Crystal Structure of the LG1-3 Region of the Laminin α2 Chain

Federico Carafoli,Naomi J Clout,Erhard Hohenester

doi:10.1074/jbc.m109.026658

Federico Carafoli, Naomi J Clout + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m109.026658

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Abstract

Laminins are large heterotrimeric glycoproteins with many essential functions in basement membrane assembly and function. Cell adhesion to laminins is mediated by a tandem of five laminin G-like (LG) domains at the C terminus of the α chain. Integrin binding requires an intact LG1-3 region, as well as contributions from the coiled coil formed by the α, β, and γ chains. We have determined the crystal structure at 2.8-Å resolution of the LG1-3 region of the laminin α2 chain (α2LG1-3). The three LG domains adopt typical β-sandwich folds, with canonical calcium binding sites in LG1 and LG2. LG2 and LG3 interact through a substantial interface, but LG1 is completely dissociated from the LG2-3 pair. We suggest that the missing γ chain tail may be required to stabilize the interaction between LG1 and LG2-3 in the biologically active conformation. A global analysis of N-linked glycosylation sites shows that the β-sandwich faces of LG1 are free of carbohydrate modifications in all five laminin α chains, suggesting that these surfaces may harbor the integrin binding site. The α2LG1-3 structure provides the first atomic view of the integrin binding region of laminins.

Highlights

The laminins constitute a major class of cell-adhesive glycoproteins that are intimately involved in basement membrane assembly and function
The LG1-3 region of laminin ␣ chains is necessary for integrin binding, but it is not sufficient: several studies have shown that the coiled coil formed by the three laminin chains and, in particular, a glutamic acid in the third position from the C terminus of the ␥ chain are required [13,14,15,16,17]
We previously proposed a model for LG1-3, in which the three LG domains are arranged like the leaves of a clover, forming three distinct interfaces between the constituent domains [7]

Summary

EXPERIMENTAL PROCEDURES

Expression Construct—DNA coding for residues 2136 –2746 (QANSI . . . MVHGP) of the mouse laminin ␣2 chain was obtained by PCR amplification from a cDNA kindly provided by Takako Sasaki. Protein Production—The laminin ␣2LG1-3 protein was purified from the conditioned medium of episomally transfected 293-EBNA cells. The conditioned medium was loaded onto a 5-ml HisTrap column (GE Healthcare) and the ␣2LG1-3 protein eluted with 0.5 M imidazole in Tris-buffered saline buffer pH 7.4 supplemented with 5 mM calcium (TBSCa). After cautious rebuilding with O [29] and refinement with CNS [30], the improved LG1-2 model gave a translation Z-score of 31.4 in PHASER, and LG3 could be located in a partial translation function using a partial poly-Ala/Ser model of laminin ␣2LG5 [31] (translation Z-score of 13.8, Rfree ϭ 0.476). The coordinates of the ␣2LG1-3 structure have been deposited in the Protein Data Bank (entry code: 2wjs)

RESULTS

Å from the carboxylate group of

DISCUSSION