Laminin, an ∼800-kDa heterotrimeric protein, is a major functional component of the extracellular matrix, contributing to tissue development and maintenance. The unique architecture of laminin is not currently amenable to determination at high resolution, as its flexible and narrow segments complicate both crystallization and single-particle reconstruction by electron microscopy. Therefore, we used cross-linking and MS, evaluated using computational methods, to address key questions regarding laminin quaternary structure. This approach was particularly well suited to the ∼750-Å coiled coil that mediates trimer assembly, and our results support revision of the subunit order typically presented in laminin schematics. Furthermore, information on the subunit register in the coiled coil and cross-links to downstream domains provide insights into the self-assembly required for interaction with other extracellular matrix and cell surface proteins.L aminins are network-forming constituents of the extracellular matrix (ECM) (1, 2). They interact with the cell surface and other ECM components to generate a physical and functional framework affecting cell viability, identity, and activity. The laminin family appears to have arisen during the evolution of multicellularity in animals (3), and laminins contribute to a diversity of basement membrane and connective tissue structures in mammals (2). Laminins are studied in the context of development (4), stem cell biology (5), tissue engineering (6), cancer (7), and aging (8). The remarkable structural organization of laminins underlies their important physiological functions.Laminins are composed of three subunits, α, β, and γ, that assemble into a roughly humanoid form as visualized using rotary shadowing electron microscopy (9). The individual subunits separately form the "head" and two "arms," which are composed of epidermal growth factor (EGF)-like cysteine-rich repeats with globular domains embedded (10) (Fig. 1). Following the head and arms, the three subunits come together to form a long coiled coil, constituting the "body." Additional globular domains unique to the α subunit are the "feet." The laminin head and arms may splay to form a tripod (2), a feature not captured in rotary shadowing images or in diagrams of domain composition. 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).Despite this progress, few insights into the overall 3D architecture of laminin have been made in the past few decades, and certain regions of the complex have been neglected as targets of structural techniques. In particular, no structure has been determined for any segment of th...

The~800 kDa laminin heterotrimer forms a distinctive cross-shaped structure that further self-assembles into networks within the extracellular matrix. The domains at the laminin chain termini, which engage in network formation and cell-surface interaction, are well understood both structurally and functionally. By contrast, the structures and roles of additional domains embedded within the limbs of the laminin cross have remained obscure. Here, we report the X-ray crystal structure, determined to 1.2 A resolution, of the human laminin a2 subunit L4b domain, site of an inframe deletion mutation associated with mild congenital muscular dystrophy. The a2 L4b domain is an irregular b-sandwich with many short and broken strands linked by extended loops. The most similar known structures are the carbohydrate-binding domains of bacterial cellulases, the ephrin-binding domain of ephrin receptors, and MAM adhesion domains in various other eukaryotic cell-surface proteins. This similarity to mammalian adhesion modules, which was not predicted on the basis of amino acid sequence alone due to lack of detectable homology, suggests that laminin internal domains evolved from a progenitor adhesion molecule and may retain a role in cell adhesion in the context of the laminin trimer. DatabaseThe atomic coordinates and structure factors have been deposited in the Protein Data Bank,