Lipid-Dependent Activation of the Orphan G Protein-Coupled Receptor, GPR3.

The class A orphan G protein-coupled receptor (GPCR), GPR3, has been implicated in a variety of conditions, including Alzheimer's and premature ovarian failure. GPR3 constitutively couples with Gαs, resulting in the production of cAMP in cells. While tool compounds and several putative endogenous ligands have emerged for the receptor, its endogenous ligand, if it exists, remains a mystery. As novel potential drug targets, the structures of orphan GPCRs have been of increasing interest, revealing distinct modes of activation, including autoactivation, presence of constitutively activating mutations, or via cryptic ligands. Here, we present a cryo-electron microscopy (cryo-EM) structure of the orphan GPCR, GPR3 in complex with DNGαs and Gß1γ2. The structure revealed clear density for a lipid-like ligand that bound within an extended hydrophobic groove, suggesting that the observed "constitutive activity" was likely due to activation via a lipid that may be ubiquitously present. Analysis of conformational variance within the cryo-EM data set revealed twisting motions of the GPR3 transmembrane helices that appeared coordinated with changes in the lipid-like density. We propose a mechanism for the binding of a lipid to its putative orthosteric binding pocket linked to the GPR3 dynamics.

A STAT3 protein complex required for mitochondrial mRNA stability and cancer.

Signal transducer and activator of transcription 3 (STAT3) is a potent transcription factor necessary for life whose activity is corrupted in diverse diseases, including cancer. STAT3 biology was presumed to be entirely dependent on its activity as a transcription factor until the discovery of a mitochondrial pool of STAT3, which is necessary for normal tissue function and tumorigenesis. However, the mechanism of this mitochondrial activity remained elusive. This study uses immunoprecipitation and mass spectrometry to identify a complex containing STAT3, leucine-rich pentatricopeptide repeat containing (LRPPRC), and SRA stem-loop-interacting RNA-binding protein (SLIRP) that is required for the stability of mature mitochondrially encoded mRNAs and transport to the mitochondrial ribosome. Moreover, we show that this complex is enriched in patients with lung adenocarcinoma and that its deletion inhibits the growth of lung cancer in vivo, providing therapeutic opportunities through the specific targeting of the mitochondrial activity of STAT3.

A structural basis for amylin receptor phenotype.

Amylin receptors (AMYRs) are heterodimers of the calcitonin (CT) receptor (CTR) and one of three receptor activity-modifying proteins (RAMPs), AMY1R, AMY2R, and AMY3R. Selective AMYR agonists and dual AMYR/CTR agonists are being developed as obesity treatments; however, the molecular basis for peptide binding and selectivity is unknown. We determined the structure and dynamics of active AMYRs with amylin, AMY1R with salmon CT (sCT), AMY2R with sCT or human CT (hCT), and CTR with amylin, sCT, or hCT. The conformation of amylin-bound complexes was similar for all AMYRs, constrained by the RAMP, and an ordered midpeptide motif that we call the bypass motif. The CT-bound AMYR complexes were distinct, overlapping the CT-bound CTR complexes. Our findings indicate that activation of AMYRs by CT-based peptides is distinct from their activation by amylin-based peptides. This has important implications for the development of AMYR therapeutics.

Epitope length variants balance protective immune responses and viral escape in HIV-1 infection.

Cytotoxic T lymphocyte (CTL) and natural killer (NK) cell responses to a single optimal 10-mer epitope (KK10) in the human immunodeficiency virus type-1 (HIV-1) protein p24Gag are associated with enhanced immune control in patients expressing human leukocyte antigen (HLA)-B∗27:05. We find that proteasomal activity generates multiple length variants of KK10 (4-14 amino acids), which bind TAP and HLA-B∗27:05. However, only epitope forms ≥8 amino acids evoke peptide length-specific and cross-reactive CTL responses. Structural analyses reveal that all epitope forms bind HLA-B∗27:05 via a conserved N-terminal motif, and competition experiments show that the truncated epitope forms outcompete immunogenic epitope forms for binding to HLA-B∗27:05. Common viral escape mutations abolish (L136M) or impair (R132K) production of KK10 and longer epitope forms. Peptide length influences how well the inhibitory NK cell receptor KIR3DL1 binds HLA-B∗27:05 peptide complexes and how intraepitope mutations affect this interaction. These results identify a viral escape mechanism from CTL and NK responses based on differential antigen processing and peptide competition.

Development of AC265347-Inspired Calcium-Sensing Receptor Ago-Positive Allosteric Modulators.

The calcium-sensing receptor (CaSR) is a clinical target in the treatment of hyperparathyroidism and related diseases. However, clinical use of approved CaSR-targeting drugs such as cinacalcet is limited due to adverse side effects including hypocalcaemia, nausea and vomiting, and in some instances, a lack of efficacy. The CaSR agonist and positive allosteric modulator (ago-PAM), AC265347, is chemically distinct from clinically-approved CaSR PAMs. AC265347 potently suppressed parathyroid hormone (PTH) release in rats with a lower propensity to cause hypocalcaemia compared to cinacalcet and may therefore offer benefits over current CaSR PAMs. Here we report a structure activity relationship (SAR) study seeking to optimise AC265347 as a drug candidate and disclose the discovery of AC265347-like compounds with diverse pharmacology and improved physicochemical and drug-like properties.

Therapeutic Opportunities of Targeting Allosteric Binding Sites on the Calcium-Sensing Receptor.

The CaSR is a class C G protein-coupled receptor (GPCR) that acts as a multimodal chemosensor to maintain diverse homeostatic functions. The CaSR is a clinical therapeutic target in hyperparathyroidism and has emerged as a putative target in several other diseases. These include hyper- and hypocalcaemia caused either by mutations in the CASR gene or in genes that regulate CaSR signaling and expression, and more recently in asthma. The development of CaSR-targeting drugs is complicated by the fact that the CaSR possesses many different binding sites for endogenous and exogenous agonists and allosteric modulators. Binding sites for endogenous and exogenous ligands are located throughout the large CaSR protein and are interconnected in ways that we do not yet fully understand. This review summarizes our current understanding of CaSR physiology, signaling, and structure and how the many different binding sites of the CaSR may be targeted to treat disease.

Structure and dynamics of the CGRP receptor in apo and peptide-bound forms.

G protein-coupled receptors (GPCRs) are key regulators of information transmission between cells and organs. Despite this, we have only a limited understanding of the behavior of GPCRs in the apo state and the conformational changes upon agonist binding that lead to G protein recruitment and activation. We expressed and purified unmodified apo and peptide-bound calcitonin gene-related peptide (CGRP) receptors from insect cells to determine their cryo-electron microscopy (cryo-EM) structures, and we complemented these with analysis of protein conformational dynamics using hydrogen-deuterium exchange mass spectrometry and three-dimensional variance analysis of the cryo-EM data. Together with our previously published structure of the active, Gs-bound CGRP receptor complex, our work provides insight into the mechanisms of class B1 GPCR activation.

International Union of Basic and Clinical Pharmacology. CVIII. Calcium-Sensing Receptor Nomenclature, Pharmacology, and Function.

The calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor that responds to multiple endogenous agonists and allosteric modulators, including divalent and trivalent cations, L-amino acids, γ-glutamyl peptides, polyamines, polycationic peptides, and protons. The CaSR plays a critical role in extracellular calcium (Ca2+ o) homeostasis, as demonstrated by the many naturally occurring mutations in the CaSR or its signaling partners that cause Ca2+ o homeostasis disorders. However, CaSR tissue expression in mammals is broad and includes tissues unrelated to Ca2+ o homeostasis, in which it, for example, regulates the secretion of digestive hormones, airway constriction, cardiovascular effects, cellular differentiation, and proliferation. Thus, although the CaSR is targeted clinically by the positive allosteric modulators (PAMs) cinacalcet, evocalcet, and etelcalcetide in hyperparathyroidism, it is also a putative therapeutic target in diabetes, asthma, cardiovascular disease, and cancer. The CaSR is somewhat unique in possessing multiple ligand binding sites, including at least five putative sites for the "orthosteric" agonist Ca2+ o, an allosteric site for endogenous L-amino acids, two further allosteric sites for small molecules and the peptide PAM, etelcalcetide, and additional sites for other cations and anions. The CaSR is promiscuous in its G protein-coupling preferences, and signals via Gq/11, Gi/o, potentially G12/13, and even Gs in some cell types. Not surprisingly, the CaSR is subject to biased agonism, in which distinct ligands preferentially stimulate a subset of the CaSR's possible signaling responses, to the exclusion of others. The CaSR thus serves as a model receptor to study natural bias and allostery. SIGNIFICANCE STATEMENT: The calcium-sensing receptor (CaSR) is a complex G protein-coupled receptor that possesses multiple orthosteric and allosteric binding sites, is subject to biased signaling via several different G proteins, and has numerous (patho)physiological roles. Understanding the complexities of CaSR structure, function, and biology will aid future drug discovery efforts seeking to target this receptor for a diversity of diseases. This review summarizes what is known to date regarding key structural, pharmacological, and physiological features of the CaSR.

Negative allosteric modulators of the human calcium-sensing receptor bind to overlapping and distinct sites within the 7-transmembrane domain.

BACKGROUND AND PURPOSE: Negative allosteric modulators (NAMs) that target the calcium-sensing receptor (CaS receptor) were originally developed for the treatment of osteoporosis by stimulating the release of endogenous parathyroid hormone, but failed in human clinical trials. Several chemically and structurally distinct NAM scaffolds have been described, but it is not known how these different scaffolds interact with the CaS receptor to inhibit receptor signalling in response to agonists. EXPERIMENTAL APPROACH: In the present study, we used a mutagenesis approach combined with analytical pharmacology and computational modelling to probe the binding sites of four distinct NAM scaffolds. KEY RESULTS: Although all four scaffolds bind to the 7-transmembrane and/or extracellular or intracellular loops, they occupy distinct regions, as previously shown for positive allosteric modulators of the CaS receptor. Furthermore, different NAM scaffolds mediate negative allosteric modulation via distinct amino acid networks. CONCLUSION AND IMPLICATIONS: These findings aid our understanding of how different NAMs bind to and inhibit the CaS receptor. Elucidation of allosteric binding sites in the CaS receptor has implications for the discovery of novel allosteric modulators.

Broad CD8+ T cell cross-recognition of distinct influenza A strains in humans.

Newly-emerged and vaccine-mismatched influenza A viruses (IAVs) result in a rapid global spread of the virus due to minimal antibody-mediated immunity. In that case, established CD8+ T-cells can reduce disease severity. However, as mutations occur sporadically within immunogenic IAV-derived T-cell peptides, understanding of T-cell receptor (TCRαß) cross-reactivity towards IAV variants is needed for a vaccine design. Here, we investigate TCRαß cross-strain recognition across IAV variants within two immunodominant human IAV-specific CD8+ T-cell epitopes, HLA-B*37:01-restricted NP338-346 (B37-NP338) and HLA-A*01:01-restricted NP44-52 (A1-NP44). We find high abundance of cross-reactive TCRαß clonotypes recognizing distinct IAV variants. Structures of the wild-type and variant peptides revealed preserved conformation of the bound peptides. Structures of a cross-reactive TCR-HLA-B37-NP338 complex suggest that the conserved conformation of the variants underpins TCR cross-reactivity. Overall, cross-reactive CD8+ T-cell responses, underpinned by conserved epitope structure, facilitates recognition of distinct IAV variants, thus CD8+ T-cell-targeted vaccines could provide protection across different IAV strains.

Identification of Global and Ligand-Specific Calcium Sensing Receptor Activation Mechanisms.

Calcium sensing receptor (CaSR) positive allosteric modulators (PAMs) are therapeutically important. However, few are approved for clinical use, in part due to complexities in assessing allostery at a receptor where the endogenous agonist (extracellular calcium) is present in all biologic fluids. Such complexity impedes efforts to quantify and optimize allosteric drug parameters (affinity, cooperativity, and efficacy) that dictate PAM structure-activity relationships (SARs). Furthermore, an underappreciation of the structural mechanisms underlying CaSR activation hinders predictions of how PAM SAR relates to in vitro and in vivo activity. Herein, we combined site-directed mutagenesis and calcium mobilization assays with analytical pharmacology to compare modes of PAM binding, positive modulation, and agonism. We demonstrate that 3-(2-chlorophenyl)-N-((1R)-1-(3-methoxyphenyl)ethyl)-1-propanamine (NPS R568) binds to a 7 transmembrane domain (7TM) cavity common to class C G protein-coupled receptors and used by (αR)-(-)-α-methyl-N-[3-[3-[trifluoromethylphenyl]propyl]-1-napthalenemethanamine (cinacalcet) and 1-benzothiazol-2-yl-1-(2,4-dimethylphenyl)-ethanol (AC265347); however, there are subtle distinctions in the contribution of select residues to the binding and transmission of cooperativity by PAMs. Furthermore, we reveal some common activation mechanisms used by different CaSR activators, but also demonstrate some differential contributions of residues within the 7TM bundle and extracellular loops to the efficacy of the PAM-agonist, AC265347, versus cooperativity. Finally, we show that PAMS potentiate the affinity of divalent cations. Our results support the existence of both global and ligand-specific CaSR activation mechanisms and reveal that allosteric agonism is mediated in part via distinct mechanisms to positive modulation.

A T Cell Receptor Locus Harbors a Malaria-Specific Immune Response Gene.

Immune response (Ir) genes, originally proposed by Baruj Benacerraf to explain differential antigen-specific responses in animal models, have become synonymous with the major histocompatibility complex (MHC). We discovered a non-MHC-linked Ir gene in a T cell receptor (TCR) locus that was required for CD8+ T cell responses to the Plasmodium berghei GAP5040-48 epitope in mice expressing the MHC class I allele H-2Db. GAP5040-48-specific CD8+ T cell responses emerged from a very large pool of naive Vß8.1+ precursors, which dictated susceptibility to cerebral malaria and conferred protection against recombinant Listeria monocytogenes infection. Structural analysis of a prototypical Vß8.1+ TCR-H-2Db-GAP5040-48 ternary complex revealed that germline-encoded complementarity-determining region 1ß residues present exclusively in the Vß8.1 segment mediated essential interactions with the GAP5040-48 peptide. Collectively, these findings demonstrated that Vß8.1 functioned as an Ir gene that was indispensable for immune reactivity against the malaria GAP5040-48 epitope.

Molecular challenges imposed by MHC-I restricted long epitopes on T cell immunity.

It has widely been accepted that major histocompatibility complex class I molecules (MHC-I) are limited to binding small peptides of 8-10 residues in length. However, this consensus has recently been challenged with the identification of longer peptides (≥11 residues) that can also elicit cytotoxic CD8+ T cell responses. Indeed, a growing number of studies demonstrate that these non-canonical epitopes are important targets for the immune system. As long epitopes represent up to 10% of the peptide repertoire bound to MHC-I molecules, here we review their impact on antigen presentation by MHC-I, TCR recognition, and T cell immunity.

Reversed T Cell Receptor Docking on a Major Histocompatibility Class I Complex Limits Involvement in the Immune Response.

The anti-viral T cell response is drawn from the naive T cell repertoire. During influenza infection, the CD8+ T cell response to an H-2Db-restricted nucleoprotein epitope (NP366) is characterized by preferential expansion of T cells bearing TRBV13+ T cell receptors (TCRs) and avoidance of TRBV17+ T cells, despite the latter dominating the naive precursor repertoire. We found two TRBV17+ TCRs that bound H-2Db-NP366 with a 180° reversed polarity compared to the canonical TCR-pMHC-I docking. The TRBV17 ß-chain dominated the interaction and, whereas the complementarity determining region-3 (CDR3) loops exclusively mediated contacts with the MHC-I, peptide specificity was attributable to germline-encoded recognition. Nevertheless, the TRBV17+ TCR exhibited moderate affinity toward H-2Db-NP366 and was capable of signal transduction. Thus, the naive CD8+ T cell pool can comprise TCRs adopting reversed pMHC-I docking modes that limit their involvement in the immune response.

Lack of Heterologous Cross-reactivity toward HLA-A*02:01 Restricted Viral Epitopes Is Underpinned by Distinct αßT Cell Receptor Signatures.

αßT cell receptor (TCR) genetic diversity is outnumbered by the quantity of pathogenic epitopes to be recognized. To provide efficient protective anti-viral immunity, a single TCR ideally needs to cross-react with a multitude of pathogenic epitopes. However, the frequency, extent, and mechanisms of TCR cross-reactivity remain unclear, with conflicting results on anti-viral T cell cross-reactivity observed in humans. Namely, both the presence and lack of T cell cross-reactivity have been reported with HLA-A*02:01-restricted epitopes from the Epstein-Barr and influenza viruses (BMLF-1 and M158, respectively) or with the hepatitis C and influenza viruses (NS31073 and NA231, respectively). Given the high sequence similarity of these paired viral epitopes (56 and 88%, respectively), the ubiquitous nature of the three viruses, and the high frequency of the HLA-A*02:01 allele, we selected these epitopes to establish the extent of T cell cross-reactivity. We combined ex vivo and in vitro functional assays, single-cell αßTCR repertoire sequencing, and structural analysis of these four epitopes in complex with HLA-A*02:01 to determine whether they could lead to heterologous T cell cross-reactivity. Our data show that sequence similarity does not translate to structural mimicry of the paired epitopes in complexes with HLA-A*02:01, resulting in induction of distinct αßTCR repertoires. The differences in epitope architecture might be an obstacle for TCR recognition, explaining the lack of T cell cross-reactivity observed. In conclusion, sequence similarity does not necessarily result in structural mimicry, and despite the need for cross-reactivity, antigen-specific TCR repertoires can remain highly specific.

Molecular basis for universal HLA-A*0201-restricted CD8+ T-cell immunity against influenza viruses.

Memory CD8(+)T lymphocytes (CTLs) specific for antigenic peptides derived from internal viral proteins confer broad protection against distinct strains of influenza A virus (IAV). However, immune efficacy can be undermined by the emergence of escape mutants. To determine how T-cell receptor (TCR) composition relates to IAV epitope variability, we used ex vivo peptide-HLA tetramer enrichment and single-cell multiplex analysis to compare TCRs targeted to the largely conserved HLA-A*0201-M158and the hypervariable HLA-B*3501-NP418antigens. The TCRαßs for HLA-B*3501-NP418 (+)CTLs varied among individuals and across IAV strains, indicating that a range of mutated peptides will prime different NP418-specific CTL sets. Conversely, a dominant public TRAV27/TRBV19(+)TCRαß was selected in HLA-A*0201(+)donors responding to M158 This public TCR cross-recognized naturally occurring M158variants complexed with HLA-A*0201. Ternary structures showed that induced-fit molecular mimicry underpins TRAV27/TRBV19(+)TCR specificity for the WT and mutant M158peptides, suggesting the possibility of universal CTL immunity in HLA-A*0201(+)individuals. Combined with the high population frequency of HLA-A*0201, these data potentially explain the relative conservation of M158 Moreover, our results suggest that vaccination strategies aimed at generating broad protection should incorporate variant peptides to elicit cross-reactive responses against other specificities, especially those that may be relatively infrequent among IAV-primed memory CTLs.

HIV-1-Specific CD8 T Cells Exhibit Limited Cross-Reactivity during Acute Infection.

Prior work has demonstrated that HIV-1-specific CD8 T cells can cross-recognize variant epitopes. However, most of these studies were performed in the context of chronic infection, where the presence of viral quasispecies makes it difficult to ascertain the true nature of the original antigenic stimulus. To overcome this limitation, we evaluated the extent of CD8 T cell cross-reactivity in patients with acute HIV-1 clade B infection. In each case, we determined the transmitted founder virus sequence to identify the autologous epitopes restricted by individual HLA class I molecules. Our data show that cross-reactive CD8 T cells are infrequent during the acute phase of HIV-1 infection. Moreover, in the uncommon instances where cross-reactive responses were detected, the variant epitopes were poorly recognized in cytotoxicity assays. Molecular analysis revealed that similar antigenic structures could be cross-recognized by identical CD8 T cell clonotypes mobilized in vivo, yet even subtle differences in a single TCR-accessible peptide residue were sufficient to disrupt variant-specific reactivity. These findings demonstrate that CD8 T cells are highly specific for autologous epitopes during acute HIV-1 infection. Polyvalent vaccines may therefore be required to provide optimal immune cover against this genetically labile pathogen.

Interspecies Variation in the Functional Consequences of Mutation of Cytochrome c.

The naturally occurring human cytochrome c variant (G41S) is associated with a mild autosomal dominant thrombocytopenia (Thrombocytopenia Cargeeg) caused by dysregulation of platelet production. The molecular basis of the platelet production defect is unknown. Despite high conservation of cytochrome c between human and mouse (91.4% identity), introducing the G41S mutation into mouse cytochrome c in a knockin mouse (CycsG41S/G41S) did not recapitulate the low platelet phenotype of Thrombocytopenia Cargeeg. While investigating the cause of this disparity we found a lack of conservation of the functional impact of cytochrome c mutations on caspase activation across species. Mutation of cytochrome c at residue 41 has distinct effects on the ability of cytochrome c to activate caspases depending on the species of both the cytochrome c and its binding partner Apaf-1. In contrast to our previous results showing the G41S mutation increases the ability of human cytochrome c to activate caspases, here we find this activity is decreased in mouse G41S cytochrome c. Additionally unlike wildtype human cytochrome c, G41S cytochrome c is unable to activate caspases in Xenopus embryo extracts. Taken together these results demonstrate a previously unreported species-specific component to the interaction of cytochrome c with Apaf-1. This suggests that the electrostatic interaction between cytochrome c and Apaf-1 is not the sole determinant of binding, with additional factors controlling binding specificity and affinity. These results have important implications for studies of the effects of cytochrome c mutations on the intrinsic apoptosis pathway.

Enhancing the peroxidase activity of cytochrome c by mutation of residue 41: implications for the peroxidase mechanism and cytochrome c release.

The peroxidase activity of cytochrome c may play a key role in the release of cytochrome c from the mitochondrial intermembrane space in the intrinsic apoptosis pathway. Induction of the peroxidase activity of cytochrome c is ascribed to partial unfolding and loss of axial co-ordination between the haem Fe and Met80, and is thought to be triggered by interaction of cytochrome c with cardiolipin (diphosphatidylglycerol) in vivo. However, the reaction mechanism for the peroxidase activity of either native or cardiolipin-bound cytochrome c is uncertain. In the present study we analyse the peroxidase activity of human and mouse cytochrome c residue 41 variants and demonstrate that stimulation of peroxidase activity can occur without prior loss of Fe-Met80 co-ordination or partial unfolding. The effects of cardiolipin and mutation of residue 41 are not additive, suggesting that cardiolipin stimulates peroxidase activity by the same mechanism as residue 41 mutation. Consistent with this, mutation of residue 41 did not enhance apoptotic release of cytochrome c from mitochondria. We propose that mutation of residue 41, and interaction with cardiolipin, increase peroxidase activity by altering the 40-57 Ω loop and its hydrogen bond network with the propionate of haem ring A. These changes enhance access of hydrogen peroxide and substrate to the haem.

Conformational change and human cytochrome c function: mutation of residue 41 modulates caspase activation and destabilizes Met-80 coordination.

Cytochrome c is a highly conserved protein, with 20 residues identical in all eukaryotic cytochromes c. Gly-41 is one of these invariant residues, and is the position of the only reported naturally occurring mutation in cytochrome c (human G41S). The basis, if any, for the conservation of Gly-41 is unknown. The mutation of Gly-41 to Ser enhances the apoptotic activity of cytochrome c without altering its role in mitochondrial electron transport. Here we have studied additional residue 41 variants and determined their effects on cytochrome c functions and conformation. A G41T mutation decreased the ability of cytochrome c to induce caspase activation and decreased the redox potential, whereas a G41A mutation had no impact on caspase induction but the redox potential increased. All residue 41 variants decreased the pK (a) of a structural transition of oxidized cytochrome c to the alkaline conformation, and this correlated with a destabilization of the interaction of Met-80 with the heme iron(III) at physiological pH. In reduced cytochrome c the G41T and G41S mutations had distinct effects on a network of hydrogen bonds involving Met-80, and in G41T the conformational mobility of two Ω-loops was altered. These results suggest the impact of residue 41 on the conformation of cytochrome c influences its ability to act in both of its physiological roles, electron transport and caspase activation.