Camelid single-domain antibody fragments (“nanobodies”) provide the remarkable specificity of antibodies within a single 15 kDa immunoglobulin VHH domain. This unique feature has enabled applications ranging from use as biochemical tools to therapeutic agents. Nanobodies have emerged as especially useful tools in protein structural biology, facilitating studies of conformationally dynamic proteins such as G protein-coupled receptors (GPCRs). Nearly all nanobodies available to date have been obtained by animal immunization, a bottleneck restricting many applications of this technology. To solve this problem, we report a fully in vitro platform for nanobody discovery based on yeast surface display. We provide a blueprint for identifying nanobodies, demonstrate the utility of the library by crystallizing a nanobody with its antigen, and most importantly, we utilize the platform to discover conformationally-selective nanobodies to two distinct human GPCRs. To facilitate broad deployment of this platform, the library and associated protocols are freely available for non-profit research.

How the cell converts graded signals into threshold-activated responses is a question of great biological relevance. Here, we uncover a nonlinear modality of epidermal growth factor receptor (EGFR)-activated signal transduction, by demonstrating that the ubiquitination of the EGFR at the PM is threshold controlled. The ubiquitination threshold is mechanistically determined by the cooperative recruitment of the E3 ligase Cbl, in complex with Grb2, to the EGFR. This, in turn, is dependent on the simultaneous presence of two phosphotyrosines, pY1045 and either one of pY1068 or pY1086, on the same EGFR moiety. The dose–response curve of EGFR ubiquitination correlate precisely with the non-clathrin endocytosis (NCE) mode of EGFR internalization. Finally, EGFR-NCE mechanistically depends on EGFR ubiquitination, as the two events can be simultaneously re-engineered on a phosphorylation/ubiquitination-incompetent EGFR backbone. Since NCE controls the degradation of the EGFR, our findings have implications for how the cell responds to increasing levels of EGFR signalling, by varying the balance of receptor signalling and degradation/attenuation.

SUMMARY G protein-coupled receptors (GPCRs) are the largest family of membrane receptors in humans, and they regulate processes ranging from neurotransmission to cardiovascular biology. Although GPCRs have been studied for decades, current methods for tracking GPCR signaling often suffer from low throughput, modification or overexpression of effector proteins, and low temporal resolution. Here, we introduce a new approach using peroxidase-catalyzed proximity labeling to track GPCR signaling and internalization in living cells. Combination of this technique with isobaric labeling and triple-stage mass spectrometry enables precise, quantitative, and time-resolved measurement of thousands of receptor-proximal proteins at their native levels to comprehensively track GPCR agonist response. Using this technique, we examine the response of the angiotensin II type 1 receptor to both balanced and biased agonists. In addition, we extend the approach to the β2 adrenergic receptor, underscoring the generalizability of this technology.

SUMMARY The immune checkpoint receptor PD-1 and its ligand, PD-L1, have emerged as key regulators of anti-tumor immunity in humans. Recently, we reported an ultra high-affinity PD-1 mutant, termed HAC PD-1, which shows superior therapeutic efficacy in mice compared to antibodies. However, the molecular details underlying the action of this agent remain incompletely understood, and a molecular view of PD-1/PD-L1 interactions in general is only beginning to emerge. Here, we report the structure of HAC PD-1 in complex with PD-L1, showing it binds PD-L1 using a unique set of polar interactions. Biophysical studies and long-timescale molecular dynamics experiments reveal the mechanisms by which ten point mutations confer a 35,000-fold enhancement in binding affinity, and offer atomic-scale views of the role of conformational dynamics in PD-1/PD-L1 interactions. Finally, we show that the HAC PD-1 exhibits pH-dependent affinity, with pseudo-irreversible binding in a low pH setting akin to the tumor microenvironment.