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. 2016 Nov 22;113(47):13384-13389.
doi: 10.1073/pnas.1608424113. Epub 2016 Nov 4.

Cross-linking Reveals Laminin Coiled-Coil Architecture

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Free PMC article

Cross-linking Reveals Laminin Coiled-Coil Architecture

Gad Armony et al. Proc Natl Acad Sci U S A. .
Free PMC article

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.

Keywords: coiled coil; cross-linking; extracellular matrix; laminin; mass spectrometry.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Laminin domains and cross-linkable side chains. (Left) Laminin subunits are labeled α, β, and γ. Domains are labeled according to convention (10). (Right) Percentages of primary amine-bearing (K, blue) and carboxylic acid-bearing (D/E, red) side chains were calculated using a sliding window of 40 amino acids, smoothed over 100 amino acids. Coiled-coil regions are shaded.
Fig. 2.
Fig. 2.
Validation of laminin cross-linking. Cross-linked positions (Cβ atoms, blue spheres) were mapped onto the structures of the β1 LN and α1 LG5 domains (PDB ID codes 4AQS and 2JD4, respectively) and by homology onto the structure of the α2 LG2 domain (PDB ID code 2WJS). Shortest SASDs are shown as magenta (BS3 and SDH links) or green tubes (zero-length links), and direct Cβ-Cβ distances are shown as black dashed lines. Measures in Ångstrom for the two routes are in the corresponding colors.
Fig. 3.
Fig. 3.
Cross-linking supports a counterclockwise arrangement of α, β, and γ, viewed from the carboxyl terminus. Schematics of the competing coiled-coil subunit arrangements are shown above. Predicted SASD values based on the likely heptad positions of BS3- and SDH-linked residues are plotted below for each model.
Fig. 4.
Fig. 4.
Map of observed cross-links in the laminin coiled coil. The α subunit is represented twice to show links to both β and γ. BS3/SDH and zero-length cross-links are represented by magenta and green lines, respectively. Orange cross-links appear reliable according to MS data evaluation but are inconsistent with the prevailing register. Figure generated using xiNET (49).
Fig. 5.
Fig. 5.
Laminin coiled-coil register obtained from molecular dynamics simulations subject to cross-linking restraints. (A) Snapshot from a simulation (last frame of Movie S1). BS3/SDH and zero-length cross-links are magenta and green, respectively. (B) Intersubunit contact map between α and β (α-γ and β-γ maps are in Fig. S2). The map is colored according to the probability for pairs of residues to be found in closest contact from the simulations. Contiguous diagonal contacts indicate a continuous register (e.g., α2016 to α2068). Parallel diagonal sets indicate alternative registers (e.g., α1619 to α1689). A register shift appears as a step between two parallel diagonals (e.g., between α1991 and α2033). (C) The uncertainty of the predicted register is estimated for each amino acid residue (x axes) by the residue range in another subunit that comprises 95% of observed nearest-neighbor contacts (y axes). Dashed lines mark one and two heptads.
Fig. S1.
Fig. S1.
Models of coiled-coil segments. (A) Three well-constrained regions of the laminin trimer. Yellow spheres are Cβ atoms of cross-linked residues (listed in Table S4). Cross-links are indicated by dashed lines (BS3 and SDH: magenta; zero-length: green). Zero-length intrasubunit cross-links are shown along with all intersubunit cross-links. The subunit register used to construct this image is given in Table S3. (B) (Left and Center) Model of residues engaging in zero-length links in the C-terminal region of the coiled coil (corresponding to the rightmost image in A; Fig. 6). (Right) Putative interactions implied by the resulting register.
Fig. S2.
Fig. S2.
Laminin coiled-coil register obtained from molecular dynamics simulations subject to cross-linking restraints. Contact maps between (A) α and γ and (B) β and γ were generated as for the contact map between α and β presented in Fig. 5B. (C) The uncertainty of the predicted register is shown as in Fig. 5C but using a range comprising 80% of the observed close contacts.
Fig. 6.
Fig. 6.
Carboxyl terminus of the laminin coiled coil. Helical wheels with residues colored according to conservation (50). The γ subunit segment downstream of the coiled coil (γ tail) is also displayed. Solid curves indicate cross-links, and dashed curves indicate proposed interactions discussed in the text. Conservation scores for the entire laminin coiled coil are in Figs. S7–S9.
Fig. S7.
Fig. S7.
Conservation scores for the laminin coiled-coil α subunit. Multiple sequence alignments were prepared and scored as described in SI Materials and Methods. The yellow glutamate is a residue for which insufficient information was available from the alignment to assign a score.
Fig. S8.
Fig. S8.
Conservation scores for the laminin coiled-coil β subunit.
Fig. S9.
Fig. S9.
Conservation scores for the laminin coiled-coil γ subunit.
Fig. S3.
Fig. S3.
MS2 spectra of β subunit K1746 linked to γ subunit E1560 and E1567.
Fig. S4.
Fig. S4.
Three possible structures of LG1, LG2, and LG3 domains as suggested by molecular docking under constraints from observed cross-links. Cβ atoms of linked side chains for cross-links between the LG domains are indicated by blue spheres (Cα for glycine). Residues found to be linked to the carboxyl-terminal region of the coiled coil are indicated by cyan spheres. Cross-links were mapped by homology onto the structure of the α2 LG domains (extracted from PDB ID code 2WJS). Solvent accessible surface distances between Cβ atoms of linked residues are shown as a magenta tube (SDH link), green tube (zero-length link), or red tube (polypeptide link). Direct Cβ-Cβ distances are indicated by dashed lines. Distances in Ångstrom calculated according to the two routes are shown for each cross-link in the appropriate color.
Fig. S5.
Fig. S5.
Sequence coverage by MS of the laminin α subunit. Amino acids observed in reliable peptide identifications, whether cross-linked or not, are highlighted in yellow. Predicted N-linked glycosylation sites that were detected as unmodified in at least one reliably identified MS spectrum are highlighted in green. Predicted N-linked glycosylation sites that were not detected in unmodified form, and thus may be glycosylated, are highlighted in cyan.
Fig. S6.
Fig. S6.
Sequence coverage by MS of the laminin β and γ subunits. See legend to Fig. S5.

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