Cross-linking reveals laminin coiled-coil architecture

Abstract

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...

Crystal Structure of the Heterotrimeric Integrin-Binding Region of Laminin-111

“…When the coiled coil is viewed from its C terminus, α1, β1, and γ1 are arranged in a counterclockwise order. Our mini-E8 structure thus confirms the register and chain order proposed in the recent crosslinking study (Armony et al., 2016). Sequence comparison shows that the very C terminus of the coiled coil is conserved in all laminin isoforms (Figure 2).…”

“…The mini-E8 structure is in excellent agreement with a recent crosslinking study of laminin-111 (Armony et al., 2016). The authors observed crosslinks from α1 Lys2119 to LG1, LG2, and the LG2-LG3 linker, consistent with the location of Lys2119 in the penultimate turn of the α1 helix and its side chain pointing toward the center of the LG1-LG3 triangle (Figure S1A).…”

Bottom-up approaches in synthetic biology and biomaterials for tissue engineering applications

“…The individual helices of coiled-coils are composed of heptad repeats and precise placement of amino acids within the coils produce high degrees of specificity between different constituents of the coils. Coiled-coil domains are found throughout nature, including within ECM that contains proteins such as collagen [224] and laminin [225]. Due to their specificity and ease of customization, coiled-coils have been employed in recombinant material design to create orthogonal protein linkages in the extracellular space.…”

Building Peptide Bonds in Haifa: The Seventh Chemical Protein Synthesis (CPS) Meeting

“…Deborah Fass (Weizmann Institute of Science, Israel) described a crosslinking mass‐spectrometry approach to obtaining structural information on proteins and protein complexes. Laminin, a ∼800 kDa heterotrimeric protein in the extracellular matrix, was treated with crosslinking chemicals to link amino acids (lysine and/or aspartate/glutamate) that are close in space, thereby providing a map of the subunit arrangements and revealing the coiled‐coil architecture of laminin …”