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Cross-Linking Reveals Laminin Coiled-Coil Architecture

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Cross-Linking Reveals Laminin Coiled-Coil Architecture Cross-linking reveals laminin coiled-coil architecture Gad Armonya, Etai Jacoba,b, Toot Morana, Yishai Levinc, Tevie Mehlmand, Yaakov Levya, and Deborah Fassa,1 aDepartment of Structural Biology, Weizmann Institute of Science, Rehovot 7610001, Israel; bThe Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel; cThe Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot 7610001, Israel; and dBiological Research Support, Weizmann Institute of Science, Rehovot 7610001, Israel Edited by Peter S. Kim, Stanford University School of Medicine, Stanford, CA, and approved September 28, 2016 (received for review May 25, 2016) Laminin, an ∼800-kDa heterotrimeric protein, is a major functional coiled coil in laminin was presented (22, 23). An analysis based on component of the extracellular matrix, contributing to tissue devel- charged residues in laminin sequences led to the proposal that α, opment and maintenance. The unique architecture of laminin is not β,andγ are arranged in a clockwise manner when viewed from the currently amenable to determination at high resolution, as its flexible carboxyl terminus (24); this order is widely adopted in seminal and narrow segments complicate both crystallization and single-particle reviews on laminin structure and function (10, 25, 26). However, reconstruction by electron microscopy. Therefore, we used cross-linking other investigators have interpreted biochemical results in the and MS, evaluated using computational methods, to address key context of a model with the opposite order (27). Clearly, gaps questions regarding laminin quaternary structure. This approach remain in our appreciation of the laminin assembly. was particularly well suited to the ∼750-Å coiled coil that mediates Cross-linking analyzed by MS is a strategy for determining the trimer assembly, and our results support revision of the subunit order spatial organization of protein quaternary structures (28, 29). This typically presented in laminin schematics. Furthermore, information technique is particularly suitable for elongated protein assemblies on the subunit register in the coiled coil and cross-links to down- such as coiled coils (30), which have high surface-to-volume ratios. stream domains provide insights into the self-assembly required for The laminin coiled coil is rich in amino acids with cross-linkable interaction with other extracellular matrix and cell surface proteins. functional groups (Fig. 1). Furthermore, coiled coils have well- defined degrees of freedom: oligomeric state, subunit register (i.e., coiled coil | extracellular matrix | laminin | cross-linking | the relative positions of the subunits along the coiled-coil axis), mass spectrometry pitch, interface angle, and the order of chains in a hetero-oligomer. Benefiting from the parameterization of coiled-coil geometry, a aminins are network-forming constituents of the extracellular computational model for laminin subunit association was evalu- Lmatrix (ECM) (1, 2). They interact with the cell surface and ated under constraints from the cross-linking data. This analysis other ECM components to generate a physical and functional illuminated previously inaccessible aspects of the laminin quater- framework affecting cell viability, identity, and activity. The nary structure. Coupled with information from low resolution laminin family appears to have arisen during the evolution of electron microscopy and laminin fragment crystallization, a com- multicellularity in animals (3), and laminins contribute to a di- plete picture of the laminin structure is beginning to emerge. versity of basement membrane and connective tissue structures in mammals (2). Laminins are studied in the context of devel- Results opment (4), stem cell biology (5), tissue engineering (6), cancer Cross-Linking of the Laminin Heterotrimer. Mouse laminin-111 [laminin (7), and aging (8). The remarkable structural organization of isotype (31) containing the α1, β1, and γ1 paralogs, hereafter laminin] laminins underlies their important physiological functions. was treated with isotopic mixtures, differing by 12.076 Da, of the Laminins are composed of three subunits, α, β,andγ,thatas- cross-linkers bis(sulfosuccinimidyl)suberate (BS3) and suberic acid semble into a roughly humanoid form as visualized using rotary dihydrazide (SDH) to target primary amine and carboxylic acid shadowing electron microscopy (9). The individual subunits sep- “ ” “ ” arately form the head and two arms, which are composed of Significance 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, Large, fibrous, and flexible extracellular matrix proteins are integral constituting the “body.” Additional globular domains unique to to development and maintenance of tissues in the body. Laminin is the α subunit are the “feet.” The laminin head and arms may splay an extracellular matrix component that provides a physical sub- to form a tripod (2), a feature not captured in rotary shadowing strate for cell adhesion and induces signaling pathways that images or in diagrams of domain composition. Due to the cen- maintain cell health and functionality. Despite the physiological trality of laminin in cell–ECM interactions, efforts have been made importance of laminin, major gaps remain in our understanding of to analyze the functional regions of the trimer, to determine which how its three subunits come together to form the characteristic α, β,andγ paralogs associate into physiological heterotrimers and cross-shaped laminin structure. Laminin was treated with chemicals to understand how trimers self-assemble into higher-order net- that link amino acids close in space, providing a map of the subunit works. Laminin fragments have been generated to assess their arrangement and correcting previous suppositions made on the binding properties (11, 12) and as targets for structure determi- basis of amino acid sequence inspection alone. nation by X-ray crystallography (13–20). Author contributions: G.A., Y. Levy, and D.F. designed research; G.A., E.J., T. Moran, Y. Levin, Despite this progress, few insights into the overall 3D archi- T. Mehlman, and D.F. performed research; G.A., E.J., Y. Levin, and D.F. analyzed data; and tecture of laminin have been made in the past few decades, and G.A. and D.F. wrote the paper. certain regions of the complex have been neglected as targets of The authors declare no conflict of interest. structural techniques. In particular, no structure has been de- This article is a PNAS Direct Submission. termined for any segment of the laminin coiled coil, which spans Freely available online through the PNAS open access option. more than 750 Å and is the backbone of the laminin quaternary α Data deposition: The mass spectrometry proteomics data have been deposited to the structure. Because the regions of high coiled-coil propensity in , ProteomeXchange Consortium via the PRIDE partner repository (dataset identifier β, and γ are similar in length, their alignment can be roughly PXD004898). surmised. However, structural deviations in the coiled coil have 1To whom correspondence should be addressed. Email: [email protected]. been suggested to occur (21). More fundamentally, the order of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the three subunits has remained ambiguous since evidence for a 1073/pnas.1608424113/-/DCSupplemental. 13384–13389 | PNAS | November 22, 2016 | vol. 113 | no. 47 www.pnas.org/cgi/doi/10.1073/pnas.1608424113 Downloaded by guest on September 27, 2021 Assignment of the Subunit Order in the Laminin Coiled Coil. The α, β, and γ subunits can be arranged either clockwise or counter clock- wise when viewed down the coiled-coil axis. The order of subunits in the trimer underlies the mechanism of combinatorial assembly of laminin isotypes (35) and therefore is key to understanding the complexity and diversification of the laminin family. We reasoned that the alternative subunit arrangements could be distinguished without knowledge of the register of the three strands. To begin, an ideal trimeric coiled coil was generated using Coiled-Coil Crick Parameterization (36), and the position of each amino acid in the heptad repeat (a, b, c, d, e, f,org) was noted. SASDs with a 34-Å upper limit then were calculated in xWALK for all possible pairs of heptad positions between helices of the model coiled coil (Table S1). In parallel, each laminin coiled-coil residue was assigned its most likely position within a heptad repeat using MARCOIL (37). According to predicted heptad positions, each intersubunit BS3 or SDH cross-link in the coiled coil was given its corresponding SASDs for the clockwise and counter clockwise arrangements (Table S2), and histograms of expected distance were generated for the two Fig. 1. Laminin domains and cross-linkable side chains. (Left) Laminin subunits competing models (Fig. 3). The cross-linking data overwhelming are labeled α, β,andγ. Domains are labeled according to convention (10). supported a counter clockwise arrangement of α, β,andγ as viewed (Right) Percentages of primary amine-bearing (K, blue) and carboxylic acid- from the carboxyl terminus. bearing (D/E, red) side chains were calculated using a sliding window of 40 Zero-length cross-linking confirmed the subunit order. Many amino acids,
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