Interactions of IGF-II with the IGF2R/cation-independent mannose-6-phosphate receptor mechanism and biological outcomes.

The cation-independent mannose-6-phosphate/insulin-like growth factor-II receptor (IGF2R) is a membrane-bound glycoprotein consisting of 15 homologous extracellular repeat domains. The major function of this receptor is trafficking of lysosomal enzymes from the trans-Golgi network to the endosomes and their subsequent transfer to lysosomes. The IGF2R also plays a major role in binding and regulating the circulating and tissue levels of IGF-II. As this ligand is important for cell growth, survival, and migration, the maintenance of correct IGF-II levels influences its actions in normal growth and development. Deregulation of IGF2R expression has therefore been associated with growth related disease and cancer. This review highlights recent advances in understanding the IGF2R structure and mechanism of interaction with its ligands, in particular IGF-II. Recent mutagenesis studies combined with the crystal structure of domains 11-14 in complex with IGF-II have mapped the sites of interaction and explain how the IGF2R specificity for IGF-II is achieved. The role of domain 13 in high-affinity IGF-II binding is also revealed. Characterization of ligand:IGF2R interactions is vital for the understanding of the mechanism of IGF2R actions and will allow the development of specific cancer therapies in the future.

First steps towards effective methods in exploiting high-throughput technologies for the determination of human protein structures of high biomedical value.

The EC 'Structural Proteomics In Europe' contract is aimed specifically at the atomic resolution structure determination of human protein targets closely linked to health, with a focus on cancer (kinesins, kinases, proteins from the ubiquitin pathway), neurological development and neurodegenerative diseases and immune recognition. Despite the challenging nature of the analysis of such targets, approximately 170 structures have been determined to date. Here, the impact of high-throughput technologies, such as parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens or the use of mass spectrometry to assist sample preparation, on the structural biology of mammalian protein targets is illustrated through selected examples.

Plexin signaling hampers integrin-based adhesion, leading to Rho-kinase independent cell rounding, and inhibiting lamellipodia extension and cell motility.

Plexins encode receptors for semaphorins, molecular signals guiding cell migration, and axon pathfinding. The mechanisms mediating plexin function are poorly understood. Plexin activation in adhering cells rapidly leads to retraction of cellular processes and cell rounding "cell collapse"). Here we show that, unexpectedly, this response does not require the activity of Rho-dependent kinase (ROCK) nor the contraction of F-actin cables. Interestingly, integrin-based focal adhesive structures are disassembled within minutes upon plexin activation; this is followed by actin depolymerization and, eventually, by cellular collapse. We also show that plexin activation hinders cell attachment to adhesive substrates, blocks the extension of lamellipodia, and thereby inhibits cell migration. We conclude that plexin signaling uncouples cell substrate-adhesion from cytoskeletal dynamics required for cell migration and axon extension.

The human low affinity Fcgamma receptors IIa, IIb, and III bind IgG with fast kinetics and distinct thermodynamic properties.

Fcgamma receptors (FcgammaRs) are expressed on all immunologically active cells. They bind the Fc portion of IgG, thereby triggering a range of immunological functions. We have used surface plasmon resonance to analyze the kinetic and thermodynamic properties of the interactions between the ectodomains of human low affinity FcgammaRs (FcgammaRIIa, FcgammaRIIb, and FcgammaRIIIb-NA2) and IgG1 or the Fc fragment of IgG1. All three receptors bind Fc or IgG with similarly low affinities (K(D) approximately 0.6-2.5 microm) and fast kinetics, suggesting that FcgammaR-mediated recognition of aggregated IgG and IgG-coated particles or cells is mechanistically similar to cell-cell recognition. Interestingly, the Fc receptors exhibit distinct thermodynamic properties. Whereas the binding of the FcgammaRIIa and FcgammaRIIb to Fc is driven by favorable entropic and enthalpic changes, the binding of FcgammaRIII is characterized by highly unfavorable entropic changes. Although the structural bases for these differences remain to be determined, they suggest that the molecular events coupled to the binding differ among the low affinity FcgammaRs.

Gene polymorphism in Netherton and common atopic disease.

Atopic dermatitis (AD) and asthma are characterized by IgE-mediated atopic (allergic) responses to common proteins (allergens), many of which are proteinases. Loci influencing atopy have been localized to a number of chromosomal regions, including the chromosome 5q31 cytokine cluster. Netherton disease is a rare recessive skin disorder in which atopy is a universal accompaniment. The gene underlying Netherton disease (SPINK5) encodes a 15-domain serine proteinase inhibitor (LEKTI) which is expressed in epithelial and mucosal surfaces and in the thymus. We have identified six coding polymorphisms in SPINK5 (Table 1) and found that a Glu420-->Lys variant shows significant association with atopy and AD in two independent panels of families. Our results implicate a previously unrecognized pathway for the development of common allergic illnesses.

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.

Crystallization and functional analysis of a soluble deglycosylated form of the human costimulatory molecule B7-1.

The interactions of B7-1 with CD28 and CTLA-4 modulate the course of human immune responses, making B7-1 an important target for developing structure-based therapeutics. B7-1 is, however, one of the most heavily glycosylated proteins found at the leukocyte cell surface, complicating the structural analysis of this molecule. Methods for the production, crystallization and selenomethionine labelling of a soluble deglycosylated form of this molecule are described. The protein readily forms both tetragonal plate and bipyramidal crystals belonging to space groups I4(1)22, with unit-cell parameters a = b = 56.9, c = 298.7 A, and P4(1)22 (or P4(3)22), with unit-cell parameters a = b = 89.0, c = 261.9 A, respectively. The I4(1)22 and primitive crystal forms diffract to 2.7 and 3.5 A, respectively. Surface plasmon resonance-based assays indicate that the ligand-binding properties of sB7-1 are unaffected by deglycosylation. Since none of the methods relied on any special structural properties of sB7-1, it is proposed that this novel combination of procedures could in principle be adapted to the systematic analysis of many other glycoproteins of structural or functional interest.

Reconstitution of antigen presentation in HLA class I-negative cancer cells with peptide-beta2m fusion molecules.

Engineered MHC-peptide targets capable of inducing recognition by CTL may prove useful in designing vaccines for infectious disease and cancer. We tested whether peptides directly linked to beta2-microglobulin (beta2m) could complex with human HLA class I heavy chain, and could be recognized by human CTL, both as soluble reagents and as cell surface constituents. An HLA-A2-restricted peptide epitope was physically linked to the N terminus of human beta2m. This fusion protein refolded efficiently in vitro with HLA-A2 heavy chain, and when multimerized, the resultant complexes ("fusamers") bound specifically to appropriate CTL clones. These fused peptide/MHC complexes were as efficient as standard tetrameric peptide/MHC complexes in recognizing antigen-specific CTL. When the fusion protein was delivered to target cells using a retroviral vector, these cells were recognized and killed by appropriate CTL clones. Efficient sensitization to CTL lysis was achieved in TAP-negative and beta2m-negative cell lines, as well as in unmutated B cell lines, proving that such constructs may be effective in inducing CTL even when the MHC class I pathway has been disrupted. Specific peptides covalently linked to beta2m and delivered via retroviral vectors may be useful reagents for in vivo priming of CTL against epitopes of clinical relevance.

The tumour necrosis factor receptor family: life or death choices.

The past twelve months have seen a renewal of interest in the therapeutic potential of members of the tumour necrosis factor receptor family and their cytokine ligands. This biomedical interest has spawned a number of structural studies, which have significantly deepened our understanding of the molecular basis for the function of these cell-surface signalling systems. The fresh data have revealed unexpected mechanisms conferring ligand-receptor specificity and have highlighted the structural requirements for the initiation of intracellular signalling.

Crystal structure and functional dissection of the cytostatic cytokine oncostatin M.

BACKGROUND: The cytokine oncostatin M (OSM) inhibits growth of certain tumour-derived cells, induces proliferation in other cell types (e.g. haemangioblasts) and is a mediator of inflammatory responses. Its mechanism of action is via specific binding to gp130 and either the leukaemia inhibitory factor receptor (LIFR) or oncostatin M receptor (OSMR) systems at the cell surface to form an active signalling complex. RESULTS: We report here the crystal structure of human oncostatin M (hOSM) along with mutagenesis data which map the receptor-binding epitopes of the molecule. The structure was determined to a resolution of 2.2 A and conforms to the haematopoietin cytokine up-up-down-down four-helix bundle topology. The site 2 epitope, responsible for gp130 binding, is centred around Gly120 which forms a 'dimple' on the surface of the molecule located on helices A and C. The site 3 motif, responsible for LIFR and OSMR binding, consists of a protruding Phe160/Lys163 pair located at the start of helix D. CONCLUSIONS: The data presented allow functional dissection of the receptor-binding interfaces to atomic resolution. Modelling suggests that the gp130 residue Phe169 packs into the site 2 dimple in an analogous fashion to structurally equivalent residues at the growth hormone-growth hormone receptor interface, implying that certain key features may underlie recognition across the whole cytokine/receptor superfamily. Conversely, detailed comparison of the available structures suggests that variations on a common theme dictate the specificity of receptor-ligand interactions within the gp130 family of cytokines.

Nonstandard peptide binding revealed by crystal structures of HLA-B*5101 complexed with HIV immunodominant epitopes.

The crystal structures of the human MHC class I allele HLA-B*5101 in complex with 8-mer, TAFTIPSI, and 9-mer, LPPVVAKEI, immunodominant peptide epitopes from HIV-1 have been determined by x-ray crystallography. In both complexes, the hydrogen-bonding network in the N-terminal anchor (P1) pocket is rearranged as a result of the replacement of the standard tyrosine with histidine at position 171. This results in a nonstandard positioning of the peptide N terminus, which is recognized by B*5101-restricted T cell clones. Unexpectedly, the P5 peptide residues appear to act as anchors, drawing the peptides unusually deeply into the peptide-binding groove of B51. The unique characteristics of P1 and P5 are likely to be responsible for the zig-zag conformation of the 9-mer peptide and the slow assembly of B*5101. A comparison of the surface characteristics in the alpha1-helix C-terminal region for B51 and other MHC class I alleles highlights mainly electrostatic differences that may be important in determining the specificity of human killer cell Ig-like receptor binding.

Definition of peptide binding motifs amongst the HLA-A*30 allelic group.

HLA class I molecules present endogenously processed peptide ligands for surveillance by the T-cell receptor. This potentially immunogenic surface of HLA and peptide is a consequence of the polymorphism found within the HLA molecule and its preference for ligand binding together with peptide conformation within the binding groove. To investigate the relation between the polymorphic differences between some closely related HLA alleles and their effect on peptide preference, transfectants were established, each containing one of four allelic variants of HLA-A*30. Peptides from all four transfectants were eluted, and both individual ligands and peptide pools were sequenced. The data shows two distinct peptide motifs which distinguish A*3001 from the other three known A*30 variants. Differences in preferences at minor positions within the peptide sequence were noted between A*3002, A*3003 and A*3004, providing additional evidence of the implications of sequence polymorphism to HLA function.

Structural basis of cell-cell adhesion by NCAM.

The neural cell adhesion molecule NCAM, a member of the immunoglobulin superfamily, mediates cell-cell recognition and adhesion via a homophilic interaction. NCAM plays a key role during development and regeneration of the nervous system and is involved in synaptic plasticity associated with memory and learning. The 1.85 A crystal structure of the two N-terminal extracellular domains of NCAM reported here provides a structural basis for the homophilic interaction. The molecular packing of the two-domain structure reveals a cross shaped antiparallel dimer, and provides fundamental insight into trans-cellular recognition mediated by NCAM.

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.

Structure and dimerization of a soluble form of B7-1.

B7-1 (CD80) and B7-2 (CD86) are glycoproteins expressed on antigen-presenting cells. The binding of these molecules to the T cell homodimers CD28 and CTLA-4 (CD152) generates costimulatory and inhibitory signals in T cells, respectively. The crystal structure of the extracellular region of B7-1 (sB7-1), solved to 3 A resolution, consists of a novel combination of two Ig-like domains, one characteristic of adhesion molecules and the other previously seen only in antigen receptors. In the crystal lattice, sB7-1 unexpectedly forms parallel, 2-fold rotationally symmetric homodimers. Analytical ultracentrifugation reveals that sB7-1 also dimerizes in solution. The structural data suggest a mechanism whereby the avidity-enhanced binding of B7-1 and CTLA-4 homodimers, along with the relatively high affinity of these interactions, favors the formation of very stable inhibitory signaling complexes.

MHC superfamily structure and the immune system.

During the past year, a plethora of structural information has provided detailed insights into the interactions between classical MHC class I molecules and their cognate receptors on T cells. Likewise, there have been major advances in our knowledge of the structures and functions of five nonclassical MHC-like molecules: HLA-DM (murine H2-M), HLA-E, HFE, ZAG and MIC-A.

Structure of the TRAIL-DR5 complex reveals mechanisms conferring specificity in apoptotic initiation.

TRAIL, an apoptosis inducing ligand, has at least four cell surface receptors including the death receptor DR5. Here we report the crystal structure at 2.2 A resolution of a complex between TRAIL and the extracellular region of DR5. TRAIL forms a central homotrimer around which three DR5 molecules bind. Radical differences in the surface charge of the ligand, together with variation in the alignment of the two receptor domains confer specificity between members of these ligand and receptor families. The existence of a switch mechanism allowing variation in receptor domain alignment may mean that it is possible to engineer receptors with multiple specificities by exploiting contact positions unique to individual receptor-ligand pairs.

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.