RESUMO
CD160 is a signaling molecule that interacts with herpes virus entry mediator (HVEM) and contributes to a wide range of immune responses, including T cell inhibition, natural killer cell activation, and mucosal immunity. GPI-anchored and transmembrane isoforms of CD160 share the same ectodomain responsible for HVEM engagement, which leads to bidirectional signaling. Despite the importance of the CD160:HVEM signaling axis and its therapeutic relevance, the structural and mechanistic basis underlying CD160-HVEM engagement has not been described. We report the crystal structures of the human CD160 extracellular domain and its complex with human HVEM. CD160 adopts a unique variation of the immunoglobulin fold and exists as a monomer in solution. The CD160:HVEM assembly exhibits a 1:1 stoichiometry and a binding interface similar to that observed in the BTLA:HVEM complex. Our work reveals the chemical and physical determinants underlying CD160:HVEM recognition and initiation of associated signaling processes.
Assuntos
Antígenos CD/química , Antígenos CD/metabolismo , Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Membro 14 de Receptores do Fator de Necrose Tumoral/química , Membro 14 de Receptores do Fator de Necrose Tumoral/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em Folha beta , Domínios Proteicos , Dobramento de ProteínaRESUMO
Chronic or persistent stimulation of the programmed cell death-1 (PD-1) pathway prevents T cells from mounting anti-tumor and anti-viral immune responses. Blockade of this inhibitory checkpoint pathway has shown therapeutic importance by rescuing T cells from their exhausted state. Cognate ligands of the PD-1 receptor include the tissue-specific PD-L1 and PD-L2 proteins. Engineering a human PD-1 interface specific for PD-L1 or PD-L2 can provide a specific reagent and therapeutic advantage for tissue-specific disruption of the PD-1 pathway. We utilized ProtLID, a computational framework, which constitutes a residue-based pharmacophore approach, to custom-design a human PD-1 interface specific to human PD-L1 without any significant affinity to PD-L2. In subsequent cell assay experiments, half of all single-point mutant designs proved to introduce a statistically significant selectivity, with nine of these maintaining a close to wild-type affinity to PD-L1. This proof-of-concept study suggests a general approach to re-engineer protein interfaces for specificity.
Assuntos
Antígeno B7-H1/química , Mutação Puntual , Receptor de Morte Celular Programada 1/química , Engenharia de Proteínas , Animais , Antígeno B7-H1/genética , Simulação por Computador , Feminino , Citometria de Fluxo , Células HEK293 , Humanos , Sistema Imunitário , Ligantes , Camundongos , Conformação Molecular , Mutagênese Sítio-Dirigida , Mutação , Receptor de Morte Celular Programada 1/genética , Domínios Proteicos , Mapeamento de Interação de Proteínas , Linfócitos T/metabolismoRESUMO
S100A4, also known as mts1, is a member of the S100 family of Ca2+-binding proteins that is directly involved in tumor invasion and metastasis via interactions with specific protein targets, including nonmuscle myosin-IIA (MIIA). Human S100A4 binds two Ca2+ ions with the typical EF-hand exhibiting an affinity that is nearly 1 order of magnitude tighter than that of the pseudo-EF-hand. To examine how Ca2+ modifies the overall organization and structure of the protein, we determined the 1.7 A crystal structure of the human Ca2+-S100A4. Ca2+ binding induces a large reorientation of helix 3 in the typical EF-hand. This reorganization exposes a hydrophobic cleft that is comprised of residues from the hinge region,helix 3, and helix 4, which afford specific target recognition and binding. The Ca2+-dependent conformational change is required for S100A4 to bind peptide sequences derived from the C-terminal portion of the MIIA rod with submicromolar affinity. In addition, the level of binding of Ca2+ to both EF-hands increases by 1 order of magnitude in the presence of MIIA. NMR spectroscopy studies demonstrate that following titration with a MIIA peptide, the largest chemical shift perturbations and exchange broadening effects occur for residues in the hydrophobic pocket of Ca2+-S100A4. Most of these residues are not exposed in apo-S100A4 and explain the Ca2+ dependence of formation of theS100A4-MIIA complex. These studies provide the foundation for understanding S100A4 target recognition and may support the development of reagents that interfere with S100A4 function.
Assuntos
Cálcio/metabolismo , Miosina não Muscular Tipo IIA/química , Miosina não Muscular Tipo IIA/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteínas S100/química , Proteínas S100/metabolismo , Cristalografia por Raios X , Dimerização , Humanos , Modelos Moleculares , Músculos/química , Músculos/metabolismo , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Quaternária de Proteína , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100/genética , TermodinâmicaRESUMO
S100A4, a member of the S100 family of Ca2+-binding proteins, displays elevated expression in malignant human tumors compared with benign tumors, and increased expression correlates strongly with poor patient survival. S100A4 has a direct role in metastatic progression, likely due to the modulation of actomyosin cytoskeletal dynamics, which results in increased cellular motility. We developed a fluorescent biosensor (Mero-S100A4) that reports on the Ca2+-bound, activated form of S100A4. Direct attachment of a novel solvatochromatic reporter dye to S100A4 results in a sensor that, upon activation, undergoes a 3-fold enhancement in fluorescence, thus providing a sensitive assay for use in vitro and in vivo. In cells, localized activation of S100A4 at the cell periphery is observed during random migration and following stimulation with lysophosphatidic acid, a known activator of cell motility and proliferation. Additionally, a screen against a library of FDA-approved drugs with the biosensor identified an array of phenothiazines as inhibitors of myosin-II associated S100A4 function. These data demonstrate the utility of the new biosensor both for drug discovery and for probing the cellular dynamics controlled by the S100A4 metastasis factor.