Biology subjects

Sarma, P.

Publications and source records attributed to Sarma, P..

8 recordsLinked to original sources

bioRxiv2023

Molecular insights into atypical modes of beta-arrestin interaction with seven transmembrane receptors

{beta}-arrestins are multifunctional proteins that are critically involved in regulating spatio-temporal aspects of GPCR signaling. The interaction of {beta}-arrestins with GPCRs is typically conceptualized in terms of receptor activation and phosphorylation primarily in the carboxyl-terminus. Interestingly however, there are several GPCRs that harbor majority of phosphorylation sites in their 3rd intracellular loop (ICL3) instead of carboxyl-terminus but still robustly engage {beta}-arrestins. Moreover, there are several 7TMRs that are now characterized as intrinsically-biased, {beta}-arrestin-coupled receptors (ACRs) due to lack of functional G-protein-coupling but robust {beta}-arrestin binding leading to functional outcomes. The molecular basis of {beta}-arrestin interaction and activation upon binding to these types of 7TMRs is currently elusive, and it represents a major knowledge gap in our current understanding of this signaling system. Here, we present seven cryo-EM structures of {beta}-arrestins in basal state, activated by the muscarinic M2 receptor (M2R) through its ICL3, and a {beta}-arrestin-coupled receptor known as decoy D6 receptor (D6R). These structural snapshots combined with biochemical, cellular, and biophysical experiments including HDX-MS and MD simulation provide novel insights into the ability of {beta}-arrestins to preferentially select specific phosphorylation patterns in the receptors, and also illuminate the structural diversity in 7TMR-{beta}-arrestin interaction. Surprisingly, we also observe that the carboxyl-terminus of {beta}-arrestin2 but not {beta}-arrestin1 undergoes structural transition from a {beta}-strand to -helix upon activation by D6R, which may preclude the core-interaction with the activated receptor. Taken together, our study elucidates previously unappreciated aspects of 7TMR-{beta}-arrestin interaction, and provides important mechanistic clues about how the two isoforms of {beta}-arrestins can recognize and regulate a large repertoire of GPCRs.

biochemistry↗

bioRxiv2023

Structure-guided engineering of biased-agonism in the human niacin receptor via single amino acid substitution

The Hydroxycarboxylic acid receptor 2 (HCA2), also known as the niacin receptor or GPR109A, is a prototypical G protein-coupled receptor that plays a central role in the inhibition of lipolytic and atherogenic activities in our body. Interestingly, GPR109A activation also results in vasodilation that is linked to the side-effect of flushing associated with dyslipidemia drugs such as niacin. This receptor continues to be a key target for developing novel pharmacophores and lead compounds as potential therapeutics in dyslipidemia with minimized flushing response, however, the lack of structural insights into agonist-binding and receptor activation has limited the efforts. Here, we present five different cryo-EM structures of the GPR109A-G-protein complexes with the receptor bound to dyslipidemia drugs, niacin or acipimox, non-flushing agonists, MK6892 or GSK256073, and recently approved psoriasis drug, monomethyl fumarate (MMF). These structures allow us to visualize the binding mechanism of agonists with a conserved molecular interaction network, and elucidate the previously lacking molecular basis of receptor activation and transducer-coupling. Importantly, cellular pharmacology experiments, guided by the structural framework determined here, elucidate pathway-selective biased signaling elicited by the non-flushing agonists. Finally, taking lead from the structural insights, we successfully engineered receptor mutants via single amino acid substitutions that either fail to elicit agonist-induced transducer-coupling or exhibits G-protein signaling bias. Taken together, our study provides previously lacking structural framework to understand the agonist-binding and activation of GPR109A, and opens up the possibilities of structure-guided novel drug discovery targeting this therapeutically important receptor.

biochemistry↗

bioRxiv2023

Structural insights into agonist-binding and activation of the human complement C3a receptor

The complement cascade is an integral part of innate immunity, and it plays a crucial role in our bodys innate immune response including combating microbial infections. Activation of the complement cascade results in the generation of multiple peptide fragments, of which complement C3a and C5a are potent anaphylatoxins. The complement C3a binds and activates a G protein-coupled receptor (GPCR) known as C3aR while C5a activates two distinct receptors namely C5aR1 and C5aR2. Our current understanding of complement peptide recognition by their corresponding receptors is limited primarily to biochemical studies, and direct structural visualization of ligand-receptor complexes is still elusive. Here, we present structural snapshots of C3aR in complex with heterotrimeric G-proteins, with the receptor in ligand-free state, activated by full-length complement C3a, and a peptide agonist EP54, derived based on the carboxyl-terminal sequence of C5a. A comprehensive analysis of these structures uncovers the critical residues involved in C3a-C3aR interaction, and also provides molecular insights to rationally design carboxyl-terminal fragments of C3a and C5a to act as potent agonists of the receptor. Surprisingly, a comparison of C3a-C3aR structure with C5a-C5aR1 structure reveals diagonally opposite placement of these two complement peptides on their respective receptors, which helps explain the subtype selectivity of these complement peptides. Finally, taking lead from the structural insights, we also identify EP141, a peptide derived from the carboxyl-terminus of C3a, as a G-protein-biased agonist at C3aR. Taken together, our study illuminates the structural mechanism of complement C3a recognition by C3aR, and it also offers the first structural template for designing novel C3aR ligands with therapeutic potential for inflammatory disorders.

biochemistry↗

bioRxiv2023

Structural insights into ligand-recognition, activation, and signaling-bias at the complement C5a receptor, C5aR1

Activation of the complement cascade is a critical part of our innate immune response against invading pathogens, and it operates in a concerted fashion with the antibodies and phagocytic cells towards the clearance of pathogens. The complement peptide C5a, generated during the activation of complement cascade, is a potent inflammatory molecule, and increased levels of C5a are implicated in multiple inflammatory disorders including the advanced stages of COVID-19 pathophysiology. The proximal step in C5a-mediated cellular and physiological responses is its interaction with two different seven transmembrane receptors (7TMRs) known as C5aR1 and C5aR2. Despite a large body of functional data on C5a-C5aR1 interaction, direct visualization of their interaction at high-resolution is still lacking, and it represents a significant knowledge gap in our current understanding of complement receptor activation and signaling. Here, we present cryo-EM structures of C5aR1 activated by its natural agonist C5a, and a G-protein-biased synthetic peptide ligand C5apep, in complex with heterotrimeric G-proteins. The C5a-C5aR1 structure reveals the ligand binding interface involving the N-terminus and extracellular loops of the receptor, and we observe that C5a exhibits a significant conformational change upon its interaction with the receptor compared to the basal conformation. On the other hand, the structural details of C5apep-C5aR1 complex provide a molecular basis to rationalize the ability of peptides, designed based on the carboxyl-terminus sequence of C5a, to act as potent agonists of the receptor, and also the mechanism underlying their biased agonism. In addition, these structural snapshots also reveal activation-associated conformational changes in C5aR1 including outward movement of TM6 and a dramatic rotation of helix 8, and the interaction interface for G-protein-coupling. In summary, this study provides previously lacking molecular basis for the complement C5a recognition and activation of C5aR1, and it should facilitate structure-based discovery of novel lead molecules to target C5aR1 in inflammatory disorders.

biophysics↗

bioRxiv2022

A key GPCR phosphorylation motif discovered in arrestin2-CCR5 phosphopeptide complexes

The two non-visual arrestin isoforms, arrestin2 and arrestin3 recognize and bind hundreds of G protein-coupled receptors (GPCRs) with different phosphorylation patterns leading to distinct functional outcomes. The impact of phosphorylation on arrestin interactions has been well studied only for very few GPCRs. Here we have characterized the interactions between the phosphorylated CC chemokine receptor 5 (CCR5) and arrestin2. We detected several new CCR5 phosphorylation sites, which are necessary for stable complex formation with arrestin2. Crystal structures of arrestin2 in apo form and in complexes with CCR5 C-terminal phosphopeptides together with NMR spectroscopy, biochemical and functional assays revealed three phosphoresidues in a pXpp motif that are essential for the arrestin2 interactions and activation. The same phosphoresidue cluster is present in other receptors, which form stable complexes with arrestin2. We propose that the identified pXpp motif is responsible for robust arrestin2 recruitment in many GPCRs. An analysis of available sequences, structural and functional information on other GPCR*arrestin interactions suggests that a particular arrangement of phosphoresidues within the GPCR intracellular loop 3 and C-terminal tail determines arrestin2 and 3 isoform specificity. Taken together, our findings demonstrate how multi-site phosphorylation controls GPCR*arrestin interactions and provide a framework to probe the intricate details of arrestin activation and signaling. One-sentence summaryA structural and functional analysis of arrestin2 in apo form and complexes with several CCR5 phosphopeptides reveals key phosphorylation sites responsible for stable GPCR*arrestin interactions and their contributions to CCR5-arrestin2 function.

biophysics↗

bioRxiv2022

Structural snapshots uncover a lock-and-key type conserved activation mechanism of β-arrestins by GPCRs

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a key determinant for the binding and activation of multifunctional regulatory proteins known as {beta}-arrestins ({beta}arrs). Although the primary sequence and phosphorylation pattern of GPCRs are poorly conserved, the downstream functional responses mediated by {beta}arrs such as receptor desensitization, endocytosis and signaling are broadly applicable across GPCRs. A conserved principle of {beta}arr activation, if any, upon their interaction with different GPCRs harboring divergent phosphorylation patterns remains to be visualized, and it represents a major knowledge gap in our current understanding of GPCR signaling and regulatory paradigms. Here, we present four structural snapshots of activated {beta}arrs, in complex with distinct phosphorylation patterns derived from the carboxyl-terminus of three different GPCRs, determined using cryogenic-electron microscopy (cryo-EM). These structures of activated {beta}arrs elucidate a "lock-and-key" type conserved mechanism of {beta}arr activation wherein a P-X-P-P phosphorylation pattern in GPCRs interacts with a spatially organized K-K-R-R-K-K sequence in the N-domain of {beta}arrs. Interestingly, the P-X-P-P pattern simultaneously engages multiple structural elements in {beta}arrs responsible for maintaining the basal conformation, and thereby, leads to efficient {beta}arr activation. The conserved nature of this lock-and-key mechanism is further illustrated by a comprehensive sequence analysis of the human GPCRome, and demonstrated in cellular context with targeted mutagenesis including "loss-of-function" and "gain-of-function" experiments with respect to {beta}arr activation measured by an intrabody-based conformational sensor. Taken together, our findings uncover previously lacking structural insights, which explain the ability of distinct GPCRs to activate {beta}arrs through a common mechanism, and a key missing link in the conceptual framework of GPCR-{beta}arr interaction and resulting functional outcomes.

biochemistry↗

bioRxiv2022

Molecular insights into intrinsic transducer-coupling bias in the CXCR4-CXCR7 system

Chemokine receptors constitute an important subfamily of G protein-coupled receptors (GPCRs), and they are critically involved in a broad range of immune response mechanisms. Ligand promiscuity among these receptors makes them an interesting target to explore novel aspects of biased agonism. Here, we comprehensively characterize two chemokine receptors namely, CXCR4 and CXCR7, which share a common chemokine agonist (CXCL12), in terms of their G-protein coupling, {beta}-arrestin ({beta}arr) recruitment, contribution of GRKs, and ERK1/2 MAP kinase activation. We observe that CXCR7 lacks G-protein coupling while maintaining robust {beta}arr recruitment with a major contribution of GRK5/6. On the other hand, CXCR4 displays robust G-protein activation as expected, however, it exhibits significantly reduced {beta}arr-coupling compared to CXCR7 in response to their shared natural agonist, CXCL12. These two receptors induce distinct {beta}arr conformations even when activated by the same agonist, and CXCR7, unlike CXCR4, fails to activate ERK1/2 MAP kinase. We further determine the crystal structure of {beta}arr2 in complex with a carboxyl-terminal phosphopeptide derived from CXCR7, which reveals a smaller interdomain rotation than observed previously for activated {beta}arrs. Importantly, structure-guided cellular experiments reveal a key contribution of a single phosphorylation site in CXCR7 on {beta}arr recruitment and endosomal trafficking. Taken together, our study provides molecular insights into intrinsic bias encoded in the CXCR4-CXCR7 system, and it has broad implications for therapeutically important framework of biased agonism.

biochemistry↗

bioRxiv2021

Preclinical Efficacy of IMM-BCP-01, a Highly Active Patient-Derived Anti-SARS-CoV-2 Antibody Cocktail

Using an unbiased interrogation of the memory B cell repertoire of convalescent COVID-19 patients, we identified human antibodies that demonstrated robust antiviral activity in vitro and efficacy in vivo against all tested SARS-CoV-2 variants. Here, we describe the pre-clinical characterization of an antibody cocktail, IMM-BCP-01, that consists of three unique, patient-derived recombinant neutralizing antibodies directed at non-overlapping surfaces on the SARS-CoV-2 spike protein. Two antibodies, IMM20184 and IMM20190 directly block spike binding to the ACE2 receptor. Binding of the third antibody, IMM20253, to its unique epitope on the outer surface of RBD, alters the conformation of the spike trimer, promoting release of spike monomers. These antibodies decreased SARS-CoV-2 infection in the lungs of Syrian golden hamsters, and efficacy in vivo efficacy was associated with broad antiviral neutralizing activity against multiple SARS-CoV-2 variants and robust antiviral effector function response, including phagocytosis, ADCC, and complement pathway activation. Our pre-clinical data demonstrate that the three antibody cocktail IMM-BCP-01 shows promising potential for preventing or treating SARS-CoV-2 infection in susceptible individuals. One sentence summaryIMM-BCP-01 cocktail triggers Spike Trimer dissociation, neutralizes all tested variants in vitro, activates a robust effector response and dose-dependently inhibits virus in vivo.

immunology↗