Structural and functional characterization of G protein–coupled receptors with deep mutational scanning
- Department of Chemistry and Biochemistry, UCLA-DOE Institute for Genomics and Proteomics, Molecular Biology Institute, Quantitative and Computational Biology Institute, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, and Jonsson Comprehensive Cancer Center, UCLA, United States
- MRC Laboratory of Molecular Biology, United Kingdom
- Department of Computer Science, Stanford University, Department of Computer Science, Institute for Computational and Mathematical Engineering, Stanford University, Department of Computer Science, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Department of Computer Science, Department of Structural Biology, Stanford University School of Medicine, United States
Figures
Figure 1 with 1 supplement
A platform for deep mutational scanning of GPCRs.
(A) Overview of the multiplexed GPCR activity assay. Plasmids encoding ADRB2 variants, a transcriptional CRE reporter of signaling activity, and 15 nucleotide barcode sequences that identify the …
Figure 1—figure supplement 1
Cellular engineering and reporter optimization for multiplexed assay.
(A) Schematic of experiment to ensure the landing pad is present at single copy in the genome and thus recombine a single donor plasmid per cell. Single copy integration is essential to prevent …
Figure 2 with 1 supplement
Variant-activity landscape for 7800 missense variants of the β2AR and multiplexed assay validation.
(A) Top: Secondary structure diagram of the β2AR: the N and C termini are black, the transmembrane helices are purple blocks, and the intra- and extracellular domains are colored blue and green, …
Figure 2—figure supplement 1
Global metrics of the multiplexed screen.
(A) The measurements between barcodes at the RNA-seq level are correlated (r = 0.89, r = 0.89, r = 0.9, r = 0.87) at all agonist concentrations (0, EC50, EC100, and EMax Iso). Similarly, the mean …
Figure 3 with 1 supplement
Individual mutations and residues reveal evolutionary and structural insights into β2AR function.
(A) Positional conservation across Class A GPCRs correlates with mutational tolerance (Spearman's ρ = −0.676, Pearson’s r = −0.681), the mean activity of all amino acid substitutions per residue at …
Figure 3—figure supplement 1
Correlation with sequence conservation and covariation and analysis of individual mutations.
(A) Mutational tolerance is highly correlated with species-level sequence conservation and is maximized at EC100 (Spearman's ρ = −0.673, Pearsons r = −0.65; ρ = −0.71, r = −0.69; ρ = −0.74, r = −0.73…
Figure 4 with 1 supplement
Unsupervised learning segregates residues into clusters with distinct responses to mutation.
(A) Amino acids were segregated into classes based on their physicochemical properties and mean activity scores were reported by class for each residue. With Uniform Manifold Approximation and …
Figure 4—figure supplement 1
Cluster assignment is robust across different UMAP embeddings.
Given the high dimensionality of the mutational responses, Uniform Manifold Approximation and Projection (UMAP) (McInnes and Healy, 2018) was used to learn lower dimension representations of the all …
Figure 5 with 2 supplements
Mutational tolerance elucidates broad structural features and critical residues of the β2AR.
(A) Residues within the transmembrane domain colored by their tolerance to particular classes of amino acid substitution. Teal residues are intolerant to both hydrophobic and charged amino acids …
Figure 5—figure supplement 1
Mutational profile suggests side chain orientation and environment.
(A) The crystal structure of the hydroxybenzyl isoproterenol-activated state of the β2AR (PDB: 4LDL) with residues colored by UMAP cluster identity. (B) Distributions of solvent-accessible surface …
Figure 5—figure supplement 2
Mutational intolerance of functionally related residues.
(A) Relative activation of an integrated CRE luciferase reporter gene for β2AR missense variants mentioned in the manuscript. (B) Functional consequences of mutation for a set of residues near the …
Figure 6 with 1 supplement
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 Class A GPCRs with a disulfide bridge between TM3 and ECL1). (B) Left: Depiction of the …
Figure 6—figure supplement 1
The WxxGxxxC motif is highly conserved across Class A GPCRs.
(A) Individual verification of the mutational intolerance of W9923x50 and G1023x21. Relative activation of an integrated CRE luciferase reporter gene for β2AR missense variants. (B) Surface …
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Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo-sapiens) | HEK293T | ATCC | CRL-3216 | |
| Cell line (Homo-sapiens) | HEK293TΔADRB2 + Landing Pad | This paper | Construction Information found in Endogenous ADRB2 Deletion using CRISPR/Cas9 and Landing Pad Genome Editing Sections | |
| Gene (Homo-sapiens) | ADRB2 | NCBI | Gene ID 154 | |
| Chemical compound, drug | Isoproterenol | Millipore Sigma | I5627 | |
| Chemical compound, drug | Forskolin | Millipore Sigma | F6886 | |
| Commercial assay or kit | Dual Glo Luciferase Assay | Promega | E2920 | |
| Recombinant DNA reagent | TALEN plasmids | Addgene | #51554 #51555 | |
| Recombinant DNA reagent | SpCas9 plasmid | Addgene | pX339 | |
| Sequence-based reagent | Oligonucleotide Microarray | Agilent | Custom Synthesis | |
| Commercial assay or kit | Nextseq Mid Output 300 cycle | Illumina | 20024905 | |
| Commercial assay or kit | Nextseq High Output 75 cycle | Illumina | 20024906 | |
| Strain, strain background (Escherichia coli) | Dh5 alpha | New England Biolabs | C2989K | |
| Antibody | AlexaFluor 488 Anti-Flag rat monoclonal | Thermo Fisher | MA1-142-A488 | (1:100) |
| Transfected construct (Homo-sapiens) | ADRB2 barcoded variant-reporter library | This paper | Reagent Construction Information found in Variant Library Generation and Cloning Section | |
| Commercial assay or kit | RNEasy Miniprep Kit | Qiagen | 74104 | |
| Commercial assay or kit | Plasmid Plus DNA Maxi Kit | Qiagen | 12963 | |
| Commercial assay or kit | Superscript IV | Thermo Fisher | 18091050 | |
| Commercial assay or kit | Lipofectamine 3000 | Thermo Fisher | L3000001 | |
| Commercial assay or kit | D1000 DNA Screen Tape | Agilent | 5067–5582 | |
| Commercial assay or kit | D1000 Reafents | Agilent | 5067–5583 | |
| Commercial assay or kit | SYBR FAST QPCR Master Mix | Roche | 07959362001 | |
| Commercial assay or kit | Zymo Clean Gel DNA Recovery Kit | Zymo Research | D4007 | |
| Commercial assay or kit | Zymo DNA Clean and Concentrator Kit | Zymo Research | D4013 | |
| Chemical compound, drug | CD293 | Thermo Fisher Scientific | 11913019 | |
| Software, algorithm | BBTools | Brian Bushnell | https://jgi.doe.gov/data-and-tools/bbtools/ | |
| Software, algorithm | Jensen-Shannon Conservation | https://doi.org/10.1093/bioinformatics/btm270 | ||
| Software, algorithm | OMA Orthology Database | https://doi.org/10.1093/nar/gkx1019 | ||
| Software, algorithm | FreeSASA | 10.12688/f1000research.7931.1 | ||
| Software, algorithm | EVmutation | doi:10.1038/nbt.3769 | ||
| Software, algorithm | Parasail | http://dx.doi.org/10.1186/s12859-016-0930-z |
Additional files
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Supplementary file 1
List of species for evolutionary analysis.
A table describing the list of 55 species used for the analysis of evolutionary constraint of residues from the OMA database.
- https://cdn.elifesciences.org/articles/54895/elife-54895-supp1-v2.xls
-
Supplementary file 2
Processed data.
A table with the processed data used in this study. Includes the position, mutation, min activity, max activity, average activity, propagated uncertainty, coefficient of variation, number of barcodes for that mutation, mutation class, and an annotation of where in the β2AR the position is.
- https://cdn.elifesciences.org/articles/54895/elife-54895-supp2-v2.xls
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Supplementary file 3
Mutational tolerance.
A table containing positions in the β2AR with their mutational tolerance, rank order, and an annotation for known positions in the receptor.
- https://cdn.elifesciences.org/articles/54895/elife-54895-supp3-v2.xls
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Supplementary file 4
List of primers used in this study.
A table documenting the DNA sequences and application of important primers in this study.
- https://cdn.elifesciences.org/articles/54895/elife-54895-supp4-v2.xls
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Transparent reporting form
- https://cdn.elifesciences.org/articles/54895/elife-54895-transrepform-v2.docx
