Rotem Rubinstein

VISTA, a novel mouse Ig superfamily ligand that negatively regulates T cell responses

VISTA suppresses T cell proliferation and cytokine production and can influence autoimmunity and antitumor responses in mice.

Single-Cell Identity Generated by Combinatorial Homophilic Interactions between α, β, and γ Protocadherins

Individual mammalian neurons stochastically express distinct repertoires of α, β, and γ protocadherin (Pcdh) proteins, which function in neural circuit assembly. We report that all three subfamilies of clustered Pcdhs can engage in specific homophilic interactions, that cell surface delivery of Pcdhα isoforms requires cis interactions with other Pcdhs, and that the extracellular cadherin domain EC6 plays a critical role in this process. Examination of homophilic interactions between specific combinations of multiple Pcdh isoforms revealed that Pcdh combinatorial recognition specificities depend on the identity of all of the expressed isoforms. A single mismatched Pcdh isoform can interfere with these combinatorial homophilic interactions. A theoretical analysis reveals that assembly of Pcdh isoforms into multimeric recognition units and the observed tolerance for mismatched isoforms can generate cell surface diversity sufficient for single-cell identity. However, the competing demands of nonself discrimination and self-recognition place limitations on the mechanisms by which homophilic recognition units can function.

Sequence, structure, function, immunity: Structural genomics of costimulation

Summary:  Costimulatory receptors and ligands trigger the signaling pathways that are responsible for modulating the strength, course, and duration of an immune response. High‐resolution structures have provided invaluable mechanistic insights by defining the chemical and physical features underlying costimulatory receptor:ligand specificity, affinity, oligomeric state, and valency. Furthermore, these structures revealed general architectural features that are important for the integration of these interactions and their associated signaling pathways into overall cellular physiology. Recent technological advances in structural biology promise unprecedented opportunities for furthering our understanding of the structural features and mechanisms that govern costimulation. In this review, we highlight unique insights that have been revealed by structures of costimulatory molecules from the immunoglobulin and tumor necrosis factor superfamilies and describe a vision for future structural and mechanistic analysis of costimulation. This vision includes simple strategies for the selection of candidate molecules for structure determination and highlights the critical role of structure in the design of mutant costimulatory molecules for the generation of in vivo structure–function correlations in a mammalian model system. This integrated ‘atoms‐to‐animals’ paradigm provides a comprehensive approach for defining atomic and molecular mechanisms.

Molecular Logic of Neuronal Self-Recognition through Protocadherin Domain Interactions.

Self-avoidance, a process preventing interactions of axons and dendrites from the same neuron during development, is mediated in vertebrates through the stochastic single-neuron expression of clustered protocadherin protein isoforms. Extracellular cadherin (EC) domains mediate isoform-specific homophilic binding between cells, conferring cell recognition through a poorly understood mechanism. Here, we report crystal structures for the EC1-EC3 domain regions from four protocadherin isoforms representing the α, β, and γ subfamilies. All are rod shaped and monomeric in solution. Biophysical measurements, cell aggregation assays, and computational docking reveal that trans binding between cells depends on the EC1-EC4 domains, which interact in an antiparallel orientation. We also show that the EC6 domains are required for the formation of cis-dimers. Overall, our results are consistent with a model in which protocadherin cis-dimers engage in a head-to-tail interaction between EC1-EC4 domains from apposed cell surfaces, possibly forming a zipper-like protein assembly, and thus providing a size-dependent self-recognition mechanism.

Predicting disulfide bond connectivity in proteins by correlated mutations analysis

MOTIVATION Prediction of disulfide bond connectivity facilitates structural and functional annotation of proteins. Previous studies suggest that cysteines of a disulfide bond mutate in a correlated manner. RESULTS We developed a method that analyzes correlated mutation patterns in multiple sequence alignments in order to predict disulfide bond connectivity. Proteins with known experimental structures and varying numbers of disulfide bonds, and that spanned various evolutionary distances, were aligned. We observed frequent variation of disulfide bond connectivity within members of the same protein families, and it was also observed that in 99% of the cases, cysteine pairs forming non-conserved disulfide bonds mutated in concert. Our data support the notion that substitution of a cysteine in a disulfide bond prompts the substitution of its cysteine partner and that oxidized cysteines appear in pairs. The method we developed predicts disulfide bond connectivity patterns with accuracies of 73, 69 and 61% for proteins with two, three and four disulfide bonds, respectively.

Structure of Nectin-2 reveals determinants of homophilic and heterophilic interactions that control cell–cell adhesion

Nectins are members of the Ig superfamily that mediate cell–cell adhesion through homophilic and heterophilic interactions. We have determined the crystal structure of the nectin-2 homodimer at 1.3 Å resolution. Structural analysis and complementary mutagenesis studies reveal the basis for recognition and selectivity among the nectin family members. Notably, the close proximity of charged residues at the dimer interface is a major determinant of the binding affinities associated with homophilic and heterophilic interactions within the nectin family. Our structural and biochemical data provide a mechanistic basis to explain stronger heterophilic versus weaker homophilic interactions among these family members and also offer insights into nectin-mediated transinteractions between engaging cells.

Structural Basis of Diverse Homophilic Recognition by Clustered α- and β-Protocadherins.

Clustered protocadherin proteins (α-, β-, and γ-Pcdhs) provide a high level of cell-surface diversity to individual vertebrate neurons, engaging in highly specific homophilic interactions to mediate important roles in mammalian neural circuit development. How Pcdhs bind homophilically through their extracellular cadherin (EC) domains among dozens of highly similar isoforms has not been determined. Here, we report crystal structures for extracellular regions from four mouse Pcdh isoforms (α4, α7, β6, and β8), revealing a canonical head-to-tail interaction mode for homophilic trans dimers comprising primary intermolecular EC1:EC4 and EC2:EC3 interactions. A subset of trans interface residues exhibit isoform-specific conservation, suggesting roles in recognition specificity. Mutation of these residues, along with trans-interacting partner residues, altered the specificities of Pcdh interactions. Together, these data show how sequence variation among Pcdh isoforms encodes their diverse strict homophilic recognition specificities, which are required for their key roles in neural circuit assembly.

Functional Classification of Immune Regulatory Proteins

The members of the immunoglobulin superfamily (IgSF) control innate and adaptive immunity and are prime targets for the treatment of autoimmune diseases, infectious diseases, and malignancies. We describe a computational method, termed the Brotherhood algorithm, which utilizes intermediate sequence information to classify proteins into functionally related families. This approach identifies functional relationships within the IgSF and predicts additional receptor-ligand interactions. As a specific example, we examine the nectin/nectin-like family of cell adhesion and signaling proteins and propose receptor-ligand interactions within this family. Guided by the Brotherhood approach, we present the high-resolution structural characterization of a homophilic interaction involving the class-I MHC-restricted T-cell-associated molecule, which we now classify as a nectin-like family member. The Brotherhood algorithm is likely to have a significant impact on structural immunology by identifying those proteins and complexes for which structural characterization will be particularly informative.

γ-Protocadherin structural diversity and functional implications

Stochastic cell-surface expression of α-, β-, and γ-clustered protocadherins (Pcdhs) provides vertebrate neurons with single-cell identities that underlie neuronal self-recognition. Here we report crystal structures of ectodomain fragments comprising cell-cell recognition regions of mouse γ-Pcdhs γA1, γA8, γB2, and γB7 revealing trans-homodimers, and of C-terminal ectodomain fragments from γ-Pcdhs γA4 and γB2, which depict cis-interacting regions in monomeric form. Together these structures span the entire γ-Pcdh ectodomain. The trans-dimer structures reveal determinants of γ-Pcdh isoform-specific homophilic recognition. We identified and structurally mapped cis-dimerization mutations to the C-terminal ectodomain structures. Biophysical studies showed that Pcdh ectodomains from γB-subfamily isoforms formed cis dimers, whereas γA isoforms did not, but both γA and γB isoforms could interact in cis with α-Pcdhs. Together, these data show how interaction specificity is distributed over all domains of the γ-Pcdh trans interface, and suggest that subfamily- or isoform-specific cis-interactions may play a role in the Pcdh-mediated neuronal self-recognition code. DOI: http://dx.doi.org/10.7554/eLife.20930.001

Structural, mutational and biophysical studies reveal a canonical mode of molecular recognition between immune receptor TIGIT and nectin-2

HighlightsT‐cell stimulatory and inhibitory signals control mammalian immune system.TIGIT:nectin‐2 interaction delivers inhibitory signals to T cell.This study reveals structural and biochemical basis of TIGIT:nectin‐2 recognition.A distinctive “lock‐and‐key” mechanism supports this molecular interaction.This study provides basis for rational manipulation of TIGIT:nectin2 interaction. ABSTRACT In addition to antigen‐specific stimulation of T cell receptor (TCR) by a peptide‐MHC complex, the functional outcome of TCR engagement is regulated by antigen‐independent costimulatory signals. Costimulatory signals are provided by an array of interactions involving activating and inhibitory receptors expressed on T cells and their cognate ligands on antigen presenting cells. T cell immunoglobulin and ITIM domain (TIGIT), a recently identified immune receptor expressed on T and NK cells, upon interaction with either of its two ligands, nectin‐2 or poliovirus receptor (PVR), inhibits activation of T and NK cells. Here we report the crystal structure of the human TIGIT ectodomain, which exhibits the classic two‐layer &bgr;‐sandwich topology observed in other immunoglobulin super family (IgSF) members. Biophysical studies indicate that TIGIT is monomeric in solution but can form a dimer at high concentrations, consistent with the observation of a canonical immunoglobulin‐like dimer interface in the crystalline state. Based on existing structural data, we present a model of the TIGIT:nectin‐2 complex and utilized complementary biochemical studies to map the nectin‐binding interface on TIGIT. Our data provide important structural and biochemical determinants responsible for the recognition of nectin‐2 by TIGIT. Defining the TIGIT:nectin‐2 binding interface provides the basis for rational manipulation of this molecular interaction for the development of immunotherapeutic reagents in autoimmunity and cancer.