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. 2018 Jul 3;115(27):7051-7056.
doi: 10.1073/pnas.1718489115. Epub 2018 Jun 18.

Computational design of orthogonal membrane receptor-effector switches for rewiring signaling pathways

M Young  1 , T Dahoun  2 , B Sokrat  3 , C Arber  4   5   6   7 , K M Chen  1 , M Bouvier  3 , P Barth  8   2   7   9   10
Affiliations

Computational design of orthogonal membrane receptor-effector switches for rewiring signaling pathways

M Young et al. Proc Natl Acad Sci U S A. .

Abstract

Membrane receptors regulate numerous intracellular functions. However, the molecular underpinnings remain poorly understood because most receptors initiate multiple signaling pathways through distinct interaction interfaces that are structurally uncharacterized. We present an integrated computational and experimental approach to model and rationally engineer membrane receptor-intracellular protein systems signaling with novel pathway selectivity. We targeted the dopamine D2 receptor (D2), a G-protein-coupled receptor (GPCR), which primarily signals through Gi, but triggers also the Gq and beta-arrestin pathways. Using this approach, we designed orthogonal D2-Gi complexes, which coupled with high specificity and triggered exclusively the Gi-dependent signaling pathway. We also engineered an orthogonal chimeric D2-Gs/i complex that rewired D2 signaling from a Gi-mediated inhibitory into a Gs-dependent activating pathway. Reinterpreting the evolutionary history of GPCRs in light of the designed proteins, we uncovered an unforeseen hierarchical code of GPCR-G-protein coupling selectivity determinants. The results demonstrate that membrane receptor-cytosolic protein systems can be rationally engineered to regulate mammalian cellular functions. The method should prove useful for creating orthogonal molecular switches that redirect signals at the cell surface for cell-engineering applications.

Keywords: G-protein–coupled receptor; cell signaling; membrane protein; protein binding; protein design.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Computational design of orthogonal GPCR–G-protein signaling pairs. (A) GPCR-mediated signaling rewiring through engineered orthogonal GPCR–G-protein pairs. A native promiscuous GPCR interacts and activates several G proteins (G1, G2), triggering distinct signaling pathways. The binding interface between the GPCR and G2 is designed to engineer an orthogonal highly selective protein pair that exclusively signals through the designed G2. (B) Integrated homology modeling, docking, and design for engineering orthogonal binding in the absence of experimental protein structures. The active state D2 structure bound to dopamine agonist ligand and the G-protein Gi is modeled by homology to the distant homologs beta2 adrenergic receptor (β2AR) and opsin, using an integrated homology modeling-docking technique (SI Appendix, Supplementary Methods and Fig. S1). The inactive-state D2 structure bound to the spiperone inverse agonist ligand is modeled starting from the close homolog dopamine D3 receptor (D3DR). Multistate design is applied to the active- and inactive-state D2 structures for engineering an orthogonal binding D2ortho–Giortho pair while ensuring that no selected mutations lead to an inactivated or constitutively active receptor (i.e., signaling without agonist stimulus).
Fig. 2.
Fig. 2.
Designed D2–Gi orthogonal pair achieves high activity and coupling selectivity. (A) Representative active-state model of D2 (D2active) bound to the C-terminal helix of Gi (GiCterm). The designed binding interface includes two hot-spot motifs (HS1 and HS2). (B) Zoomed view of the D2–Gi interface at HS1. Predicted energetic contribution of designed residues at HS1 to the binding affinity of the designed D2ortho–Giortho and the noncognate ortho/WT pairs. D2 and Gi residues are colored in green and gold, respectively. The highlighted designed D2 residue HS1L (mutation K204L) is located on TM6 at the Ballesteros Weinstein (BW) position 6.29. Stabilization and destabilization effects correspond to negative and positive energy differences from WT, respectively. Energy differences are in Rosetta Energy Units and shown on top of the black arrows. (C) Similar to B but for HS2. The highlighted designed D2 residue HS2F (mutation V100F) is located on TM3 at the BW position 3.54. (D) TrpC4β channel activation upon D2 activation by dopamine through the Gi signaling pathway. Fluorescence changes are measured for cells stably expressing TrpC4β channels cotransfected with plasmids coding the D2 and Gi variants indicated on each curve (SI Appendix, Supplementary Methods). Under the experimental conditions, the D2 variants were expressed at the same level on the cell surface as measured by ELISA (SI Appendix, Fig. S3A).
Fig. 3.
Fig. 3.
Designed D2–Gi orthogonal pair achieves high signaling pathway selectivity. (A) Calcium release and CREB phosphorylation upon D2 activation by dopamine through the Gq signaling pathway. (B) After addition of 20 μM of dopamine (arrow), change in intracellular concentration of calcium ion was measured using a Ca2+-sensitive fluorescent probe on cells transiently transfected with either pcDNA3.1-D2WT (solid line) or pcDNA3.1-D2ORTHO (dashed line). (C) pCREB levels are measured by ELISA on cells transiently transfected with the above-mentioned constructs (SI Appendix, Supplementary Methods). pCREB levels are normalized to those obtained upon β2AR activation by isoproterenol through the Gs signaling pathway. Signaling through GiWT is abrogated with pertussis toxin (SI Appendix, Supplementary Methods). **P < 0.05. (D and E) β-Arrestin recruitment by D2 receptors upon stimulation by dopamine using ebBRET on cells transiently transfected with the above-mentioned constructs. Signals are normalized to that obtained with D2WT and β-arrestin 2. Under the experimental conditions, the D2 variants were expressed at the same level on the cell surface as measured by ELISA (SI Appendix, Figs. S3B, S7B, and S8D).
Fig. 4.
Fig. 4.
Two classes of contact selectivity determinants of GPCR–G-protein coupling. (A) Schematic representation of the contact selectivity determinants at the GPCR–G-protein–binding interface. Primary determinants are located at highly conserved positions on naturally evolved G proteins. Modifying the primary contacts results in major selectivity switches. Native GPCR–G-protein systems differ in the secondary contact determinants that fine-tune their binding specificity profile, enabling GPCRs to recognize multiple G proteins with distinct affinities. Muscarinic M1, dopamine D2, and beta2 adrenergic receptors are provided as examples with their corresponding G-protein–coupling profiles. (B) Sequence-structure relationships at the binding interface of three major classes of GPCR–G-protein systems. C-terminal residues of the G protein are separated in two sets of positions: positions selected for design based on D2–Gi models (Bottom) and nondesigned positions (Top). G-protein position numbering is given based on the consensus G-protein aligned sequences (within ovals) and based on the exact residue type and number in Gi2 (above or below ovals). Each G-protein position is connected to a GPCR position based on the number of interaction contacts observed at the D2–Gi interface. Each position on the GPCR is represented as a pie divided into three regions that describe the chemical property of the consensus amino acid sequence coupling to each family of G proteins (i.e., Gs, Gi, Gq) (SI Appendix, Supplementary Methods). Sixty percent of the GPCR residues contacting G proteins do not share the same chemical properties in different GPCR–G-protein classes. Compared with previous studies (3), the amino acid type of residues contacting G proteins differs even between GPCR subfamilies coupling to the same G-protein class.
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