Reconciling the regulatory role of Munc18 proteins in SNARE-complex assembly.

Membrane fusion is essential for human health, playing a vital role in processes as diverse as neurotransmission and blood glucose control. Two protein families are key: (1) the Sec1p/Munc18 (SM) and (2) the soluble N-ethylmaleimide-sensitive attachment protein receptor (SNARE) proteins. Whilst the essential nature of these proteins is irrefutable, their exact regulatory roles in membrane fusion remain controversial. In particular, whether SM proteins promote and/or inhibit the SNARE-complex formation required for membrane fusion is not resolved. Crystal structures of SM proteins alone and in complex with their cognate SNARE proteins have provided some insight, however, these structures lack the transmembrane spanning regions of the SNARE proteins and may not accurately reflect the native state. Here, we review the literature surrounding the regulatory role of mammalian Munc18 SM proteins required for exocytosis in eukaryotes. Our analysis suggests that the conflicting roles reported for these SM proteins may reflect differences in experimental design. SNARE proteins appear to require C-terminal immobilization or anchoring, for example through a transmembrane domain, to form a functional fusion complex in the presence of Munc18 proteins.

SNARE-ing the structures of Sec1/Munc18 proteins.

Membrane fusion is essential for cellular transport in eukaryotes. Abnormalities contribute to a wide range of diseases including diabetes and neurological disorders. A key regulator of SNARE-mediated membrane fusion is the Sec1/Munc18 (SM) protein family. Universal structural features of SM proteins have been identified that affect the way these interact with their partner Syntaxin SNARE proteins. Whilst the molecular basis for SM-regulated SNARE complex formation has been extensively studied, it remains poorly understood. Recent crystal structures of SM proteins alone or in complex have provided new insight. Here we examine the available structural information on SM proteins for clues to how these enigmatic proteins might regulate SNARE complex assembly and membrane fusion.

Towards selective lysophospholipid GPCR modulators.

G-protein-coupled receptors (GPCRs) that recognize the lysophospholipids (LPLs) are grouped into two phylogenetically distinct families: the endothelial differentiation gene (Edg) and non-Edg GPCRs. Owing to their more recent identification, and hindered by a lack of selective pharmacological tools, our understanding of the functions and signaling pathways of the non-Edg GPCRs is still in its infancy. Targeting the non-conserved allosteric binding sites of the LPL GPCRs shows particular promise for the development of selective modulators by structure-based drug design. However, only one Edg GPCR (S1PR1) structure has been determined to date, and it has low sequence identity with the non-Edg GPCRs (<20%). Thus, a representative structure of a non-Edg GPCR remains a pressing objective for selective structure-based drug design. Obtaining selective modulators targeting the non-Edg receptors would help to unravel the biology behind these novel GPCRs and potentially will support therapeutic treatment of diseases such as cancer, inflammation, and neuropsychiatric disorders.

The α-proteobacteria Wolbachia pipientis protein disulfide machinery has a regulatory mechanism absent in γ-proteobacteria.

The α-proteobacterium Wolbachia pipientis infects more than 65% of insect species worldwide and manipulates the host reproductive machinery to enable its own survival. It can live in mutualistic relationships with hosts that cause human disease, including mosquitoes that carry the Dengue virus. Like many other bacteria, Wolbachia contains disulfide bond forming (Dsb) proteins that introduce disulfide bonds into secreted effector proteins. The genome of the Wolbachia strain wMel encodes two DsbA-like proteins sharing just 21% sequence identity to each other, α-DsbA1 and α-DsbA2, and an integral membrane protein, α-DsbB. α-DsbA1 and α-DsbA2 both have a Cys-X-X-Cys active site that, by analogy with Escherichia coli DsbA, would need to be oxidized to the disulfide form to serve as a disulfide bond donor toward substrate proteins. Here we show that the integral membrane protein α-DsbB oxidizes α-DsbA1, but not α-DsbA2. The interaction between α-DsbA1 and α-DsbB is very specific, involving four essential cysteines located in the two periplasmic loops of α-DsbB. In the electron flow cascade, oxidation of α-DsbA1 by α-DsbB is initiated by an oxidizing quinone cofactor that interacts with the cysteine pair in the first periplasmic loop. Oxidizing power is transferred to the second cysteine pair, which directly interacts with α-DsbA1. This reaction is inhibited by a non-catalytic disulfide present in α-DsbA1, conserved in other α-proteobacterial DsbAs but not in γ-proteobacterial DsbAs. This is the first characterization of the integral membrane protein α-DsbB from Wolbachia and reveals that the non-catalytic cysteines of α-DsbA1 regulate the redox relay system in cooperation with α-DsbB.

Calcitonin and calcitonin receptor-like receptors: common themes with family B GPCRs?

The calcitonin receptor (CTR) and calcitonin receptor-like receptor (CLR) are two of the 15 human family B (or Secretin-like) GPCRs. CTR and CLR are of considerable biological interest as their pharmacology is moulded by interactions with receptor activity-modifying proteins. They also have therapeutic relevance for many conditions, such as osteoporosis, diabetes, obesity, lymphatic insufficiency, migraine and cardiovascular disease. In light of recent advances in understanding ligand docking and receptor activation in both the family as a whole and in CLR and CTR specifically, this review reflects how applicable general family B GPCR themes are to these two idiosyncratic receptors. We review the main functional domains of the receptors; the N-terminal extracellular domain, the juxtamembrane domain and ligand interface, the transmembrane domain and the intracellular C-terminal domain. Structural and functional findings from the CLR and CTR along with other family B GPCRs are critically appraised to gain insight into how these domains may function. The ability for CTR and CLR to interact with receptor activity-modifying proteins adds another level of sophistication to these receptor systems but means careful consideration is needed when trying to apply generic GPCR principles. This review encapsulates current thinking in the realm of family B GPCR research by highlighting both conflicting and recurring themes and how such findings relate to two unusual but important receptors, CTR and CLR.

Structural insights into RAMP modification of secretin family G protein-coupled receptors: implications for drug development.

Secretin family G protein-coupled receptors (GPCRs) are important therapeutic targets for migraine, diabetes, bone disorders, inflammatory disorders and cardiovascular disease. They possess a large N-terminal extracellular domain (ECD) known to be the primary ligand-binding determinant. Structural determination of several secretin family GPCR ECDs in complex with peptide ligands has been achieved recently, providing insight into the molecular determinants of hormone binding. Some secretin family GPCRs associate with receptor activity-modifying proteins (RAMPs), resulting in changes to receptor pharmacology. Recently, the first crystal structure of a RAMP ECD in complex with a secretin family GPCR was solved, revealing the elegant mechanism governing receptor selectivity of small molecule antagonists of the calcitonin gene-related peptide (CGRP) receptor. Here we review the structural basis of ligand binding to secretin family GPCRs, concentrating on recent progress made on the structural basis of RAMP-modified GPCR pharmacology and its implications for rational drug design.

Hard wiring of T cell receptor specificity for the major histocompatibility complex is underpinned by TCR adaptability.

alphabeta T cell receptors (TCRs) are genetically restricted to corecognize peptide antigens bound to self-major histocompatibility complex (pMHC) molecules; however, the basis for this MHC specificity remains unclear. Despite the current dogma, evaluation of the TCR-pMHC-I structural database shows that the nongermline-encoded complementarity-determining region (CDR)-3 loops often contact the MHC-I, and the germline-encoded CDR1 and -2 loops frequently participate in peptide-mediated interactions. Nevertheless, different TCRs adopt a roughly conserved docking mode over the pMHC-I, in which three MHC-I residues (65, 69, and 155) are invariably contacted by the TCR in one way or another. Nonetheless, the impact of mutations at these three positions, either individually or together, was not uniformly detrimental to TCR recognition of pHLA-B*0801 or pHLA-B*3508. Moreover, when TCR-pMHC-I recognition was impaired, this could be partially restored by expression of the CD8 coreceptor. The structure of a TCR-pMHC-I complex in which these three (65, 69, and 155) MHC-I positions were all mutated resulted in shifting of the TCR footprint relative to the cognate complex and formation of compensatory interactions. Collectively, our findings reveal the inherent adaptability of the TCR in maintaining peptide recognition while accommodating changes to the central docking site on the pMHC-I.

To be gammadelta or not to be gammadelta? Signaling pathways in alphabeta versus gammadelta T cell maturation.

Maturation of T cells in the thymus involves input from a number of signaling pathways; their combined input determines whether thymic precursor cells will differentiate into mature alphabeta or gammadelta T cells. This Journal Club article highlights recent research showing that the role of Notch signaling in human T cell maturation differs from that in mice. In mice, reducing Notch gene dosage in vivo promotes gammadelta T cell differentiation. In humans, an increase in Notch activity early in development will trigger gammadelta T cell development. This research emphasizes how the molecular events controlling T cell development are fundamentally different in humans and mice.

Natural micropolymorphism in human leukocyte antigens provides a basis for genetic control of antigen recognition.

Human leukocyte antigen (HLA) gene polymorphism plays a critical role in protective immunity, disease susceptibility, autoimmunity, and drug hypersensitivity, yet the basis of how HLA polymorphism influences T cell receptor (TCR) recognition is unclear. We examined how a natural micropolymorphism in HLA-B44, an important and large HLA allelic family, affected antigen recognition. T cell-mediated immunity to an Epstein-Barr virus determinant (EENLLDFVRF) is enhanced when HLA-B*4405 was the presenting allotype compared with HLA-B*4402 or HLA-B*4403, each of which differ by just one amino acid. The micropolymorphism in these HLA-B44 allotypes altered the mode of binding and dynamics of the bound viral epitope. The structure of the TCR-HLA-B*4405(EENLLDFVRF) complex revealed that peptide flexibility was a critical parameter in enabling preferential engagement with HLA-B*4405 in comparison to HLA-B*4402/03. Accordingly, major histocompatibility complex (MHC) polymorphism can alter the dynamics of the peptide-MHC landscape, resulting in fine-tuning of T cell responses between closely related allotypes.

T cell allorecognition via molecular mimicry.

T cells often alloreact with foreign human leukocyte antigens (HLA). Here we showed the LC13 T cell receptor (TCR), selected for recognition on self-HLA-B( *)0801 bound to a viral peptide, alloreacts with B44 allotypes (HLA-B( *)4402 and HLA-B( *)4405) bound to two different allopeptides. Despite extensive polymorphism between HLA-B( *)0801, HLA-B( *)4402, and HLA-B( *)4405 and the disparate sequences of the viral and allopeptides, the LC13 TCR engaged these peptide-HLA (pHLA) complexes identically, accommodating mimicry of the viral peptide by the allopeptide. The viral and allopeptides adopted similar conformations only after TCR ligation, revealing an induced-fit mechanism of molecular mimicry. The LC13 T cells did not alloreact against HLA-B( *)4403, and the single residue polymorphism between HLA-B( *)4402 and HLA-B( *)4403 affected the plasticity of the allopeptide, revealing that molecular mimicry was associated with TCR specificity. Accordingly, molecular mimicry that is HLA and peptide dependent is a mechanism for human T cell alloreactivity between disparate cognate and allogeneic pHLA complexes.

Human leukocyte antigen class I-restricted activation of CD8+ T cells provides the immunogenetic basis of a systemic drug hypersensitivity.

The basis for strong immunogenetic associations between particular human leukocyte antigen (HLA) class I allotypes and inflammatory conditions like Behçet's disease (HLA-B51) and ankylosing spondylitis (HLA-B27) remain mysterious. Recently, however, even stronger HLA associations are reported in drug hypersensitivities to the reverse-transcriptase inhibitor abacavir (HLA-B57), the gout prophylactic allopurinol (HLA-B58), and the antiepileptic carbamazepine (HLA-B*1502), providing a defined disease trigger and suggesting a general mechanism for these associations. We show that systemic reactions to abacavir were driven by drug-specific activation of cytokine-producing, cytotoxic CD8+ T cells. Recognition of abacavir required the transporter associated with antigen presentation and tapasin, was fixation sensitive, and was uniquely restricted by HLA-B*5701 and not closely related HLA allotypes with polymorphisms in the antigen-binding cleft. Hence, the strong association of HLA-B*5701 with abacavir hypersensitivity reflects specificity through creation of a unique ligand as well as HLA-restricted antigen presentation, suggesting a basis for the strong HLA class I-association with certain inflammatory disorders.

T-cell allorecognition: a case of mistaken identity or déjà vu?

T cells bearing alphabeta T-cell receptors (TCRs) are selected by a subset of peptide-laden major histocompatibility (pMHC) molecules in the thymus and in the periphery and therefore are restricted to recognising host or 'self' MHC molecules. Nevertheless, T cells are inherently cross-reactive and often react with 'foreign' allogeneic MHC molecules (direct T-cell alloreactivity), manifested clinically as organ transplant rejection. Although the basis of T-cell alloreactivity has remained a puzzle to immunologists for decades, studies on alloreactive TCRs have begun to shed light on the basic mechanisms underpinning this 'mistaken identity'. Here we review recent advances in the field, focusing on structural and cellular studies, showing that alloreactivity may sometimes result from cross-reactivity without molecular mimicry and at other times may result directly from TCR interactions with allogeneic pMHC surfaces that mimic the cognate ligand.

Impact of clonal competition for peptide-MHC complexes on the CD8+ T-cell repertoire selection in a persistent viral infection.

CD8(+) T-cell responses to persistent viral infections are characterized by the accumulation of an oligoclonal T-cell repertoire and a reduction in the naive T-cell pool. However, the precise mechanism for this phenomenon remains elusive. Here we show that human cytomegalovirus (HCMV)-specific CD8(+) T cells recognizing distinct epitopes from the pp65 protein and restricted through an identical HLA class I allele (HLA B*3508) exhibited either a highly conserved public T-cell repertoire or a private, diverse T-cell response, which was uniquely altered in each donor following in vitro antigen exposure. Selection of a public T-cell receptor (TCR) was coincident with an atypical major histocompatibility complex (MHC)-peptide structure, in that the epitope adopted a helical conformation that bulged from the peptide-binding groove, while a diverse TCR profile was observed in response to the epitope that formed a flatter, more "featureless" landscape. Clonotypes with biased TCR usage demonstrated more efficient recognition of virus-infected cells, a greater CD8 dependency, and were more terminally differentiated in their phenotype when compared with the T cells expressing diverse TCR. These findings provide new insights into our understanding on how the biology of antigen presentation in addition to the structural features of the pMHC-I might shape the T-cell repertoire and its phenotype.

Polydiscamides B-D from a marine sponge Ircinia sp. as potent human sensory neuron-specific G protein coupled receptor agonists.

Polydiscamides B, C, and D (1-3) were isolated from a sponge Ircinia sp. The structures of 1 to 3 were elucidated by the comparison of their NMR and HRESIMS spectroscopic data with that of a structurally related compound, polydiscamide A. All compounds showed potent agonist activity against human sensory neuron-specific G protein couple receptor (SNSR), a receptor involved in the modulation of pain, and they are the first examples of nonendogenous human SNSR agonists.

T cell allorecognition and MHC restriction--A case of Jekyll and Hyde?

A great paradox in cellular immunology is how T cell allorecognition exists at high frequencies (up to 10%) despite the stringent requirements of discriminating 'self' from 'non-self' imposed by MHC restriction. Thus, in tissue transplantation, a substantial proportion of the recipient's T cells will have the ability to recognize the graft and instigate an immune response against the transplanted tissue, ultimately resulting in graft rejection--a manifestation of T cell alloreactivity. Transplantation of human organs and lymphoid cells as treatment for otherwise life-threatening diseases has become a more routine medical procedure making this problem of great importance. Immunologists have gained important insights into the mechanisms of T cell alloreactivity from cytotoxic T cell assays, affinity-avidity studies, and crystal structures of peptide-MHC (pMHC) molecules and T cell receptors (TCRs) both alone and in complex. Despite the clinical significance of alloreactivity, the crystal structure of an alloreactive human TCR in complex with both cognate pMHC and an allogeneic pMHC complex has yet to be determined. This review highlights some of the important findings from studies characterizing the way in which alloreactive T cell receptors and pMHC molecules interact in an attempt to resolve this great irony of the cellular immune response.

The impact of HLA-B micropolymorphism outside primary peptide anchor pockets on the CTL response to CMV.

The factors controlling epitope selection in the T cell response to persistent viruses are not fully understood, and we have examined this issue in the context of four HLA-B*35-binding peptides from the pp65 antigen of human cytomegalovirus, two of which are previously undescribed. Striking differences in the hierarchy of immunodominance between these four epitopes were observed in healthy virus carriers expressing HLA-B*3501 versus B*3508, two HLA-B allotypes that differ by a single amino acid at position 156 (HLA-B*3501, (156)Leucine; HLA-B*3508, (156)Arginine) that projects from the alpha2 helix into the centre of the peptide-binding groove. While HLA-B*3501(+) individuals responded most strongly to the (123)IPSINVHHY(131) and (366)HPTFTSQY(373) epitopes, HLA-B*3508(+) individuals responded preferentially to (103)CPSQEPMSIYVY(114) and (188)FPTKDVAL(195). By comparing peptide-MHC association and disassociation rates with peptide immunogenicity, it was clear that dissociation rates correlate more closely with the hierarchy of immunodominance among the four pp65 peptides. These findings demonstrate that MHC micropolymorphism at positions outside the primary anchor residue binding pockets can have a major impact on determinant selection in antiviral T cell responses. Such influences may provide the evolutionary pressure that maintains closely related MHC molecules in diverse human populations.

Alloreactivity between disparate cognate and allogeneic pMHC-I complexes is the result of highly focused, peptide-dependent structural mimicry.

Our understanding of the molecular mechanisms of T cell alloreactivity remains limited by the lack of systems for which both the T cell receptor allo- and cognate ligand are known. Here we provide evidence that a single alloreactive T cell receptor interacts with analogous structural regions of its cognate ligand, HLA-B*0801(FLRGRAYGL), as its allogeneic ligand, HLA-B*3501(KPIVVLHGY). The crystal structures of the binary peptide-major histocompatibility complexes show marked differences in the conformation of the heavy chains as well as the bound peptides. Nevertheless, both epitopes possess a prominent solvent-exposed aromatic residue at position 7 flanked by a small glycine at position 8 of the peptide determinant. Moreover, regions of close structural homology between the heavy chains of HLA B8 and HLA B35 coincided with regions that have previously been implicated in "hot spots" of T cell receptor recognition. The avidity of this human T cell receptor was also comparable for the allo- and cognate ligand, consistent with the modes of T cell receptor binding being broadly similar for these complexes. Collectively, it appears that highly focused structural mimicry against a diverse structural background provides a basis for the observed alloreactivity in this system. This cross-reactivity underpins the T cell degeneracy inherent in the limited mature T cell repertoire that must respond to a vast diversity of microbial antigens.

Molecular recognition of sub-micromolar inhibitors by the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase.

The crystal structures of human phenylethanolamine N-methyltransferase in complex with S-adenosyl-l-homocysteine (7, AdoHcy) and either 7-iodo-1,2,3,4-tetrahydroisoquinoline (2) or 8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine (3, LY134046) were determined and compared with the structure of the enzyme complex with 7 and 7-aminosulfonyl-1,2,3,4-tetrahydroisoquinoline (1, SK&F 29661). The enzyme is able to accommodate a variety of chemically disparate functional groups on the aromatic ring of the inhibitors through adaptation of the binding pocket for this substituent and by subtle adjustments of the orientation of the inhibitors within the relatively planar binding site. In addition, the interactions formed by the amine nitrogen of all three inhibitors reinforce the hypothesis that this functional group mimics the beta-hydroxyl of norepinephrine rather than the amine. These studies provide further clues for the development of improved inhibitors for use as pharmacological probes.