Structural and Functional Characterization of G Protein-Coupled Receptors with Deep Mutational Scanning

Abstract

In humans, the 813 G protein-coupled receptors (GPCRs) are responsible for transducing diverse chemical stimuli to alter cell state, and are the largest class of drug targets. Their myriad structural conformations and various modes of signaling make it challenging to understand their structure and function. Here we developed a platform to characterize large libraries of GPCR variants in human cell lines with a barcoded transcriptional reporter of G-protein signal transduction. We tested 7,800 of 7,828 possible single amino acid substitutions to the beta-2 adrenergic receptor ( β 2 AR) at four concentrations of the agonist isoproterenol. We identified residues specifically important for β 2 AR signaling, mutations in the human population that are potentially loss of function, and residues that modulate basal activity. Using unsupervised learning, we resolve residues critical for signaling, including all major structural motifs and molecular interfaces. We also find a previously uncharacterized structural latch spanning the first two extracellular loops that is highly conserved across Class A GPCRs and is conformationally rigid in both the inactive and active states of the receptor. More broadly, by linking deep mutational scanning with engineered transcriptional reporters, we establish a generalizable method for exploring pharmacogenomics, structure and function across broad classes of drug receptors. Fig. 6 A Conserved Extracellular Tryptophan-Disulfide "Structural Latch" in Class A GPCRs is Mutationally Intolerant and Conformation-Independent. A.Sequence conservation of extracellular loop 1 (ECL1) and the extracellular interface of TM3 (202 GPCRs). B. Left: Depiction of the interaction of W99 23x50 , G102 3x21 , and C106 3x25 in ECL1 of the β 2 AR. Top Right: Conservation of the structure of the ECL1 region across functionally different class A GPCRs. Bottom Right: Activity of all 19 missense variants assayed for each of the three conserved residues, with the mean activity (mutational tolerance) shown as a blue bar. The shaded bars represent the mean mutational tolerance ± 1 SD of residues in the tolerant cluster 6 (green) and the intolerant clusters 1 and 2 (red). C. A hydrogen bond network between mutationally intolerant positions W99 23x50 , G102 3x21 , and C106 3x25 . Representative examples of the structural latch are shown. D. This structural latch is maintained in both the inactive and active state structures for the β 2 AR (inactive: 2RH1, active: 3P0G), the M2 muscarinic receptor (inactive: 3UON, active: 4MQS), the kappa-opioid receptor (inactive: 4DJH, active: 6B73), and the mu-opioid receptor (inactive: 4DKL, active: 5C1M)

Genome-wide Functional Characterization of Escherichia coli Promoters and Sequence Elements Encoding Their Regulation

“…Prior to genome integration, DNA-sequencing was performed to computationally map barcodes to sequences. A custom barcode mapper developed by Nathan Lubock (Jones et al , 2019) was used to collapse reads into a barcode-sequence map. We used two filtering steps for barcode quality.…”

Dissection of c-AMP Response Element Architecture by Using Genomic and Episomal Massively Parallel Reporter Assays

“…We cloned these libraries into a reporter construct we engineered to maximize signal to noise when integrated into the genome (Figure S1C) and then cloned a minimal promoter and luciferase gene between variant and barcode, placing the barcode in the 3 0 UTR of the luciferase gene. The assays were conducted at varying induction conditions, either episomally by transient transfection (Episomal MPRA), or singly integrated into the intergenic H11 safe-harbor locus (Zhu et al, 2014) by using BxBI-mediated recombination (Genomic MPRA) (Duportet et al, 2014;Jones et al, 2019;Matreyek et al, 2017;Xu et al, 2013) (Figures 1B, S1A, and S1B). The episomal MPRAs were run in biological duplicate across 8 different concentrations of forskolin (Figures 1D and S1E), which stimulates phosphorylation and activation of the CREB protein by activating adenylyl (Gonzalez and Montminy, 1989).…”

“…Hek293T H11 landing pad cells (Female), originally generated in (Jones et al, 2019), were grown in DMEM with 1% Penicillin-streptomycin and 10% FBS (Thermo Fisher Scientific) at 37 C and 5 % CO 2 . Cells were grown to confluence, unless otherwise stated, and passaged with Trypsin-EDTA 0.25% (Thermo Fisher Scientific) once over 2-4 day periods.…”

Evolutionary history of ATP‐binding cassette proteins

“…Finally, it is possible to make all combinations of residues in a very small region‐of‐interest (≤5 residues). Experimentally selecting for a specific protein function (e.g., folding and localization, complex formation, transport, or substrate selectivity) increases the abundance of functional mutants in the library [112,114,115]. Deep sequencing the library before and after selection identifies positions that positively or negatively impact function and thus identifies the available evolutionary trajectories.…”