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...