Crystal Structure of the V Domain of Human Nectin-like Molecule-1/Syncam3/Tsll1/Igsf4b, a Neural Tissue-specific Immunoglobulin-like Cell-Cell Adhesion Molecule

Tao Chen,Xiaozhong Peng,Boqin Qiang,Jing Gao,Feng Xu,Ying Peng,Peng Lin,Jiangang Yuan,Xiuhua Dong,Zihe Rao,Yanhua Gong

doi:10.1074/jbc.m513459200

Abstract

Nectins are Ca(2+)-independent immunoglobulin (Ig) superfamily proteins that participate in the organization of epithelial and endothelial junctions. Nectins have three Ig-like domains in the extracellular region, and the first one is essential in cell-cell adhesion and plays a central role in the interaction with the envelope glycoprotein D of several viruses. Five Nectin-like molecules (Necl-1 through -5) with similar domain structures to those of Nectins have been identified. Necl-1 is specifically expressed in neural tissue, has Ca(2+)-independent homophilic and heterophilic cell-cell adhesion activity, and plays an important role in the formation of synapses, axon bundles, and myelinated axons. Here we report the first crystal structure of its N-terminal Ig-like V domain at 2.4 A, providing insight into trans-cellular recognition mediated by Necl-1. The protein crystallized as a dimer, and the dimeric form was confirmed by size-exclusion chromatography and chemical cross-linking experiments, indicating this V domain is sufficient for homophilic interaction. Mutagenesis work demonstrated that Phe(82) is a key residue for the adhesion activity of Necl-1. A model for homophilic adhesion of Necl-1 at synapses is proposed based on its structure and previous studies.

Highlights

The first Ig-like domain is necessary and sufficient for the formation of homo-trans-dimers, whereas the second Ig-like domain is necessary for the formation of homo-cis-dimers [5, 7,8,9]
The protein was crystallized as a dimer, and a dimeric form was observed in solution by size-exclusion chromatography and chemical cross-linking experiments, suggesting the first Ig-like domain of Nectin-like molecules (Necls)-1 is sufficient for the homophilic interaction
Based on the crystal structure and previous studies, we propose a V to V model for homophilic adhesion of Necl-1 at synapses

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—The human necl-1 gene was identified from a large-scale sequencing of human brain. The L-SeMet-labeled Ig-like V domain protein was expressed in E. coli strain BL21(DE3). The L-SeMet-labeled Ig-like domain protein was purified and stored under the same condition as the native protein. Chemical Cross-linking of the Ig-like V Domain Protein—The gelfiltration purified proteins (including wild-type and mutant proteins) were concentrated to ϳ2 mg/ml by ultrafiltration (5-kDa cut-off). Crystallization and Data Collection—The protein solutions of Ig-like V domain of Necl-1 and L-SeMet-labeled protein used for crystallization contained 20 mM HEPES, pH 7.5, 100 mM NaCl, and 20 mg mlϪ1 protein. Multiwavelength anomalous diffraction (MAD) data for L-SeMetlabeled Ig-like V domain protein were collected using radiation of wavelengths 0.9788, 0.9793, and 0.9700 Å on beamline 3W1A at the Beijing Synchrotron Radiation Facility. MAD phasing and refinement statistics Numbers in parentheses correspond to the highest resolution shell

Unique reflections

RESULTS

Atom from monomer B

DISCUSSION