Computational rewiring of allosteric pathways reprograms GPCR selective responses to ligands

G-protein-coupled receptors (GPCRs) are the largest class of cell surface receptors and drug targets, and respond to a wide variety of chemical stimuli to activate diverse cellular functions. Understanding and predicting how ligand binding triggers a specific signaling response is critical for drug discovery and design but remains a major challenge. Here, computational design of GPCR allosteric functions is used to uncover the mechanistic relationships between agonist ligand chemistry, receptor sequence, structure, dynamics and allosteric signaling in the dopamine D2 receptor. Designed gain of function D2 variants for dopamine displayed very divergent G-protein signaling responses to other ligand agonists that strongly correlated with ligand structural similarity. Consistent with these observations, computational analysis revealed distinct topologies of allosteric signal transduction pathways for each ligand-bound D2 pair that were perturbed differently by the designs. We leveraged these findings by rewiring ligand-specific pathways and designed receptors with highly selective ligand responses. Overall, our study suggests that distinct ligand agonists can activate a given signaling effector through specific “allosteric activator” moieties that engage partially independent signal transmission networks in GPCRs. The results provide a mechanistic framework for understanding and predicting the impact of sequence polymorphism on receptor pharmacology, informing selective drug design and rationally designing receptors with highly selective ligand responses for basic and therapeutic applications.

Reprogramming G Protein-Coupled Receptor Structure, Function And Signaling By Computational Design

Dániel Kéri

G protein-coupled receptors (GPCRs) are 7-transmembrane alpha-helical integral membrane proteins on which cells heavily rely to receive information regarding their external environment. These receptors are able to transfer information to intracellular downstream effectors upon stimulation from their cognate ligands, which can be considerably diverse. They can include light, small molecules, peptides, protons; Due to its evolutionary success, over millions of years of evolution, this protein family has expanded to include over 800 members. Despite the large size of the family and the diverse inputs they accept, the structure and the mechanism for relaying signals to their downstream partners, G proteins and B-arrestins, remains largely the same. As these receptors are involved in a great deal of biological processes such as sight (rhodopsin), cognition (dopamine, serotonin, opioid receptors), immunity (chemokine receptors), heart function (adrenergic, angiotensin receptors), etc.; a great deal of effort has gone into understanding their structure and function. Our efforts have gone into understanding their signaling properties and rationally modifying them. Most proteins are fairly flexible entities and their structures tend to oscillate and move around, sampling a number of conformations. Nature has taken advantage of these natural motions to regulate protein function from distant binding sites, in a phenomenon called allostery. GPCRs are unique in the sense that their primary function relies on allostery. By tweaking the allosteric signaling of GPCRs, we hope to have a better understand allostery, use the principles we learn to create designer GPCR biosensors with the desired sensitivity, and to eventually create de novo allosteric proteins. In this work, I present our progress in this effort, which has been significant. We have created a highly accurate in silico method to allosterically alter relative stabilities of the active and inactive states of the GPCR dopamine D2 receptor, but also to independently control its allosteric signaling strength. With our methodology we have been able to create receptors which have high basal activities by selectively stabilizing the active state of the receptor. This has already facilitated the structure determination of the active, G protein-bound dopamine D2 receptor; a first for this GPCR. Additionally, we have created a number of dopamine D2 variants with roughly 100 times higher sensitivity to dopamine than the WT receptor. These efforts continue to characterize the functional effects of these allosteric mutations. Furthermore, we are also working on a collaboration to rewire the signaling pathway of the adenosine A2A receptor. Adenosine is a common immunosuppresant in solid tumor micro-environments (TMEs). By rewiring the downstream signaling of the A2A receptor, we hope to reverse the response of T-cells in these TMEs to activate and attack the solid tumor.

EPFL2021

Computational design of G Protein-Coupled Receptor allosteric signal transductions

Patrick Daniel Barth, Dániel Kéri

Membrane receptors sense and transduce extracellular stimuli into intracellular signaling responses but the molecular underpinnings remain poorly understood. We report a computational approach for designing protein allosteric signaling functions. By combining molecular dynamics simulations and design calculations, the method engineers amino-acid ‘microswitches’ at allosteric sites that modulate receptor stability or long-range coupling, to reprogram specific signaling properties. We designed 36 dopamine D2 receptor variants, whose constitutive and ligand-induced signaling agreed well with our predictions, repurposed the D2 receptor into a serotonin biosensor and predicted the signaling effects of more than 100 known G-protein-coupled receptor (GPCR) mutations. Our results reveal the existence of distinct classes of allosteric microswitches and pathways that define an unforeseen molecular mechanism of regulation and evolution of GPCR signaling. Our approach enables the rational design of allosteric receptors with enhanced stability and function to facilitate structural characterization, and reprogram cellular signaling in synthetic biology and cell engineering applications.

2019

Investigating molecular interactions of G protein coupled receptors by fluorescence techniques

Jean-Baptiste Perez

G Protein coupled receptors (GPCRs) constitute an abundant family of membrane proteins which play a central role in cellular signaling and are important targets for modern medicine. Despite intensive research, central questions of the molecular mechanism of GPCR mediated signal transduction remain unresolved. This thesis concerns α1b-adrenergic receptor (α1b-AR) as a prototypic GPCR. The question whether ARs form homo- or hetero-oligomers was investigated using fluorescence resonance energy transfer (FRET). An important finding was that both α1a and α1b-AR subtypes form homo-oligomers. Also hetero-oligomers have been observed between the α1b- and the α1a-AR subtypes, but not with less related receptors. Another interesting finding concerns α1-AR co-internalization which correlates with the ability of the receptor to hetero-oligomerize. Investigating particular processes in living cells is often complicated by the complex cellular environment. Here we have developed a rather simple approach to study transmembrane signaling by using supported cell membrane sheets. They were prepared by pressing a glass coverslip on the apical part of cultured cells, ripping off large regions of the native plasma membrane. For the α1b-AR we still could observe ligand binding to such sheets, indicating that GPCR functionality was at least partly conserved. Due to the absence of cytosolic autofluorescence of the cells, supported membrane sheets were also found to be suited for single-molecule microscopy. Because both leaflets of the supported membranes remained fluid, it was possible to follow the diffusion of certain membrane proteins. For example using fluorescence recovery after photobleaching (FRAP) and single-molecule microscopy, we investigated how G proteins diffused along the membrane and compartmentalized. The predominant role of lipid anchors in G protein localization was highlighted by measuring the diffusion of two Gαq proteins fused with citrine at two different positions and citrine based constructs designed to mimic the monomeric and the trimeric form of a G protein. Finally, first results were obtained showing the feasibility to form supported cell membrane sheets on microstructured devices consisting of a planar silicon support perforated with arrays of holes. This geometry provides a full accessibility to both intra- and extracellular sides of the bilayer. This concept could potentially be applied for the development of chip-based screening assays.