Abstract
An increasing number of surface-exposed ligands and receptors acting on immune cells are being considered as a starting point in drug development applications. As they are dedicated to manipulating a wide range of immune responses, accurately predicting their molecular interactions will be necessary for the development of safe and effective therapeutics to enhance immune responses and vaccination. Here, we focused on the characterization of human CD160 and HVEM immune receptors, whose mutual engagement leads to bidirectional signaling (e.g., T cell inhibition, natural killer cell activation or mucosal immunity). In particular, our study reports on the molecule preparation, characterization and initial crystallographic analysis of the CD160–HVEM complex and both HVEM and CD160 in the absence of their binding partner. Despite the importance of the CD160–HVEM immune signaling and its therapeutic relevance, the structural and mechanistic basis underlying CD160–HVEM engagement has some controversial evidence. On one hand, there are studies reporting on the CD160 molecule in monomeric form that was produced by refolding from bacterial cells, or as a covalently linked single-chain complex with its ligand HVEM in insect cells. On the other hand, there are older reports providing evidence on the multimeric form of CD160 that acts directly on immune cells. In our study, the native non-linked CD160–HVEM complex was co-expressed in the baculovirus insect host, purified to homogeneity by anion-exchange chromatography to provide missing evidence of the trimeric form in solution. Its trimeric existence was also confirmed by the initial crystallographic analysis. The native CD160–HVEM complex crystallized in the orthorhombic space group with unit cell parameters that could accommodate one trimeric complex (3:3) in an asymmetric unit, thus providing ample space for the multimeric form. Crystals of the CD160–HVEM complex, CD160 trimer and HVEM monomer (reported in two space groups) diffracted to a minimum Bragg spacing of 2.8, 3.1 and 1.9/2.1 Å resolution, respectively. The obtained data will lead to elucidating the native structure of the complex.
Highlights
Introduction distributed under the terms andThe complexity of the ligand–receptor interactions of the immediate TNF superfamily (SF) members engaging HVEM provides an unprecedented challenge in defining their mechanism of action and physiologic functions
High-affinity ligand binding induces clustering of receptors expressed in the cognate target cell that in turn initiates signal transduction pathways, culminating in cellular responses
Immune T cell-activating molecules can be divided into TNFRSF (TNF receptor SF) and IgSF
Summary
The complexity of the ligand–receptor interactions of the immediate TNF (tumor necrosis factor) superfamily (SF) members engaging HVEM (herpes virus entry mediator; TNFRSF14) provides an unprecedented challenge in defining their mechanism of action and physiologic functions. High-affinity ligand binding induces clustering of receptors expressed in the cognate target cell that in turn initiates signal transduction pathways, culminating in cellular responses. HVEM was identified as the signaling ligand for CD160 by a proteomics screening of a human B cell cDNA expression library using CD160-Ig [8]. In human NK cells, HVEM can promote cytolytic and pro-inflammatory pathways through CD160 as a host counter measure to human cytomegalovirus (CMV) [9]. In these studies, the viral glycoprotein UL144 (an HVEM orthologue) that mimics HVEM binding was examined for its selectivity for BTLA.
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