Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jul 6;24(7):1130-41.
doi: 10.1016/j.str.2016.04.016. Epub 2016 Jun 2.

LD Motif Recognition by Talin: Structure of the Talin-DLC1 Complex

Thomas Zacharchenko  1 , Xiaolan Qian  2 , Benjamin T Goult  3 , Devina Jethwa  4 , Teresa B Almeida  1 , Christoph Ballestrem  4 , David R Critchley  5 , Douglas R Lowy  2 , Igor L Barsukov  6
Affiliations

LD Motif Recognition by Talin: Structure of the Talin-DLC1 Complex

Thomas Zacharchenko et al. Structure. .

Abstract

Cell migration requires coordination between integrin-mediated cell adhesion to the extracellular matrix and force applied to adhesion sites. Talin plays a key role in coupling integrin receptors to the actomyosin contractile machinery, while deleted in liver cancer 1 (DLC1) is a Rho GAP that binds talin and regulates Rho, and therefore actomyosin contractility. We show that the LD motif of DLC1 forms a helix that binds to the four-helix bundle of the talin R8 domain in a canonical triple-helix arrangement. We demonstrate that the same R8 surface interacts with the paxillin LD1 and LD2 motifs. We identify key charged residues that stabilize the R8 interactions with LD motifs and demonstrate their importance in vitro and in cells. Our results suggest a network of competitive interactions in adhesion complexes that involve LD motifs, and identify mutations that can be used to analyze the biological roles of specific protein-protein interactions in cell migration.

PubMed Disclaimer

Figures

None
Graphical abstract
Figure 1
Figure 1
DLC1(467–489) Interacts with the Talin R8 Domain (A) Model of the talin rod based on the structures of individual domains. Domain R8 interacts with DLC1. (B) Domain composition of DLC1. The location of the talin binding site (TBS) in the largely unstructured serine-rich linker region is indicated. (C) Secondary structure prediction for the TBS in DLC1, which includes an LD motif marked by the red box. “h” denotes a region of high helical propensity and “c” a random coil region. Fragments used in this study are indicated by the thick blue lines. (D) Superposition of the 1H,15N-HSQC spectra (298 K, 800 MHz) of 100 μM talin R8 domain in the free form (blue) and in the presence of 4-fold excess of DLC1(467–489) (red). See also Figure S3.
Figure 2
Figure 2
Structure of the Talin/DLC1 Complex (A) Cartoon representation of the X-ray structure of the talin R7R8 fragment (green) in complex with DLC1(467–489) (orange). (B) Superposition of the crystal structure of R7R8 in the free form (cyan) and in complex with DLC1(467–489) (green) aligned on the R7 domain. Residues at the ends of the linker regions between R7 and R8 are shown in stick representation (red) and labeled. (C) Two-stranded anti-parallel twisted β sheet formed in the linker region. Side chains of the residues highlighted in (B) are shown in the stick representation and labeled. (D) Comparison of the structure of the talin R8/DLC1(467–489) complex (left) and the talin R10 domain (PDB: 2KVP; right). The DLC1 helix and α0 helix of talin R10 are highlighted in orange. (E) Topology of the talin R8/DLC1(467–489) complex (left) and talin R10 (right). See also Figure S1.
Figure 3
Figure 3
Recognition of the DLC1(467–489) Helix by the Talin R8 Domain (A) Position of the DLC1(467–489) helix (orange) relative to the α2 and α3 helices of talin R8 (green). (B) DLC1 and talin residues that make contacts in the complex. Side chains of the residues involved in hydrophobic interactions are shown as balls; charged and hydrophilic interactions are shown as balls and sticks. Blue rectangle identifies the “polar ridge” of the complex. (C) DLC1-interacting residues on the talin surface. LD-recognition box is marked by a red rectangle. (D) Talin-interacting residues on the surface of DLC1 helix. The helix is rotated by 180° around the vertical axis relative to the orientation in (B). (E) Sequence alignment of DLC1 with RIAM TBS and paxillin LD domains. Peptide fragments used to solve the structures of the complexes are underlined. Residues involved in the interactions with the corresponding proteins are highlighted in magenta (hydrophobic interactions) and orange (charged and hydrophilic interactions). Red box indicates the DLC1 LD-motif identified from sequence comparison. For paxillin LD1 the underlined region corresponds to the LD motif. Positions of the coiled-coil heptad repeat are shown above the sequences. The underlined positions “a” and “d” correspond to the interacting hydrophobic residues in coiled coils. (F) Comparison of the positions of DLC1 and RIAM helices in the complexes with the talin R8 domain. (G) Locations of the hydrophobic residues on the surfaces of the DLC1 and RIAM helices involved in the interaction with talin R8. The helices are rotated by 180° around the horizontal axis relative to the orientation in (F). See also Figure S1.
Figure 4
Figure 4
Interactions of Charge-Reversal Mutations of Talin R7R8 and DLC1(461–489) (A) Location of the mutated residues in the structure of talin R8/DLC1(467–489) complex. (B–F) Superposition of the HSQC spectra of 0.2 mM talin R7R8 free (blue) and in the presence of 0.8 mM DLC1(461–489) (red). Mutations are marked on the spectra. wt, wild-type form of the protein. See also Figure S2.
Figure 5
Figure 5
Interaction of Paxillin LD Motifs with Talin R8 (A) Superposition of the HSQC spectra of 0.1 mM talin R8 free (red) and in the presence of 0.4 mM paxillin LD1 (blue). (B) Comparison of structures of talin R8/DLC1 and FAK/paxillin complexes. From left to right: side view of the R8/DLC1 complex—the DLC1 helix is in orange with the LD motif highlighted in red; front view of the R8/DLC1 complex—largest chemical-shift perturbations caused by LD1 binding are highlighted in purple; structure of the FAK complex with LD2 bound to the 2–3 site (helices α2 and α3) and LD4 bound to the 1–4 site (helices α1 and α4) (PDB: 1OW7). (C) Superposition of the HSQC spectra of 0.2 mM talin R7R8 K1544E mutant free (red) and in the presence of 0.8 mM paxillin LD1 (blue). (D) Ratio imaging was used to determine the proportion of endogenous paxillin and DLC1 present at FA in TKOs expressing either talin FL or talin ΔR8. Quantitative analysis shows that both paxillin and DLC1 are markedly reduced in adhesions when talin R8 is deleted (n = 20 cells from three independent experiments). Error bars are ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001 (ANOVA). White line indicates cell margin. Scale bar, 10 μm. See also Figures S2 and S3.
Figure 6
Figure 6
Talin R8 Mutations Disrupt the Interaction with DLC1 and Affect Its Biological Activity (A) Wild-type GST-talin fragments pull down more DLC1 than the 2E mutants. Extracts of HEK293T cells transfected with GFP-DLC1 and GST-talin constructs were subjected to pull-down assays with glutathione beads followed by immunoblotting with anti-GST and anti-DLC1 on the same membrane (top). The transfected GFP-DLC1 in each sample is shown by the anti-DLC1 blot (bottom) as a loading control. (B) Wild-type GST-talin fragments compete efficiently with endogenous talin to form a complex with DLC1. The supernatants collected after pull-down assay from (A) were reused for co-immunoprecipitation with an anti-talin antibody and blotted with anti-DLC1 (top). A small aliquot from each lane was blotted for endogenous talin as a loading control (bottom). (C) Co-expression of GST or GST-talin fragments (wild-type or 2E mutant) with GFP DLC1 in A549 cells. Six days after transfection, A549 cell lysates were blotted with anti-DLC1 (top) and anti-GST (bottom) to conform equal protein expression. (D) G418 colony growth assay. Transfected A549 cells were cultured in G418 for 3 weeks, and colonies counted and quantitated (top). Representative stained colonies are shown (bottom). (E) Growth in soft agar. Transfected A549 cells were grown for 3 weeks in soft agar, and colonies counted and quantitated (top). Representative stained whole dishes are shown (bottom). (F) Transwell cell migration assay. Lysates from migrated cells were quantitated (top), and representative microscopic images of the migrated cells are shown (bottom). The results in (D–F) are represented as means over three experiments ± SD. See also Figures S4–S6.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alam T., Alazmi M., Gao X., Arold S.T. How to find a leucine in a haystack? Structure, ligand recognition and regulation of leucine-aspartic acid (LD) motifs. Biochem. J. 2014;460:317–329. - PubMed
    1. Atherton P., Stutchbury B., Wang D.Y., Jethwa D., Tsang R., Meiler-Rodriguez E., Wang P., Bate N., Zent R., Barsukov I.L. Vinculin controls talin engagement with the actomyosin machinery. Nat. Commun. 2015;6:10038. - PMC - PubMed
    1. Calderwood D.A., Campbell I.D., Critchley D.R. Talins and kindlins: partners in integrin-mediated adhesion. Nat. Rev. Mol. Cell Biol. 2013;14:503–517. - PMC - PubMed
    1. Carisey A., Tsang R., Greiner A.M., Nijenhuis N., Heath N., Nazgiewicz A., Kemkemer R., Derby B., Spatz J., Ballestrem C. Vinculin regulates the recruitment and release of core focal adhesion proteins in a force-dependent manner. Curr. Biol. 2013;23:271–281. - PMC - PubMed
    1. Chang Y.C., Zhang H., Franco-Barraza J., Brennan M.L., Patel T., Cukierman E., Wu J.H. Structural and mechanistic insights into the recruitment of talin by RIAM in integrin signaling. Structure. 2014;22:1810–1820. - PMC - PubMed

Publication types