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 GPCR that plays a central role in the inhibition of lipolytic and atherogenic activities. Its activation also results in vasodilation that is linked to the side-effect of flushing associated with dyslipidemia drugs such as niacin. GPR109A continues to be a target for developing potential therapeutics in dyslipidemia with minimized flushing response. Here, we present cryo-EM structures of the GPR109A in complex with dyslipidemia drugs, niacin or acipimox, non-flushing agonists, MK6892 or GSK256073, and recently approved psoriasis drug, monomethyl fumarate (MMF). These structures elucidate the binding mechanism of agonists, molecular basis of receptor activation, and insights into biased signaling elicited by some of the agonists. The structural framework also allows us to engineer receptor mutants that exhibit G-protein signaling bias, and therefore, our study may help in structure-guided drug discovery efforts targeting this receptor.

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

ß-arrestins (ßarrs) are multifunctional proteins involved in signaling and regulation of seven transmembrane receptors (7TMRs), and their interaction is driven primarily by agonist-induced receptor activation and phosphorylation. Here, we present seven cryo-electron microscopy structures of ßarrs either in the basal state, activated by the muscarinic receptor subtype 2 (M2R) through its third intracellular loop, or activated by the ßarr-biased decoy D6 receptor (D6R). Combined with biochemical, cellular, and biophysical experiments, these structural snapshots allow the visualization of atypical engagement of ßarrs with 7TMRs and also reveal a structural transition in the carboxyl terminus of ßarr2 from a ß strand to an α helix upon activation by D6R. Our study provides previously unanticipated molecular insights into the structural and functional diversity encoded in 7TMR-ßarr complexes with direct implications for exploring novel therapeutic avenues.

Molecular basis of anaphylatoxin binding, activation, and signaling bias at complement receptors.

The complement system is a critical part of our innate immune response, and the terminal products of this cascade, anaphylatoxins C3a and C5a, exert their physiological and pathophysiological responses primarily via two GPCRs, C3aR and C5aR1. However, the molecular mechanism of ligand recognition, activation, and signaling bias of these receptors remains mostly elusive. Here, we present nine cryo-EM structures of C3aR and C5aR1 activated by their natural and synthetic agonists, which reveal distinct binding pocket topologies of complement anaphylatoxins and provide key insights into receptor activation and transducer coupling. We also uncover the structural basis of a naturally occurring mechanism to dampen the inflammatory response of C5a via proteolytic cleavage of the terminal arginine and the G-protein signaling bias elicited by a peptide agonist of C3aR identified here. In summary, our study elucidates the innerworkings of the complement anaphylatoxin receptors and should facilitate structure-guided drug discovery to target these receptors in a spectrum of disorders.

Structural snapshots uncover a key phosphorylation motif in GPCRs driving ß-arrestin activation.

Agonist-induced GPCR phosphorylation is a key determinant for the binding and activation of ß-arrestins (ßarrs). However, it is not entirely clear how different GPCRs harboring divergent phosphorylation patterns impart converging active conformation on ßarrs leading to broadly conserved functional responses such as desensitization, endocytosis, and signaling. Here, we present multiple cryo-EM structures of activated ßarrs in complex with distinct phosphorylation patterns derived from the carboxyl terminus of different GPCRs. These structures help identify a P-X-P-P type phosphorylation motif in GPCRs that interacts with a spatially organized K-K-R-R-K-K sequence in the N-domain of ßarrs. Sequence analysis of the human GPCRome reveals the presence of this phosphorylation pattern in a large number of receptors, and its contribution in ßarr activation is demonstrated by targeted mutagenesis experiments combined with an intrabody-based conformational sensor. Taken together, our findings provide important structural insights into the ability of distinct GPCRs to activate ßarrs through a significantly conserved mechanism.

Structural snapshot of a ß-arrestin-biased receptor.

Atypical chemokine receptor subtype 3 (ACKR3), a chemokine receptor, couples selectively to ß-arrestins (ßarrs) but not to G proteins despite having seven transmembrane (7TM) helix architecture. Yen et al. present cryogenic-electron microscopy (cryo-EM) structures of agonist-bound ACKR3, elucidating a distinct chemokine-binding mechanism, and offering a structural template to probe the transducer-coupling bias at this receptor.

Allosteric modulation of GPCR-induced ß-arrestin trafficking and signaling by a synthetic intrabody.

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of ß-arrestin (ßarr) recruitment and trafficking. For several GPCRs such as the vasopressin receptor subtype 2 (V2R), agonist-stimulation first drives the translocation of ßarrs to the plasma membrane, followed by endosomal trafficking, which is generally considered to be orchestrated by multiple phosphorylation sites. We have previously shown that mutation of a single phosphorylation site in the V2R (i.e., V2RT360A) results in near-complete loss of ßarr translocation to endosomes despite robust recruitment to the plasma membrane, and compromised ERK1/2 activation. Here, we discover that a synthetic intrabody (Ib30), which selectively recognizes activated ßarr1, efficiently rescues the endosomal trafficking of ßarr1 and ERK1/2 activation for V2RT360A. Molecular dynamics simulations reveal that Ib30 enriches active-like ßarr1 conformation with respect to the inter-domain rotation, and cellular assays demonstrate that it also enhances ßarr1-ß2-adaptin interaction. Our data provide an experimental framework to positively modulate the receptor-transducer-effector axis for GPCRs using intrabodies, which can be potentially integrated in the paradigm of GPCR-targeted drug discovery.

Emerging structural insights into GPCR-ß-arrestin interaction and functional outcomes.

Agonist-induced recruitment of ß-arrestins (ßarrs) to G protein-coupled receptors (GPCRs) plays a central role in regulating the spatio-temporal aspects of GPCR signaling. Several recent studies have provided novel structural and functional insights into our understanding of GPCR-ßarr interaction, subsequent ßarr activation and resulting functional outcomes. In this review, we discuss these recent advances with a particular emphasis on recognition of receptor-bound phosphates by ßarrs, the emerging concept of spatial positioning of key phosphorylation sites, the conformational transition in ßarrs during partial to full-engagement, and structural differences driving functional outcomes of ßarr isoforms. We also highlight the key directions that require further investigation going forward to fully understand the structural mechanisms driving ßarr activation and functional responses.

Intrinsic bias at non-canonical, ß-arrestin-coupled seven transmembrane receptors.

G-protein-coupled receptors (GPCRs), also known as seven transmembrane receptors (7TMRs), typically interact with two distinct signal-transducers, i.e., G proteins and ß-arrestins (ßarrs). Interestingly, there are some non-canonical 7TMRs that lack G protein coupling but interact with ßarrs, although an understanding of their transducer coupling preference, downstream signaling, and structural mechanism remains elusive. Here, we characterize two such non-canonical 7TMRs, namely, the decoy D6 receptor (D6R) and the complement C5a receptor subtype 2 (C5aR2), in parallel with their canonical GPCR counterparts. We discover that D6R and C5aR2 efficiently couple to ßarrs, exhibit distinct engagement of GPCR kinases (GRKs), and activate non-canonical downstream signaling pathways. We also observe that ßarrs adopt distinct conformations for D6R and C5aR2, compared to their canonical GPCR counterparts, in response to common natural agonists. Our study establishes D6R and C5aR2 as ßarr-coupled 7TMRs and provides key insights into their regulation and signaling with direct implication for biased agonism.

Functional competence of a partially engaged GPCR-ß-arrestin complex.

G Protein-coupled receptors (GPCRs) constitute the largest family of cell surface receptors and drug targets. GPCR signalling and desensitization is critically regulated by ß-arrestins (ßarr). GPCR-ßarr interaction is biphasic where the phosphorylated carboxyl terminus of GPCRs docks to the N-domain of ßarr first and then seven transmembrane core of the receptor engages with ßarr. It is currently unknown whether fully engaged GPCR-ßarr complex is essential for functional outcomes or partially engaged complex can also be functionally competent. Here we assemble partially and fully engaged complexes of a chimeric ß2V2R with ßarr1, and discover that the core interaction is dispensable for receptor endocytosis, ERK MAP kinase binding and activation. Furthermore, we observe that carvedilol, a ßarr biased ligand, does not promote detectable engagement between ßarr1 and the receptor core. These findings uncover a previously unknown aspect of GPCR-ßarr interaction and provide novel insights into GPCR signalling and regulatory paradigms.

A conserved tryptophan (W91) at the barrel-lid junction modulates the packing and stability of Kunitz (STI) family of inhibitors.

ß-trefoil fold, consisting of a six stranded ß-barrel capped at one end by a lid comprising of another six ß-strands, is one of the most important folds among proteins. Important classes of proteins like Interleukins (ILs), Fibroblast Growth Factors (FGFs), Kunitz (STI) family of inhibitors etc. belong to this fold. Their core is packed by hydrophobic residues contributed by the 6 stranded ß-barrel and three ß-hairpins that make essential contacts with each other and keep the protein in 'topologically minimal frustrated state'. A complete database analysis of the core residues of the ß-trefoil fold proteins presented here identified a conserved tryptophan (W91) residue in the Kunitz (STI) family of inhibitors that projects from the lid and interacts with the bottom layer residues of the barrel. This kind of interactions is unique in Kunitz (STI) family because no other families of ß-trefoil fold have such a shear sized residue at the barrel lid junction; suggesting its possible importance in packing and stability. We took WCI as a representative of this family and prepared four cavity creating mutants W91F-WCI, W91M-WCI, W91I-WCI & W91A-WCI. CD experiments show that the secondary structure of the mutants remains indistinguishable with the wild type. Crystal structures of the mutants W91F-WCI, W91M-WCI & W91A-WCI also show the same feature. However, slight readjustments of the side chains around the site of mutation have been observed so as to minimize the cavity created due to mutation. Comparative stability of these mutants, estimated using heat denaturation CD spectroscopy, indicates that stability of the mutants inversely correlates with the size of the cavity inside the core. Interestingly, although we mutated at the core, mutants show varying susceptibility against tryptic digestion that grossly follow their instability determined by CD. Our findings suggest that the W91 residue plays an important role in determining the stability and packing of the core of WCI.

Crystallization and preliminary X-ray analysis of a ribokinase from Vibrio cholerae O395.

Ribokinase (RK) is one of the principal enzymes in carbohydrate metabolism, catalyzing the reaction of D-ribose and adenosine triphosphate to produce ribose-5-phosphate and adenosine diphosphate (ADP). To provide further insight into the catalytic mechanism, the rbsK gene from Vibrio cholerae O395 encoding ribokinase was cloned and the protein was overexpressed in Escherichia coli BL21 (DE3) and purified using Ni(2+)-NTA affinity chromatography. Crystals of V. cholerae RK (Vc-RK) and of its complex with ribose and ADP were grown in the presence of polyethylene glycol 6000 and diffracted to 3.4 and 1.75 Šresolution, respectively. Analysis of the diffraction data showed that both crystals possess symmetry consistent with space group P1. In the Vc-RK crystals, 16 molecules in the asymmetric unit were arranged in a spiral fashion, leaving a large empty space inside the crystal, which is consistent with its high Matthews coefficient (3.9 Å(3) Da(-1)) and solvent content (68%). In the Vc-RK co-crystals four molecules were located in the asymmetric unit with a Matthews coefficient of 2.4 Å(3) Da(-1), corresponding to a solvent content of 50%.

Atomic resolution crystal structure of VcLMWPTP-1 from Vibrio cholerae O395: insights into a novel mode of dimerization in the low molecular weight protein tyrosine phosphatase family.

Low molecular weight protein tyrosine phosphatase (LMWPTP) is a group of phosphotyrosine phosphatase ubiquitously found in a wide range of organisms ranging from bacteria to mammals. Dimerization in the LMWPTP family has been reported earlier which follows a common mechanism involving active site residues leading to an enzymatically inactive species. Here we report a novel form of dimerization in a LMWPTP from Vibrio cholera 0395 (VcLMWPTP-1). Studies in solution reveal the existence of the dimer in solution while kinetic study depicts the active form of the enzyme. This indicates that the mode of dimerization in VcLMWPTP-1 is different from others where active site residues are not involved in the process. A high resolution (1.45Å) crystal structure of VcLMWPTP-1 confirms a different mode of dimerization where the active site is catalytically accessible as evident by a tightly bound substrate mimicking ligand, MOPS at the active site pocket. Although being a member of a prokaryotic protein family, VcLMWPTP-1 structure resembles very closely to LMWPTP from a eukaryote, Entamoeba histolytica. It also delineates the diverse surface properties around the active site of the enzyme.

A novel 8-nm protein cage formed by Vibrio cholerae acylphosphatase.

Here we show the formation of an ~8-nm cage formed by the self-assembly of acylphosphatase from Vibrio cholerae O395 (Vc-AcP). The 12-subunit cage structure forms spontaneously and is stabilized through binding of sulfate ions at its exterior face and interfacial regions. Crystal structure and studies in solutions illuminate the basis for the formation of the cage, while a single (Cys20→Arg) mutation (Vc-AcP-C20R) transforms Vc-AcP to a potent enzyme but disrupts the assembly into a trimer.

The moonlighting function of bacteriophage P4 capsid protein, Psu, as a transcription antiterminator.

Psu, a 20-kD bacteriophage P4 capsid decorating protein moonlights as a transcription antiterminator of the Rho-dependent termination. Psu forms specific complex with E.coli Rho protein, and affects the latter's ATP-dependent translocase activity along the nascent RNA. It forms a unique knotted dimer to take a V-shaped structure. The C-terminal helix of Psu makes specific contacts with a disordered region of Rho, encompassing the residues 139-153. An energy minimized structural model of the Rho-Psu complex reveals that the V-shaped Psu dimer forms a lid over the central channel of the Rho hexamer. This configuration of Psu causes a mechanical impediment to the translocase activity of Rho. The knowledge of structural and mechanistic basis of inhibition of Rho action by Psu may help to design peptide inhibitors for the conserved Rho-dependent transcription termination process of bacteria.

Structural and mechanistic basis of anti-termination of Rho-dependent transcription termination by bacteriophage P4 capsid protein Psu.

The conserved bacterial transcription terminator, Rho, is a potent target for bactericidal agents. Psu, a bacteriophage P4 capsid protein, is capable of inducing anti-termination to the Rho-dependent transcription termination. Knowledge of structural and mechanistic basis of this anti-termination is required to design peptide-inhibitor(s) of Rho from Psu. Using suppressor genetics, cross-linking, protein foot-printing and FRET analyses, we describe a conserved disordered structure, encompassing 139-153 amino acids of Rho, as the primary docking site for Psu. Also a neighbouring helical structure, comprising 347-354 amino acids, lining its central channel, plays a supportive role in the Rho-Psu complex formation. Based on the crystal structure of Psu, its conformation in the capsid of the P4 phage, and its interacting regions on Rho, we build an energy-minimized structural model of the Rho:Psu complex. In this model, a V-shaped dimer of Psu interacts with the two diagonally opposite subunits of a hexameric Rho, enabling Psu to form a 'lid' on the central channel of the latter. We show that this configuration of Psu makes the central channel of Rho inaccessible, and it causes a mechanical impediment to its translocase activity.

The first structure of polarity suppression protein, Psu from enterobacteria phage P4, reveals a novel fold and a knotted dimer.

Psu is a capsid decoration protein of bacteriophage P4 and acts as an antiterminator of Rho-dependent transcription termination in bacteria. So far, no structures have been reported for the Psu protein or its homologues. Here, we report the first structure of Psu solved by the Hg(2+) single wavelength anomalous dispersion method, which reveals that Psu exists as a knotted homodimer and is first of its kind in nature. Each monomer of Psu attains a novel fold around a tight coiled-coil motif. CD spectroscopy and the structure of an engineered disulfide-bridged Psu derivative reveal that the protein folds reversibly and reassembles by itself into the knotted dimeric conformation without the requirement of any chaperone. This structure would help to explain the functional properties of the protein and can be used as a template to design a minimal peptide fragment that can be used as a drug against Rho-dependent transcription termination in bacteria.

Cloning, purification, crystallization and preliminary X-ray analysis of two low-molecular-weight protein tyrosine phosphatases from Vibrio cholerae.

Low-molecular-weight protein tyrosine phosphatases (LMWPTPs) are small cytoplasmic enzymes of molecular weight ∼18 kDa that belong to the large family of protein tyrosine phosphatases (PTPs). Despite their wide distribution in both prokaryotes and eukaryotes, their exact biological role in bacterial systems is not yet clear. Two low-molecular-weight protein tyrosine phosphatases (VcLMWPTP-1 and VcLMWPTP-2) from the Gram-negative bacterium Vibrio cholerae have been cloned, overexpressed, purified by Ni(2+)-NTA affinity chromatography followed by gel filtration and used for crystallization. Crystals of VcLMWPTP-1 were grown in the presence of ammonium sulfate and glycerol and diffracted to a resolution of 1.6 Å. VcLMWPTP-2 crystals were grown in PEG 4000 and diffracted to a resolution of 2.7 Å. Analysis of the diffraction data showed that the VcLMWPTP-1 crystals had symmetry consistent with space group P3(1) and that the VcLMWPTP-2 crystals had the symmetry of space group C2. Assuming the presence of four molecules in the asymmetric unit, the Matthews coefficient for the VcLMWPTP-1 crystals was estimated to be 1.97 Å(3) Da(-1), corresponding to a solvent content of 37.4%. The corresponding values for the VcLMWPTP-2 crystals, assuming the presence of two molecules in the asymmetric unit, were 2.77 Å(3) Da(-1) and 55.62%, respectively.

Identification of two differentially expressed mitochondrial genes in a methotrexate-resistant Chinese hamster cell strain derived from v79 cells using RNA fingerprinting by arbitrary primed polymerase chain reaction.

To identify genes that are differentially expressed in a methotrexate (MTX)-resistant cell strain designated as M5 that exhibits resistance to gamma radiation and a number of chemotherapeutic drugs compared to the parental Chinese hamster V79 cells, we used RNA fingerprinting by arbitrary primed polymerase chain reaction (RAP-PCR). By comparative analysis, we identified six differentially expressed transcripts that were cloned and sequenced. Two of these partial cDNA clones showed high homology to the mitochondrial genes NADH dehydrogenase subunit 1 and subunit 4. The steady-state mRNA level of both the NADH dehydrogenase subunits was about twofold higher in the M5 cell strain compared to V79 cells. Moreover, the expression of both the subunits decreased in gamma-irradiated Chinese hamster V79 cells. Cytochrome oxidase, another enzyme of the mitochondrial electron transport chain encoded in the mitochondrial genome, was also found to be overexpressed in M5 cells. All three genes are under the control of the same promoter. However, no amplification of DNA was observed. These data indicate that the alterations in mitochondrial gene expression may be involved in the recovery of irradiated cells, which may arise from transcriptional modulation of the mitochondria from the nucleus.