PDBsum entry 2vaj

Cell adhesion

PDB id

2vaj

Contents

			Protein chain

					93 a.a.

			Waters ×6

References listed in PDB file

Key reference

Title

Crystal structure of the ig1 domain of the neural cell adhesion molecule ncam2 displays domain swapping.

Authors

K.K.Rasmussen, N.Kulahin, O.Kristensen, J.C.Poulsen, B.W.Sigurskjold, J.S.Kastrup, V.Berezin, E.Bock, P.S.Walmod, M.Gajhede.

Ref.

J Mol Biol, 2008, 382, 1113-1120. [DOI no: 10.1016/j.jmb.2008.07.084]

PubMed id

18706912

Abstract

The crystal structure of the first immunoglobulin (Ig1) domain of neural cell adhesion molecule 2 (NCAM2/OCAM/RNCAM) is presented at a resolution of 2.7 A. NCAM2 is a member of the immunoglobulin superfamily of cell adhesion molecules (IgCAMs). In the structure, two Ig domains interact by domain swapping, as the two N-terminal beta-strands are interchanged. beta-Strand swapping at the terminal domain is the accepted mechanism of homophilic interactions amongst the cadherins, another class of CAMs, but it has not been observed within the IgCAM superfamily. Gel-filtration chromatography demonstrated the ability of NCAM2 Ig1 to form dimers in solution. Taken together, these observations suggest that beta-strand swapping could have a role in the molecular mechanism of homophilic binding for NCAM2.



	Figure 1. Fig. 1. (a) A cartoon of the structure of the domain-swapped dimer of NCAM2 Ig1. Hinge region residues Leu7-Ser8 and other residues (Val10 and F19) that participate in stabilization of the dimer are shown as red sticks. The NCAM2 Ig1 β-strands are labelled (A, A', B, C, C', D, E, F, and G) according to the nomenclature used for NCAM Ig1.^5 Strand C' is in a perturbed β-strand conformation. (b) A stereo view of the strand-exchange region of the NCAM2 dimer. Omit-map electron density is shown as chickenwire (grey) around the swapped β-strand. The hydrophobic cluster of residues assumed to facilitate domain swapping and to stabilize dimer formation is shown as red sticks. The human Ig1 domain of NCAM2 used in this study was prepared with a C-terminal His tag using PCR amplified cDNA (Ensembl Gene ID ENSG00000154654; RZPD, Germany) for sub-cloning into the ClaI/NotI sites of the pPICZα C plasmid (Invitrogen). The protein consists of two N-terminal residues (Ser and Met) remaining from the cloning procedure, followed by the amino acids (19–114) of human NCAM2 (Swiss-Prot code O15394) and six C-terminal histidine residues. The first four amino acids (SMAL) and the last seven amino acids (KHHHHHH) are not defined by electron density due to flexibility. The construct was verified by DNA sequencing. The recombinant plasmid was linearized with SacI enzyme and used for transformation of Pichia pastoris strain KM71H (Invitrogen). Transformation and selection were performed by the protocol supplied by the manufacturer. A pre-induction culture was grown for 48 h in BMGH medium before transfer and continued growth and induction in BMMH medium for 24 h. The secreted protein was purified using Ni-NTA (Qiagen) affinity chromatography followed by gel-filtration chromatography in phosphate-buffered saline (PBS) on a Superdex 75 column (GE Healthcare). Fractions corresponding to the monomeric form of the protein were collected and stored for two days at 4 °C, and the protein was subsequently used for crystallization experiments. All figures were prepared using the program PyMOL (http://www.pymol.org).		Figure 2. Fig. 2. (a) Superposition of the NCAM2 Ig1 (blue) and NCAM Ig1^5 (yellow) domains shown in stereo view. The β-strands are labelled as in Fig. 1a; residues in the NCAM2 hinge region and residues around the NCAM β-bulge that separates the A/A' β-strands are numbered. The absence of proline and glycine from NCAM2 Ig1 at positions 7 and 10, respectively, may reduce β-bulge stability and contribute to the domain swapping observed for the NCAM2 Ig1 domain. (b) Superposition of the domain-swapped dimer of NCAM2 Ig1 (blue/grey) and the domain-swapped dimer of E-cadherin EC1 (yellow/magenta). Despite the differences between cadherin and Ig domains, the similarity between the domain-swapped dimers is clearly visible.

PROCHECK

	Headers