides a molecular understanding of B cell co-receptor function and offers new insight into approaches to modulate the B cell signaling pathway.