Robert Everett Jefferson

I am engineering GPCRs to alter their ligand binding properties, creating variants that have specific and novel signaling responses. Before working in the Barth lab, I studied membrane protein folding in James Bowie's lab at UCLA, where I probed the rather interesting folding of a chloride transporter from E. coli. Before that I employed the steric trap method (developed in the Bowie lab) to study membrane protein folding under native conditions and measured the extremely slow unfolding kinetics of the E. coli membrane enzyme diacylglycerol kinase. Before that I dabbled in membrane protein crystallography and methods to stabilize membrane proteins. Before that I was an undergraduate at Whitman College where I worked in Doug Juers lab studying the structure of myelin basic protein. Before that I was a youth of minor consequence.

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Computational design of dynamic receptor—peptide signaling complexes applied to chemotaxis

Patrick Daniel Barth, Andreas Füglistaler, Mahdi Hijazi, Robert Everett Jefferson, Aurélien Laurent Jean-Charles Oggier, Ana Paola Rico Villarreal

Engineering protein biosensors that sensitively respond to specific biomolecules by triggering precise cellular responses is a major goal of diagnostics and synthetic cell biology. Previous biosensor designs have largely relied on binding structurally well-defined molecules. In contrast, approaches that couple the sensing of flexible compounds to intended cellular responses would greatly expand potential biosensor applications. Here, to address these challenges, we develop a computational strategy for designing signaling complexes between conformationally dynamic proteins and peptides. To demonstrate the power of the approach, we create ultrasensitive chemotactic receptor—peptide pairs capable of eliciting potent signaling responses and strong chemotaxis in primary human T cells. Unlike traditional approaches that engineer static binding complexes, our dynamic structure design strategy optimizes contacts with multiple binding and allosteric sites accessible through dynamic conformational ensembles to achieve strongly enhanced signaling efficacy and potency. Our study suggests that a conformationally adaptable binding interface coupled to a robust allosteric transmission region is a key evolutionary determinant of peptidergic GPCR signaling systems. The approach lays a foundation for designing peptide-sensing receptors and signaling peptide ligands for basic and therapeutic applications.

2023

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Computational design of ultrasensitive flexible peptide:receptor signaling complexes for enhanced chemotaxis

Patrick Daniel Barth, Andreas Füglistaler, Mahdi Hijazi, Robert Everett Jefferson, Aurélien Laurent Jean-Charles Oggier, Ana Paola Rico Villarreal

Engineering biosensors that sensitively recognize specific biomolecules and trigger functional cellular responses is a holy grail of diagnostics and synthetic cell biology. Biosensor design approaches have mostly focused on binding structurally well-defined molecules. Coupling the sensing of flexible compounds to complex cellular responses would considerably expand biosensor applications, but remains challenging. We developed a computational strategy for designing highly sensitive receptor biosensors of flexible peptides. Using the method, we created ultrasensitive chemotactic GPCR:peptide pairs capable of eliciting potent chemotaxis in human primary T cells. Through mutual induced fit, our flexible structure design approach enhanced peptide contacts with binding and allosteric sites in the ligand pocket to achieve unprecedented signaling efficacy and potency. The approach paves the road for designing peptide-sensing receptors and expanding the biosensor toolbox for basic and therapeutic applications.

bioRxiv2022

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Computationally designed GPCR quaternary structures bias signaling pathway activation

Patrick Daniel Barth, Robert Everett Jefferson

Communication across membranes controls critical cellular processes and is achieved by receptors translating extracellular signals into selective cytoplasmic responses. While receptor tertiary structures can be readily characterized, receptor associations into quaternary structures are challenging to study and their implications in signal transduction remain poorly understood. Here, we report a computational approach for predicting receptor self-associations, and designing receptor oligomers with various quaternary structures and signaling properties. Using this approach, we designed chemokine receptor CXCR4 dimers with reprogrammed binding interactions, conformations, and abilities to activate distinct intracellular signaling proteins. In agreement with our predictions, the designed CXCR4s dimerized through distinct conformations and displayed different quaternary structural changes upon activation. Consistent with the active state models, all engineered CXCR4 oligomers activated the G protein Gi, but only specific dimer structures also recruited β-arrestins. Overall, we demonstrate that quaternary structures represent an important unforeseen mechanism of receptor biased signaling and reveal the existence of a bias switch at the dimer interface of several G protein-coupled receptors including CXCR4, mu-Opioid and type-2 Vasopressin receptors that selectively control the activation of G proteins vs β-arrestin-mediated pathways. The approach should prove useful for predicting and designing receptor associations to uncover and reprogram selective cellular signaling functions.

2022

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