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. 2012 May 25;149(5):1035-47.
doi: 10.1016/j.cell.2012.03.046.

LINC complexes form by binding of three KASH peptides to domain interfaces of trimeric SUN proteins

Brian A Sosa  1 , Andrea Rothballer, Ulrike Kutay, Thomas U Schwartz
Affiliations

LINC complexes form by binding of three KASH peptides to domain interfaces of trimeric SUN proteins

Brian A Sosa et al. Cell. .

Abstract

Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the nuclear envelope and are composed of KASH and SUN proteins residing in the outer and inner nuclear membrane, respectively. LINC formation relies on direct binding of KASH and SUN in the perinuclear space. Thereby, molecular tethers are formed that can transmit forces for chromosome movements, nuclear migration, and anchorage. We present crystal structures of the human SUN2-KASH1/2 complex, the core of the LINC complex. The SUN2 domain is rigidly attached to a trimeric coiled coil that prepositions it to bind three KASH peptides. The peptides bind in three deep and expansive grooves formed between adjacent SUN domains, effectively acting as molecular glue. In addition, a disulfide between conserved cysteines on SUN and KASH covalently links both proteins. The structure provides the basis of LINC complex formation and suggests a model for how LINC complexes might arrange into higher-order clusters to enhance force-coupling.

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Figures

Figure 1
Figure 1. A Minimal Coiled-coil and the SUN Domain are Required for KASH Binding
(A) Schematic representation of zz-KASH fusion proteins. The luminal peptides of Nesprin-1 and Nesprin-2 (29 aa) were fused to an N-terminal protein A tag (zz-KASH1 and zz-KASH2). zz-KASH2Δ lacks the C-terminal amino acids PPPT. (B) SUN1 and SUN2 from HeLa cells bind to zz-KASH peptides in vitro. HeLa cells were lysed in RIPA buffer and the extract was incubated with immobilized zz-KASH fusion proteins. Bound proteins were eluted and analyzed by immunoblotting using α-SUN1 and α-SUN2 antibodies. Loads in input and pulldown lanes correspond to 2.5% and 20% of the total, respectively. (C) Schematic representation of SUN2 constructs used in pulldown experiments in (D). Coiled-coil regions were predicted using Parcoil2 (McDonnell et al., 2006). (D) KASH binding of recombinant SUN2 fragments. His-tagged SUN2 fragments were expressed in E. coli, purified and added at 0.25 μg/μl to E. coli lysate supplemented with RIPA detergents. Mixtures were incubated with immobilized zz-KASH fusion proteins. Bound proteins were eluted and analyzed by SDS-PAGE and Coomassie staining. Loads in the input and pulldown lanes correspond to 1.25% and 10% of the total, respectively. See also Figure S1.
Figure 2
Figure 2. Structural Analysis of the SUN-KASH Interaction
(A) Overview of a SUN2522–717 protomer isolated from its binding partners in the trimeric SUN-KASH complex. The protein is organized around a compact β-sandwich core, decorated with features important for function (labeled). Bound cation in green. (B) Top view of the SUN2522–717 protomer (facing the outer nuclear membrane), rotated by 90° around the horizontal axis relative to the view in (A). The apo-protomer of SUN2522–717 is superimposed in red. Only the region with significant change is shown, coinciding largely with the KASH-lid. (C) Top view of the trimeric SUN2522–717-KASH2 complex. The trimerizing SUN2 domains in cartoon representation and colored in shades of blue, the KASH2 peptide in orange. The peptide is covalently bound via a disulfide bridge between KASH2-C6862 and SUN2-C563. (D) Same view as (C), but SUN2522–717 in surface representation, illustrating how each bound KASH2 peptide is clamped between two SUN2 protomers. (E) Side view of the SUN2-KASH2 complex, illustrating the deep binding pocket on SUN2 into which the terminal four residues of the KASH peptide bind. (F) Sedimentation equilibrium ultracentrifugation analysis of apo-SUN2335–717. The experiment was performed at two protein concentrations and two centrifugation speeds. Data was fitted for a single species. Fitted curves overlaid over primary data (dots) in red and black for the two speeds. Residuals in the upper panel. The mass was determined to be 131.3 ± 6.1 kDa, the calculated mass is 138 kDa for the trimer. See also Figures S2, S4, S5.
Figure 3
Figure 3. Details of the SUN-KASH Interaction
(A) Close-up view of the SUN2-KASH2 interaction. Two neighboring SUN protomers are shown in two shades of blue, with the KASH2 peptides in between in orange. Surface of the SUN2 binding area is half-transparent. KASH residues crucial for interaction are numbered. ‘0’ denotes the C-terminal residue of the peptide. Pocket residues that abolish KASH-binding if mutated are labeled. (B) Same view of the SUN2-KASH1 interaction. Residues that differ between KASH1 and KASH2 are colored in green. (C) Multiple sequence alignment of the four identified human KASH proteins, followed by a list of KASH peptides from highly diverged eukaryotes. The numbers match residues important for SUN binding. See also Figure S3.
Figure 4
Figure 4. Surface Analysis of the SUN protomer
The SUN protomer in three different orientations, related to one another by 120° rotations around the vertical axis. (A) Cartoon representation, coloring as in Figure 2A. (B) Surface representation, gradient-colored to illustrate the conservation. (C) Surface representation, colored to show the different binding interfaces to the two neighboring SUN protomers (shades of blue), and the two KASH peptides (shades of orange). See also Figure S2.
Figure 5
Figure 5. KASH Interaction Depends on Both the Terminal Binding Pocket and the β-Hairpin of the SUN Domain
(A) The C-terminal 14 aa of KASH2 are sufficient for interaction with the SUN domain. The depicted zz-KASH2 derivatives were bound to beads and incubated with E. coli lysate of cells expressing His-SUN2335–717. Bound proteins were eluted and analyzed by SDS-PAGE and Coomassie staining. Loads in the input and pulldown lanes correspond to 1.25% and 10% of the total, respectively. (B) Contribution of conserved residues within the C-terminal 14 aa of KASH2 to SUN binding. Binding of His-SUN2335–717 to the depicted zz-KASH2 derivatives was analyzed as in (A). Note that the extension of the KASH peptide by one residue abolishes SUN interaction. (C) KASH binding of SUN domain mutants. Wildtype His-SUN2335–717 or mutant derivatives were added to E. coli lysate (1.6 μM of trimer) and incubated with immobilized zz-KASH2 as in Figure 1D. Bound proteins were eluted and analyzed as in (A). (D) SUN domain mutants deficient in KASH interaction fail to mediate NE targeting of a SUN domain-dependent reporter construct in vivo. The localization of SPAG41–189-SUN2507–717-GFP wildtype or indicated SUN domain mutants was analyzed after transfection of HeLa cells. Cells were analyzed by confocal fluorescence microscopy.
Figure 6
Figure 6. Model for the LINC-Complex Bridging the Nuclear Envelope
The LINC complex is displayed to scale within the perinuclear space between ONM and INM. The trimeric SUN2 is modeled based on our experimental structure, with a theoretically calculated trimeric coiled-coil extending N-terminally to the INM (http://arteni.cs.dartmouth.edu/cccp/). The N-terminal, nucleoplasmic domain is indicated as an oval sphere of approximate size assuming a globular shape. Atomic coordinates for the lipid bilayers and the transmembrane segments are from public sources.
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