UCSF

Proteins are the primary mediators of cellular activity and play a critical role in disease etiology and treatment. The conformation of proteins, either intrinsic or induced by other interacting partners, can inform their localization, their capacity to transmit signaling cascades, and, ultimately, their function. Immunoglobulins, or antibodies, are glycoproteins generated by the immune system with the primary function of protecting the host by blocking or neutralizing the spread of pathogens. The blocking activity of antibodies can proceed via simple mechanisms such as occlusion of interacting interfaces or through more complex allosteric pathways for blocking pathogenic protein function. Paradoxically, immune system dysregulation can also produce self-reactive, pathogenic antibodies causative for disease. Amongst these disparate examples is a shared feature: the capacity for antibodies to effect protein function through conformational control of their interacting partner. This dissertation uses a combination of cryo-electron microscopy, pharmacological signaling studies, and biochemical reconstitutions to understand how natural, patient-derived, self-reactive antibodies can select for active state conformations of the thyrotropin G protein-coupled receptor, and how synthetic single-domain antibodies can restrict SARS-CoV-2 S protein conformation and subsequent viral entry.

In the first chapter, I and others structurally describe the activation mechanism of the thyrotropin receptor both in the context of normal, hormone-mediated signaling and in pathogenic, autoantibody-induced activation. These results describe the hormone mimicry and ensuing conformational selection employed by autoantibodies in the pathogenesis of Graves’ disease in their action at the thyrotropin receptor. This work is also the first to structurally describe how autoantibodies can activate G protein-coupled receptors and further refines the model for glycoprotein hormone receptor activation by the eponymous glycoprotein hormones.

In the second chapter, I and others discover and biophysically characterize single-domain antibodies directed against the SARS-CoV-2 S protein. The single-domain antibodies we initially describe only inhibit viral entry through steric occlusion of the angiotensin-converting enzyme II binding interface on the viral S protein. In a structure-guided manner, we further engineer one single-domain antibody into a multivalent construct that restricts the viral S protein into a closed conformation and show enhanced potency in viral entry inhibition after additional affinity maturation. We demonstrate that this construct is stable to common routes of aerosolized delivery and propose its use as a viral prophylactic. This work also describes single-domain antibodies with allosteric, yet incompletely described mechanisms of S protein entry inhibition.