Multimerin-2 is a ligand for group 14 family C-type lectins CLEC14A, CD93 and CD248 spanning the endothelial pericyte interface.

The C-type lectin domain containing group 14 family members CLEC14A and CD93 are proteins expressed by endothelium and are implicated in tumour angiogenesis. CD248 (alternatively known as endosialin or tumour endothelial marker-1) is also a member of this family and is expressed by tumour-associated fibroblasts and pericytes. Multimerin-2 (MMRN2) is a unique endothelial specific extracellular matrix protein that has been implicated in angiogenesis and tumour progression. We show that the group 14 C-type lectins CLEC14A, CD93 and CD248 directly bind to MMRN2 and only thrombomodulin of the family does not. Binding to MMRN2 is dependent on a predicted long-loop region in the C-type lectin domain and is abrogated by mutation within the domain. CLEC14A and CD93 bind to the same non-glycosylated coiled-coil region of MMRN2, but the binding of CD248 occurs on a distinct non-competing region. CLEC14A and CD248 can bind MMRN2 simultaneously and this occurs at the interface between endothelium and pericytes in human pancreatic cancer. A recombinant peptide of MMRN2 spanning the CLEC14A and CD93 binding region blocks CLEC14A extracellular domain binding to the endothelial cell surface as well as increasing adherence of human umbilical vein endothelial cells to the active peptide. This MMRN2 peptide is anti-angiogenic in vitro and reduces tumour growth in mouse models. These findings identify novel protein interactions involving CLEC14A, CD93 and CD248 with MMRN2 as targetable components of vessel formation.

Secondary structure and (1)H, (13)C and (15)N resonance assignments of Skint-1: a selecting ligand for a murine γδ T cell subset implicated in tumour suppression.

A study describing the (1)H, (13)C and (15)N backbone and side chain chemical shift assignments and secondary structure of Skint-1 a prototypic member of a family of mouse genes, of which Skint-1 is involved in the development of the dendritic epidermal T cell (DETC) subset of γδ T cells.

Structures and functions of microbial lipid antigens presented by CD1.

The CD1 family of proteins has evolved to bind a range of endogenous and foreign lipids and present these at the cell surface for antigen-specific recognition by T cells. The distinct intracellular trafficking pathways of CD 1 molecules indicate that collectively, they have the potential to survey the endocytic system widely for antigen, consistent with a role in the presentation of lipids derived from intracellular microbial pathogens. In keeping with this idea, CDla, CDlb, CDlc and CDld have now been shown to present foreign lipid antigens derived from mycobacteria, Gram-negative bacteria and also protozoan species to T cells. These antigens are extremely diverse chemically, and include naturally occurring lipopeptide, glycolipid and phospholipid structures that are distinct from mammalian lipids. CD1-restricted mycobacterial lipids defined to date derive from the highly complex microbial cell envelope. They play a variety of physiological roles for the microbe, including formation of the plasma membrane and protective cell wall and as metabolic intermediates in iron-scavenging pathways. In each case, alkyl chains of CD 1-restricted lipid antigens are accommodated within a deep hydrophobic groove in the membrane-distal alphal-alpha2 domains of the CD1 molecule, with hydrophilic elements solvent-exposed and accessible for recognition by the T cell receptor. Variation in the number, length and saturation of alkyl chains, and the precise chemistry and chirality of the lipid headgroup, clearly exert dominant influences on antigenicity, mediated by effects on CD1 binding and T cell receptor recognition. In the context of structural studies of CD1-lipid complexes, these data suggest that the CD1 isoforms have evolved binding specificities for different classes of foreign lipids, and strongly support a model for antigen recognition involving fine discrimination of lipid headgroup components by the alpha beta T cell receptor. In this review, we summarise our current knowledge of foreign lipid antigens bound by CD 1, focusing on the roles their distinct structural features play in presentation and T cell antigen recognition, and their likely function in antimicrobial T cell responses.

Molecular competition for NKG2D: H60 and RAE1 compete unequally for NKG2D with dominance of H60.

NKG2D is a potent activating receptor on natural killer cells, T cells, and macrophages. Mouse NKG2D interacts with two cell surface ligands related to class I MHC molecules: RAE1 and H60. We used soluble versions of NKG2D, RAE1, and H60 to characterize their interactions. RAE1 and H60 each bind NKG2D with nanomolar affinities, indicating tighter binding than most cell surface immune interactions, but NKG2D binds to H60 with approximately 25-fold higher affinity than to RAE1. RAE1 and H60 compete directly for occupancy of NKG2D, and, thus, NKG2D can be occupied by only one ligand at a time. The NKG2D-H60 interaction is more temperature dependent and makes greater use of electrostatic interactions than the NKG2D-RAE1 interaction. The distinct thermodynamic profiles provide insights into the different molecular mechanisms of the binding interactions.

Cytotoxic T lymphocytes recognize structurally diverse, clade-specific and cross-reactive peptides in human immunodeficiency virus type-1 gag through HLA-B53.

Human immunodeficiency virus type-1 (HIV-1) cytotoxic T lymphocyte (CTL) epitopes have largely been defined in Caucasian populations infected with clade B virus. Identification of potentially protective CTL epitopes in non-B clade-infected African subjects is important for vaccine development. In a study of CTL responses in clade A-infected Gambians, using cytotoxicity, interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISpot) and HLA-B53-peptide tetramer assays, we identified three HLA-B53-restricted epitopes in HIV-1 gag p24. CTL specific for an epitope in a highly immunogenic region of the p24 protein showed no cross-reactivity to other HIV-1 clades. Two of the epitopes would not have been predicted from the peptide-binding motif due to the absence of a proline anchor at position 2. Structural analysis of HLA-B53 and its relative, HLA B35, enabled us to re-define the peptide-binding motif to include other P2 anchors. These results demonstrate the value of combined immunological and structural analyses in defining novel CTL epitopes and have implications for HIV-1 vaccine design.

Complex structure of the activating immunoreceptor NKG2D and its MHC class I-like ligand MICA.

The major histocompatibility complex (MHC) class I homolog, MICA, is a stress-inducible ligand for NKG2D, a C-type lectin-like activating immunoreceptor. The crystal structure of this ligand-receptor complex that we report here reveals an NKG2D homodimer bound to a MICA monomer in an interaction that is analogous to that seen in T cell receptor-MHC class I protein complexes. Similar surfaces on each NKG2D monomer interact with different surfaces on either the alpha1 or alpha2 domains of MICA. The binding interactions are large in area and highly complementary. The central section of the alpha2-domain helix, disordered in the structure of MICA alone, is ordered in the complex and forms part of the NKG2D interface. The extensive flexibility of the interdomain linker of MICA is shown by its altered conformation when crystallized alone or in complex with NKG2D.

Classical and nonclassical class I major histocompatibility complex molecules exhibit subtle conformational differences that affect binding to CD8alphaalpha.

The cell surface molecules CD4 and CD8 greatly enhance the sensitivity of T-cell antigen recognition, acting as "co-receptors" by binding to the same major histocompatibility complex (MHC) molecules as the T-cell receptor (TCR). Here we use surface plasmon resonance to study the binding of CD8alphaalpha to class I MHC molecules. CD8alphaalpha bound the classical MHC molecules HLA-A*0201, -A*1101, -B*3501, and -C*0702 with dissociation constants (K(d)) of 90-220 microm, a range of affinities distinctly lower than that of TCR/peptide-MHC interaction. We suggest such affinities apply to most CD8alphaalpha/classical class I MHC interactions and may be optimal for T-cell recognition. In contrast, CD8alphaalpha bound both HLA-A*6801 and B*4801 with a significantly lower affinity (>/=1 mm), consistent with the finding that interactions with these alleles are unable to mediate cell-cell adhesion. Interestingly, CD8alphaalpha bound normally to the nonclassical MHC molecule HLA-G (K(d) approximately 150 microm), but only weakly to the natural killer cell receptor ligand HLA-E (K(d) >/= 1 mm). Site-directed mutagenesis experiments revealed that variation in CD8alphaalpha binding affinity can be explained by amino acid differences within the alpha3 domain. Taken together with crystallographic studies, these results indicate that subtle conformational changes in the solvent exposed alpha3 domain loop (residues 223-229) can account for the differential ability of both classical and nonclassical class I MHC molecules to bind CD8.

Production of soluble alphabeta T-cell receptor heterodimers suitable for biophysical analysis of ligand binding.

A method to produce alphabeta T-cell receptors (TCRs) in a soluble form suitable for biophysical analysis was devised involving in vitro refolding of a TCR fusion protein. Polypeptides corresponding to the variable and constant domains of each chain of a human and a murine receptor, fused to a coiled coil heterodimerization motif from either c-Jun (alpha) or v-Fos (beta), were overexpressed separately in Escherichia coli. Following recovery from inclusion bodies, the two chains of each receptor were denatured, and then refolded together in the presence of denaturants. For the human receptor, which is specific for the immunodominant influenza A HLA-A2-restricted matrix epitope (M58-66), a heterodimeric protein was purified in milligram yields and found to be homogeneous, monomeric, antibody-reactive, and stable at concentrations lower than 1 microM. Using similar procedures, analogous results were obtained with a murine receptor specific for an influenza nucleoprotein epitope (366-374) restricted by H2-Db. Production of these receptors has facilitated a detailed analysis of viral peptide-Major Histocompatibility Complex (peptide-MHC) engagement by the TCR using both surface plasmon resonance (SPR) and, in the case of the human TCR, isothermal titration calorimetry (ITC) (Willcox et al., 1999). The recombinant methods described should enable a wide range of TCR-peptide-MHC interactions to be studied and may also have implications for the production of other heterodimeric receptor molecules.

TCR binding to peptide-MHC stabilizes a flexible recognition interface.

The binding of TCRs to their peptide-MHC ligands is characterized by a low affinity, slow kinetics, and a high degree of cross-reactivity. Here, we report the results of a kinetic and thermodynamic analysis of two TCRs binding to their peptide-MHC ligands, which reveal two striking features. First, significant activation energy barriers must be overcome during both association and dissociation, suggesting that conformational adjustments are required. Second, the low affinity of binding is a consequence of highly unfavorable entropic effects, indicative of a substantial reduction in disorder upon binding. This is evidence that the TCR and/or peptide-MHC have flexible binding surfaces that are stabilized upon binding. Such conformational flexibility, which may also be a feature of primary antibodies, is likely to contribute to cross-reactivity in antigen recognition.

T cell receptor and coreceptor CD8 alphaalpha bind peptide-MHC independently and with distinct kinetics.

The T cell surface glycoprotein CD8 enhances T cell antigen recognition by binding to MHC class I molecules. We show that human CD8 alphaalpha binds to the MHC class I molecule HLA-A2 with an extremely low affinity (Kd approximately 0.2 mM at 37 degrees C) and with kinetics that are between 2 and 3 orders of magnitude faster than reported for T cell receptor/peptide-MHC interactions. Furthermore, CD8 alphaalpha had no detectable effect on a T cell receptor (TCR) binding to the same peptide-MHC class I complex. These binding properties provide an explanation as to why the CD8/MHC class I interaction is unable to initiate cell-cell adhesion and how it can enhance TCR recognition without interfering with its specificity.

BirA enzyme: production and application in the study of membrane receptor-ligand interactions by site-specific biotinylation.

The enzyme BirA is a key reagent because of its ability to biotinylate proteins at a specific residue in a recognition sequence. We report a rapid, efficient, and economical method for the production, purification, and application of this enzyme. The method is easily scaled up and the protein produced is of high purity and can be stored for many months with retention of activity. We have used this enzyme to biotinylate the C termini of membrane proteins, allowing these proteins to be tetramerized by binding to streptavidin. Because of the specificity of the biotinylation at the C terminus, the orientation of the membrane proteins on the streptavidin is equivalent to that of the native protein on the cell surface. These tetrameric proteins can be used to study protein receptor-ligand interactions at the cell surface, and site-specific biotinylation can be used to study proteins in vitro using a defined orientation.

Structural features impose tight peptide binding specificity in the nonclassical MHC molecule HLA-E.

The crystal structure of the nonclassical human class lb MHC molecule HLA-E has been determined in complex with a prototypic ligand, the nonamer peptide (VMAPRTVLL), derived from the highly conserved residues 3-11 of the human MHC class la leader sequence. The mode of peptide binding retains some of the standard features observed in MHC class la complexes, but novel features imply that HLA-E has evolved to mediate specific binding to a tightly defined set of almost identical hydrophobic peptides from the highly conserved class l leader sequences. These molecular adaptations make HLA-E a rigorous checkpoint at the cell surface reporting on the integrity of the antigen processing pathway to CD94/NKG2 receptor-bearing natural killer cells.

Assembly and crystallization of the complex between the human T cell coreceptor CD8alpha homodimer and HLA-A2.

A strategy for overexpression in Escherichia coli of the extracellular immunoglobulin domain of human CD8alpha was devised using codon usage alterations in the 5' region of the gene, designed so as to prevent the formation of secondary structures in the mRNA. A fragment of CD8alpha, comprising residues 1-120 of the mature protein, excluding the signal peptide and the membrane-proximal stalk region, was recovered from bacterial inclusion bodies and refolded to produce a single species of homodimeric, soluble receptor. HLA-A2 heavy chain, beta2-microglobulin and a synthetic peptide antigen corresponding to the pol epitope from HIV-1 were also expressed in E. coli, refolded and purified. CD8alpha/HLA-A2 complexes were formed in solution and by co-crystallization with a stoichiometry of one CD8alpha alpha dimer to one HLA-A2-peptide unit.

Production, crystallization, and preliminary X-ray analysis of the human MHC class Ib molecule HLA-E.

HLA-E is the first human class Ib major histocompatibility complex molecule to be crystallized. HLA-E is highly conserved and almost nonpolymorphic, and has recently been shown to be the first specialized ligand for natural killer cell receptors. In functional studies, HLA-E is unlike the class Ia MHC molecules in having tightly restricted peptide binding specificity. HLA-E binds a limited set of almost identical leader sequence peptides derived from class Ia molecules and presents these at the cell surface for recognition by natural killer cell receptors. We now show that the extracellular region of HLA-E forms a stable complex with beta2 microglobulin and can be refolded around synthetic peptide. Crystals of this complex formed slowly over four to six months in the presence of ammonium sulphate. The crystals diffract to 2.85 A with space group P3(1)21 and unit cell dimensions a = 182.2 A, b = 182.2 A, c = 88.4 A.

Complexes of HIV-1 reverse transcriptase with inhibitors of the HEPT series reveal conformational changes relevant to the design of potent non-nucleoside inhibitors.

Crystal structures of HIV-1 reverse transcriptase (RT) complexed with a range of chemically diverse non-nucleoside inhibitors (NNIs) have shown a single pocket in which the inhibitors bind and details of the inhibitor-protein interactions. To delineate the structural requirements for an effective inhibitor, we have determined the structures of three closely related NNIs which vary widely in their potencies. Crystal structures of HIV-1 RT complexed with two very potent inhibitors, MKC-442 and TNK-651, at 2.55 angstroms resolution complement our previous analysis of the complex with the less effective inhibitor, HEPT. These structures reveal conformational changes which correlate with changes in potency. We suggest that a major determinant of increased potency in the analogues of HEPT is an improved interaction between residue Tyr181 in the protein and the 6-benzyl ring of the inhibitors which stabilizes the structure of the complex. This arises through a conformational switching of the protein structure triggered by the steric bulk of the 5-substituent of the inhibitor pyrimidine ring.