Structure of a TCR with high affinity for self-antigen reveals basis for escape from negative selection

The failure to eliminate self-reactive T cells during negative selection is a prerequisite for autoimmunity. To escape deletion, autoreactive T-cell receptors (TCRs) may form unstable complexes with self-peptide-MHC by adopting suboptimal binding topologies compared with anti-microbial TCRs. Alternatively, escape can occur by weak binding between self-peptides and MHC. We determined the structure of a human autoimmune TCR (MS2-3C8) bound to a self-peptide from myelin basic protein (MBP) and the multiple sclerosis-associated MHC molecule HLA-DR4. MBP is loosely accommodated in the HLA-DR4-binding groove, accounting for its low affinity. Conversely, MS2-3C8 binds MBP-DR4 as tightly as the most avid anti-microbial TCRs. MS2-3C8 engages self-antigen via a docking mode that resembles the optimal topology of anti-foreign TCRs, but is distinct from that of other autoreactive TCRs. Combined with a unique CDR3β conformation, this docking mode compensates for the weak binding of MBP to HLA-DR4 by maximizing interactions between MS2-3C8 and MBP. Thus, the MS2-3C8-MBP-DR4 complex reveals the basis for an alternative strategy whereby autoreactive T cells escape negative selection, yet retain the ability to initiate autoimmunity.     

Three-dimensional structural location and molecular functional effects of missense SNPs in the T cell receptor Vbeta domain

The mechanisms by which human single nucleotide polymorphisms (SNPs) influence susceptibility to disease are not yet well understood. In a previous study, we developed a structure-based model that may be used to identify which missense SNPs are neutral and which are deleterious to protein function and so potentially involved in disease (Wang and Moult, Hum Mutat 2001;263-270). The model has now been applied to a set of 54 missense cSNPs in the 46 functional T-cell receptor Vbeta-genes. Most of these missense cSNPs are found to be neutral, but 10 are identified as likely deleterious to protein function. Only one was previously associated with disease. We suggest that the others may be disease related but that redundancy in the T-cell response prevents any simple, monogenic effect. Therefore, these SNPs are the most likely contributors to complex, polygenic disease traits. It has been noted that there is a surprisingly high (74%) fraction of nonsynonymous SNPs in these genes. Contrary to expectation, the analysis shows that these are not associated with an unusually high fraction of deleterious SNPs, nor do they significantly contribute to a larger range of antigen recognition or a reduced superantigen-binding repertoire.     

Unique "delta lock" structure of telmisartan is involved in its strongest binding affinity to angiotensin II type 1 receptor

Angiotensin II type 1 receptor (AT1 receptor) blockers (ARBs) are one of the most popular anti-hypertensive agents. Control of blood pressure (BP) by ARBs is now a therapeutic target for the organ protection in patients with hypertension. Recent meta-analysis demonstrated the possibility that telmisartan was the strongest ARB for the reduction of BP in patients with essential hypertension. However, which molecular interactions of telmisartan with the AT1 receptor could explain its strongest BP lowering activity remains unclear. To address the issue, we constructed models for the interaction between commonly used ARBs and AT1 receptor and compared the docking model of telmisartan with that of other ARBs. Telmisartan has a unique binding mode to the AT1 receptor due to its distal benzimidazole portion. This unique portion could explain the highest molecular lipophilicity, the greatest volume distribution and the strongest binding affinity of telmisartan to AT1 receptor. Furthermore, telmisartan was found to firmly bind to the AT1 receptor through the unique “delta lock” structure. Our present study suggests that due to its “delta lock” structure, telmisartan may be superior to other ARBs in halting cardiovascular disease in patients with hypertension.     

The chemical synthesis and binding affinity to the EGF receptor of the EGF-like domain of heparin-binding EGF-like growth factor (HB-EGF).

Heparin-binding EGF-like growth factor (HB-EGF), which belongs to the EGF-family of growth factors, was isolated from the conditioned medium of macrophage-like cells. To investigate the effect of N- and C-terminal residues of the EGF-like domain of HB-EGF in the binding affinity to the EGF receptor on A431 cell. We synthesized HB-EGF(44-86) corresponding to the EGF-like domain of HB-EGF and its N- or C-terminal truncated peptides. Thermolytic digestion demonstrated three disulfide bond pairings of the EGF-like domain in HB-EGF is consistent with that of human-EGF and human-TGF-alpha. HB-EGF(44-86) showed high binding affinity to EGF-receptor, like human-EGF. The truncation of the C-terminal Leu86 residue from HB-EGF(44-86), HB-EGF(45-86) or HB-EGF(46-86) caused a drastic reduction in the binding affinity to the EGF receptor. These results suggest that the EGF-like domain of HB-EGF plays an important role in the binding to the EGF receptor, and its C-terminal Leu86 residue is necessary for binding with the EGF-receptor. In addition, the deletion of the two N-terminal residues (Asp44-Pro45) from HB-EGF(44-86) caused a 10-fold decrease in relative binding affinity to the EGF receptor. This indicates that the two N-terminal residues of the EGF-like domain of HB-EGF are necessary for its optimal binding affinity to the EGF receptor.     

Investigation of the binding characteristics between ligands and epidermal growth factor receptor by cell membrane chromatography

The binding property between a ligand and its receptor is very important for numerous biological processes. In this study, we developed a high epidermal growth factor receptor (EGFR)‐expression cell membrane chromatography (CMC) method to investigate the binding characteristics between EGFR and the ligands gefitinib, erlotinib, canertinib, afatinib, and vandetanib. Competitive binding analysis using gefitinib as the marker was used to investigate the interactions that occurred at specific binding sites on EGFR. The ability of displacement was measured from the HEK293‐EGFR/CMC column on the binding sites occupied by gefitinib for these ligands, which revealed the following order: gefitinib (K_D, 8.49 ± 0.11 × 10^?7 M) > erlotinib (K_D, 1.07 ± 0.02 × 10^?6 M) > canertinib (K_D, 1.41 ± 0.07 × 10^?6 M) > afatinib (K_D, 1.80 ± 0.12 × 10^?6 M) > vandetanib (K_D, 1.99 ± 0.03 × 10^?6 M). This order corresponded with the values estimated by frontal displacement analysis and the scores obtained with molecular docking. Furthermore, thermodynamic analysis indicated that the hydrogen bond or Van der Waals force was the main interaction force in the process of EGFR binding to all 5 ligands. Overall, these results demonstrate that a CMC method could be an effective tool to investigate the binding characteristics between ligands and receptors.     

Inhibition of the binding of low-density lipoprotein to its cell surface receptor in human fibroblasts by positively charged proteins

A group of proteins and polyamino acids with positively charged domains were shown to inhibit the binding of ¹²⁵I-LDL to its receptor on the surface of human fibroblasts. The list of inhibitory proteins included platelet factor 4 (which has a cluster of lysine residues at its carboxyl terminus), two lysinerich histones, poly-L-lysines of chain length greater than 4, and protamine. These proteins were effective in the concentration range of 5–50 μg/ml. Two other positively charged proteins, lysozyme and avidin, did not inhibit ¹²⁵I-LDL binding. Kinetic studies suggested that protamine was not acting simply as a competitive inhibitor with regard to the LDL receptor. In light of previous data showing that polyanions such as heparin and polyphosphates also inhibit ¹²⁵I-LDL binding to its cell surface receptor, the current findings suggest that charge interactions are important in this binding reaction. In a related series of studies, a number of glycoproteins and their asialo derivatives as well as a number of sugar phosphates failed to inhibit ¹²⁵I-LDL binding to its receptor in fibroblasts.     

The crystal structure of CD8 in complex with YTS156.7.7 Fab and interaction with other CD8 antibodies define the binding mode of CD8 alphabeta to MHC class I

The CD8αβ heterodimer interacts with class I pMHC on antigen-presenting cells as a co-receptor for TCR-mediated activation of cytotoxic T cells. To characterize this immunologically important interaction, we used monoclonal antibodies (mAbs) specific to either CD8α or CD8β to probe the mechanism of CD8αβ binding to pMHCI. The YTS156.7 mAb inhibits this interaction and blocks T cell activation. To elucidate the molecular basis for this inhibition, the crystal structure of the CD8αβ immunoglobulin-like ectodomains were determined in complex with mAb YTS156.7 Fab at 2.7 Å resolution. The YTS156.7 epitope on CD8β was identified and implies that residues in the CDR1 and CDR2-equivalent loops of CD8β are occluded upon binding to class I pMHC. To further characterize the pMHCI/CD8αβ interaction, binding of class I tetramers to CD8αβ on the surface of T cells was assessed in the presence of anti-CD8 mAbs. In contrast to YTS156.7, mAb YTS105.18, which is specific for CD8α, does not inhibit binding of CD8αβ to class I tetramers, indicating the YTS105.18 epitope is not occluded in the pMHCI/CD8αβ complex. Together, these data indicate a model for the pMHCI/CD8αβ interaction similar to that observed for CD8αα in the CD8αα/pMHCI complex, but in which CD8α occupies the lower orientation (membrane proximal to the antigen presenting cell), and CD8β occupies the upper position (membrane distal). The implication of this molecular assembly for the function of CD8αβ in T cell activation is discussed.     

Replacement of lysine-181 by aspartic acid in the third transmembrane region of endothelin type B receptor reduces its affinity to endothelin peptides and sarafotoxin 6c without affecting G protein coupling.

A conserved aspartic acid residue in the third transmembrane region of many of the G protein-coupled receptors has been shown to play a role in ligand binding. In the case of endothelin receptors, however, a lysine residue replaces this conserved aspartic acid residue. To access the importance of this residue in ligand binding, we have replaced it with an aspartic acid in the rat endothelin type B (ETb) receptor by PCR mediated mutagenesis. The binding characteristics and functional properties of both the wild type and mutant receptors were determined in COS-7 cells transiently expressing the cloned receptor cDNAs. Using 125I-ET-1 as the radioactive peptide ligand in displacement binding studies, the wild type receptor displayed a typical non-isopeptide-selective binding profile with similar IC50 values (0.2-0.6 nM) for all three endothelin peptides (ET-1, ET-2, and ET-3) and sarafotoxin 6c (SRTX 6c). Interestingly, the mutant receptor showed an increase in IC50 values for ET-1 (5 nM), ET-2 (27 nM), and ET-3 (127 nM) but displayed a much larger increase in IC50 value for SRTX 6c (> 10 uM). The lysine mutant receptor still elicited full inositol phosphate (IP) turnover responses in the presence of saturating concentrations of endothelins (10 nM of ET-1, 100 nM of ET-2, or 1 uM of ET-3), indicating that the mutation (K181D) did not affect the coupling of mutant receptor to the appropriate G protein. These results demonstrate that lysine-181 on the receptor is important for binding ET peptides; however, it is required for binding the ETb selective agonist-SRTX 6c.     

Binding site and interlobe interactions of the ionotropic glutamate receptor GluK3 ligand binding domain revealed by high resolution crystal structure in complex with (S)-glutamate

Ionotropic glutamate receptors (iGluRs) are involved in excitatory signal transmission throughout the central nervous system and their malfunction is associated with various health disorders. GluK3 is a subunit of iGluRs, belonging to the subfamily of kainate receptors (GluK1–5). Several crystal structures of GluK1 and GluK2 ligand binding domains have been determined in complex with agonists and antagonists. However, little is known about the molecular mechanisms underlying GluK3 ligand binding properties and no compounds displaying reasonable selectivity towards GluK3 are available today. Here, we present the first X-ray crystal structure of the ligand binding domain of GluK3 in complex with glutamate, determined to 1.6 Å resolution. The structure reveals a conserved glutamate binding mode, characteristic for iGluRs, and a water molecule network in the glutamate binding site similar to that seen in GluK1. In GluK3, a slightly lower degree of domain closure around glutamate is observed compared to most other kainate receptor structures with glutamate. The volume of the GluK3 glutamate binding cavity was found to be of intermediate size between those of GluK1 and GluK2. The residues in GluK3 contributing to the subfamily differences in the binding sites are primarily: Thr520, Ala691, Asn722, Leu736 and Thr742. The GluK3 ligand binding domain seems to be less stabilized through interlobe interactions than GluK1 and this may contribute to the faster desensitization kinetics of GluK3.     

Insights revealed by the co‐crystal structure of the Saccharomyces cerevisiae histidine phosphotransfer protein Ypd1 and the receiver domain of its downstream response regulator Ssk1

Two‐component signaling systems are the primary means by which bacteria, archaea, and certain plants and fungi react to their environments. The model yeast, Saccharomyces cerevisiae, uses the Sln1 signaling pathway to respond to hyperosmotic stress. This pathway contains a hybrid histidine kinase (Sln1) that autophosphorylates and transfers a phosphoryl group to its own receiver domain (R1). The phosphoryl group is then transferred to a histidine phosphotransfer protein (Ypd1) that finally passes it to the receiver domain (R2) of a downstream response regulator (Ssk1). Under normal conditions, Ssk1 is constitutively and preferentially phosphorylated in the phosphorelay. Upon detecting hyperosmotic stress, Ssk1 rapidly dephosphorylates and activates the high‐osmolarity glycerol (HOG) pathway, initiating a response. Despite their distinct physiological roles, both Sln1 and Ssk1 bind to Ypd1 at a common docking site. Co‐crystal structures of response regulators in complex with their phosphorelay partners are scarce, leaving many mechanistic and structural details uncharacterized for systems like the Sln1 pathway. In this work, we present the co‐crystal structure of Ypd1 and a near wild‐type variant of the receiver domain of Ssk1 (Ssk1‐R2‐W638A) at a resolution of 2.80 Å. Our structural analyses of Ypd1‐receiver domain complexes, biochemical determination of binding affinities for Ssk1‐R2 variants, in silico free energy estimates, and sequence comparisons reveal distinctive electrostatic properties of the Ypd1/Ssk1‐R2‐W638A complex that may provide insight into the regulation of the Sln1 pathway as a function of dynamic osmolyte concentration.     

Crystal structure of the streptococcal superantigen SpeI and functional role of a novel loop domain in T cell activation by group V superantigens

Superantigens (SAgs) are potent microbial toxins that bind simultaneously to T cell receptors (TCRs) and class II major histocompatibility complex molecules, resulting in the activation and expansion of large T cell subsets and the onset of numerous human diseases. Within the bacterial SAg family, streptococcal pyrogenic exotoxin I (SpeI) has been classified as belonging to the group V SAg subclass, which are characterized by a unique, relatively conserved ∼15 amino acid extension (amino acid residues 154 to 170 in SpeI; herein referred to as the α3-β8 loop), absent in SAg groups I through IV. Here, we report the crystal structure of SpeI at 1.56 Å resolution. Although the α3-β8 loop in SpeI is several residues shorter than that of another group V SAg, staphylococcal enterotoxin serotype I, the C-terminal portions of these loops, which are located adjacent to the putative TCR binding site, are structurally similar. Mutagenesis and subsequent functional analysis of SpeI indicates that TCR β-chains are likely engaged in a similar general orientation as other characterized SAgs. We show, however, that the α3-β8 loop length, and the presence of key glycine residues, are necessary for optimal activation of T cells. Based on Vβ-skewing analysis of human T cells activated with SpeI and structural models, we propose that the α3-β8 loop is positioned to form productive intermolecular contacts with the TCR β-chain, likely in framework region 3, and that these contacts are required for optimal TCR recognition by SpeI, and likely all other group V SAgs.     

Molecular basis for herpesvirus entry mediator recognition by the human immune inhibitory receptor CD160 and its relationship to the cosignaling molecules BTLA and LIGHT

CD160 was recently identified as a T cell coinhibitory molecule that interacts with the herpesvirus entry mediator (HVEM) on antigen-presenting cells to deliver a potent inhibitory signal to CD4⁺ T cells. HVEM also binds to the coinhibitory receptor BTLA (B- and T-lymphocyte attenuator) and the costimulatory receptor LIGHT (which is homologous to lymphotoxins, exhibits inducible expression, and competes with the herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes, or TNFSF14), thus regulating the CD160/BTLA/LIGHT/HVEM signaling pathway. To date, the detailed properties of the formation of these complexes, especially HVEM binding to the newly identified receptor CD160, and the relationship of CD160 with BTLA and LIGHT are still unclear. We performed N-terminal sequencing and a mass spectrometric analysis, which revealed that the extracellular domain of CD160 exists primarily in the monomeric form. The surface plasmon resonance analysis revealed that CD160 binds directly to the cysteine-rich domain 1-3 of HVEM with a similar affinity to, but slower dissociation rate than, that of BTLA. Notably, CD160 competed with BTLA for binding to HVEM; in contrast, LIGHT did not affect HVEM binding to either CD160 or BTLA. The results of a mutagenesis study of HVEM also suggest that the CD160 binding region on HVEM was slightly different from, but overlapped with, the BTLA binding site. Interestingly, an anti-CD160 antibody exhibiting antiangiogenic properties blocked CD160/HVEM binding. These results provide insight into the molecular architecture of the CD160/BTLA/LIGHT/HVEM signaling complex that regulates immune function.     

A large‐scale expression strategy for multimeric extracellular protein complexes using Drosophila S2 cells and its application to the recombinant expression of heterodimeric ligand‐binding domains of taste receptor

Many of the extracellular proteins or extracellular domains of plasma membrane proteins exist or function as homo‐ or heteromeric multimer protein complexes. Successful recombinant production of such proteins is often achieved by co‐expression of the components using eukaryotic cells via the secretory pathway. Here we report a strategy addressing large‐scale expression of hetero‐multimeric extracellular domains of plasma membrane proteins and its application to the extracellular domains of a taste receptor. The target receptor consists of a heterodimer of T1r2 and T1r3 proteins, and their extracellular ligand binding domains (LBDs) are responsible for the perception of major taste substances. However, despite the functional importance, recombinant production of the heterodimeric proteins has so far been unsuccessful. We achieved the successful preparation of the heterodimeric LBD by use of Drosophila S2 cells, which have a high secretory capacity, and by the establishment of a stable high‐expression clone producing both subunits at a comparable level. The method overcame the problems encountered in the conventional transient expression of the receptor protein in insect cells using baculovirus or vector lipofection, which failed in the proper heterodimer production because of the biased expression of T1r3LBD over T1r2LBD. The large‐scale expression methodology reported here may serve as one of the considerable strategies for the preparation of multimeric extracellular protein complexes.     

Single‐nucleotide polymorphism (A118G) in exon 1 of OPRM1 gene causes alteration in downstream signaling by mu‐opioid receptor and may contribute to the genetic risk for addiction

The opioid receptor mu1 (OPRM1) mediates the action of morphine. Although genetic background plays an important role in the susceptibility toward abuse of drugs as evident from familial, adoption and twin studies, association of specific single‐nucleotide polymorphisms of OPRM1 gene with narcotic addiction is to be established. Here, we demonstrate the involvement of A118G polymorphism of exon1 of human OPRM1 gene (hOPRM1), with heroin and alcohol addiction, in a population in eastern India. Statistical analysis exhibited a significant association of G allele with both heroin and alcohol addiction with a risk factor of P_trend < 0.05. The functional significance of G allele in A118G single‐nucleotide polymorphisms was evaluated by studying the regulation of protein kinase A (PKA), pCREB, and pERK1/2 by morphine in Neuro 2A cells, stably transfected with either wild type or A118G mutant hOPRM1. Unlike acute morphine treatment, both chronic morphine exposure and withdrawal precipitated by naloxone were differentially regulated by A118 and G118 receptor isoforms when both PKA and pERK1/2 activities were compared. Results suggest that the association of A118G polymorphism to heroin and alcohol addiction may be because of the altered regulation of PKA and pERK1/2 during opioid and alcohol exposures.