Crystal Structure and Cell Surface Anchorage Sites of Laminin α1LG4-5

Abstract

The laminin G-like (LG) domains of laminin-111, a glycoprotein widely expressed during embryogenesis, provide cell anchoring and receptor binding sites that are involved in basement membrane assembly and cell signaling. We now report the crystal structure of the laminin ␣1LG4-5 domains and provide a mutational analysis of heparin, ␣-dystroglycan, and galactosylsulfatide binding. The two domains of ␣1LG4-5 are arranged in a V-shaped fashion similar to that observed with laminin ␣2LG4-5 but with a substantially different interdomain angle. Recombinant ␣1LG4-5 binding to heparin, ␣-dystroglycan, and sulfatides was dependent upon both shared and unique contributions from basic residues distributed in several clusters on the surface of LG4. For heparin, the greatest contribution was detected from two clusters, 2719 Laminin-111, recently renamed from laminin-1 to better reflect its ␣1␤ 1␥1 subunit composition, is one of the first two laminins to be expressed during embryonic development, appearing in the peri-implantation period in the basement membrane of the embryonic plate along with laminin-511 (laminin-10) and in the absence of other laminins in Reichert's membrane (1, 2). Later in development, laminin-111 is strongly expressed in placenta, liver, kidney, and testis, where it is thought to play a role in organogenesis (3). In the adult, the laminin ␣1 chain has very limited expression and is largely supplanted by the laminin ␣5 chain. Targeted inactivation of the LAMA1 gene coding for the ␣1 chain was found to result in a failure of Reichert's membrane with developmental arrest by embryonic day 6.5 in the mouse (4). In-frame deletion of the mouse laminin ␣1 exons corresponding to laminin G-like (LG) 3 domains 4 and 5 was found to result in similar stage lethality accompanied by defective epiblast differentiation without loss of basement membrane (5), the last possibly a result of the partially redundant expression of laminin-511 (4).The ␣1 subunit provides most of the unique characteristics of laminin-111. The N-terminal LN domain participates in polymerization by interacting with the LN domains of the ␤1 and ␥1 chains. The C-terminal LG domains, LG1-3, bind to the ␣ 6 ␤ 1 integrin, whereas LG4-5 bind to heparin, sulfated glycolipids, and ␣-dystroglycan (␣-DG). The polymerization and cell-anchoring activities are thought to act in concert to assemble a functional basement membrane on a cell surface (6).Our earlier understanding of the laminin ␣1 LG1-5 structure was based on crystal structures of LG4 and LG4-5 from the related ␣2 chain (ϳ40% sequence identity). The LG domain fold was revealed as a multistranded ␤-sandwich with one bound calcium ion. In the LG4-5 pair, the two domains are connected in a V-shaped arrangement, in which LG5 is disulfide-bonded to the linker preceding LG4 (7,8). Although the structure of LG1-3 remains to be elucidated, it is thought that these three domains form a closed arrangement with similar angles between domains and separated from the LG4-5 pair by a hinge-like region (9).Analy...

Cross-linking reveals laminin coiled-coil architecture

“…Most of the cross-linked peptides were in the coiled coil, but some were within or between globular domains and in the EGF-like repeats. Crystal structures of laminin fragments (14,15,17,18) were used to assess the quality of the cross-linking results (Fig. 2).…”

“…Due to the centrality of laminin in cell-ECM interactions, efforts have been made to analyze the functional regions of the trimer, to determine which α, β, and γ paralogs associate into physiological heterotrimers and to understand how trimers self-assemble into higher-order networks. Laminin fragments have been generated to assess their binding properties (11,12) and as targets for structure determination by X-ray crystallography (13)(14)(15)(16)(17)(18)(19)(20).…”

Basement Membranes: Cell Scaffoldings and Signaling Platforms

“…Laminins differ based on their complement of domains, ability to polymerize, proteolytic processing, receptor-binding repertoire, and receptor affinities. Relative strong (heavy solid and dashed lines) and weak (thin dashed lines) interactions are indicated with heavy and thin lines with approximate dissociation constants are indicated where known (small numbers in nM values) (Denzer et al 1998;Gesemann et al 1998;Hopf et al 1999;Talts et al 1999;Talts et al 2000;Hopf et al 2001;Nielsen and Yamada 2001;Ries et al 2001;Garbe et al 2002;Smirnov et al 2002;Nishiuchi et al 2006;Harrison et al 2007). …”

“…Binding occurs among the mucinous O-linked carbohydrate chains located in the mid-neck region of a-dystroglycan with the laminin, perlecan, and agrin LG domains. The LG domains bind to sulfatides and heparin in addition to dystroglycan through clusters of lysine and arginine residues present in overlapping surface patches of the b-sandwich globular domains (Wizemann et al 2003;Harrison et al 2007). In some tissues such as Reichert's membrane, breast epithelium, and skeletal muscle, a-dystroglycan may provide the primary anchorage needed for laminin accumulation and basement membrane assembly (Williamson et al 1997;Henry and Campbell 1998;Weir et al 2006).…”

“…A surprising recent set of findings is that the mannose residue can possess an internal phosphoryl linkage insensitive to alkaline phosphatase digestion, that formation of the modified mannose requires the putative glycosyl-transferase LARGE, and that LG-domain binding requires the presence of the phosphoryl group (YoshidaMoriguchi et al 2010). This carbohydrate modification may help explain why dystroglycan, which does not require the terminal sialic acid for binding, interacts with many of the charged lysine and arginine residues in the LG4/5 domains of the laminin a1 and a2 subunits involved in heparin and sulfatide binding (Wizemann et al 2003;Harrison et al 2007).…”