The crystal structure of SUN1-KASH6 reveals an asymmetric LINC complex architecture compatible with nuclear membrane insertion

Abstract

The LINC complex transmits cytoskeletal forces into the nucleus to control the structure and movement of nuclear contents. It is formed of nuclear SUN and cytoplasmic KASH proteins, which interact within the nuclear lumen, immediately below the outer nuclear membrane. However, the symmetrical location of KASH molecules within SUN-KASH complexes in previous crystal structures has been difficult to reconcile with the steric requirements for insertion of their immediately upstream transmembrane helices into the outer nuclear membrane. Here, we report the crystal structure of the SUN-KASH complex between SUN1 and JAW1/LRMP (KASH6) in an asymmetric 9:6 configuration. This intertwined assembly involves two distinct KASH conformations such that all six KASH molecules emerge on the same molecular surface. Hence, they are ideally positioned for insertion of upstream sequences into the outer nuclear membrane. Thus, we report a SUN-KASH complex architecture that appears to be directly compatible with its biological role.

Fig. 1. Models of the LINC complex.

a Schematic of the LINC complex topology (top), and structure-based alignment of KASH domains from human KASH proteins in which sequences observed in their crystal structures are highlighted (bottom). The KASH domain of JAW1/LRMP is referred to as KASH6 throughout this manuscript. Inner and outer nuclear membranes, INM and ONM, respectively. b Schematic highlighting the steric challenge of inserting transmembrane helices of all six KASH molecules into the outer nuclear membrane from the symmetric 6:6 hetero-oligomer observed in crystal structures of the SUN-KASH complex. The KASH-lids of each SUN trimer are coloured blue, yellow and pink. c, d Proposed models of LINC complex structure in vivo. c Linear model in which SUN-KASH 6:6 complexes are disrupted into 3:3 structures that are oriented vertically in the nuclear lumen^,. d Hinged model in which SUN-KASH 6:6 complexes are angled to facilitate insertion of their bound KASH molecules into the ONM.

Fig. 2. Crystal structure of SUN1-KASH6.

a–c Crystal structure of SUN1-KASH6 (1.7 Å resolution) in a ‘trimer-of-trimers’ configuration. a, b Overall structure with SUN domains shown as (a) surfaces and (b) cartoons. The KASH6α and KASH6β molecules bound to the three SUN1 trimers interact extensive with each other, and their associated KASH-lids at the centre of the structure, and emerge on the upper molecular surface. c The trimer-of-trimers interface is mediated by interactions between KASH6α and KASH6β molecules, and their associated KASH-lids, from adjacent SUN1 trimers. In this interface, KASH-lidα and KASH-lidβ are sandwiched between KASH6α and KASH6β in a six-stranded β-sheet. The N-termini of KASH6α and KASH6β are directed towards the upper surface of the molecule, whilst SUN1 trimers are angled with their N-terminal trimer coiled-coiled pointing downwards towards the lower surface of the molecule.

Fig. 3. Unique conformations of KASH6 peptides within the SUN1-KASH6 structure.

a–c Alternative conformations adopted by KASH6 peptides and their associated SUN domain KASH-lids for (a) KASH6α, (b) KASH6β and (c) KASH6γ. d–f Superposition of the KASH peptide and its associated SUN1 protomer from previous crystal structures of SUN1-KASH1 (green; PDB accession 6R15), SUN1-KASH4 (brown; PDB accession 6R16) and SUN1-KASH5 (purple; PDB accession 6R2I), with (d) SUN1-KASH6α (red), (e) SUN1-KASH6β (blue) and (f) SUN1-KASH6γ (yellow). d KASH6α follows the same initial path, undergoing β-interaction with its canonically-oriented KASH-lid, as in all previous SUN1-KASH structures. It then hooks below KASH-lidα, undergoing a 90° bend to form an α-helix that is oriented in the opposite direction to the partial α-helical end of KASH1. e KASH6β forms the same β-interaction with its KASH-lid as in all other SUN1-KASH structures, but with an unusually high angulation of both KASH6β and KASH6-lidβ. f KASH6γ was only observed as three amino-acids at the KASH-binding pocket, with partial occupancy, and in only a subset of crystal structures. In absence of the β-interaction, its KASH-lid adopts a low angulation in which its first β-strand overlays with the KASH-lid-interacting β-strand of other KASH peptides.

Fig. 4. Structural roles of KASH6 peptides and KASH-lids within the trimer-of-trimers assembly.

a, b Location of interacting KASH6 peptides and associated SUN domains within the trimer-of-trimers structure (left), and their molecular details (right). (a) The trimer-of-trimers interface is mediated by an interaction between KASH6α (and associated SUN domain KASH-lidα) and KASH6β (and associated SUN domain KASH-lidβ) of adjacent SUN1 trimers. The KASH6α and KASH6β peptides sandwich their KASH-lids together in a β-sheet structure that is stabilised by hydrophobic packing between the KASH6α helix and the β-sheet. This results in the N-termini of KASH6α and KASH6β peptides being oriented close together on the upper molecular surface. b KASH-lidγ of the KASH6γ-associated or unbound SUN1 protomer interacts with the KASH6α helix and KASH-lidβ of the same SUN1 trimer, and KASH-lidα of the adjacent SUN1 trimer. Its interaction with KASH-lidβ involves the same molecular contacts as the head-to-head interfaces of SUN1-KASH1 and SUN1-KASH5 6:6 complexes.

Fig. 5. Assembly of SUN1-KASH6 9:6 complexes in solution.

a, b Size-exclusion chromatography coupled to multi-angle light scattering showing that (a) SUN1-KASH6 and (b) SUN1-KASH6 core are 6:6 complexes that assemble into 9:6 complexes following treatment at pH 5.0 (theoretical 9:6–232 kDa and 222 kDa; theoretical 6:6–165 kDa and 155 kDa). c, d Small-angle X-ray scattering of SUN1-KASH6 core 9:6 and 6:6 species. c SAXS scattering data overlaid with the theoretical scattering curves of the 9:6 and 6:6 crystal structures of SUN1-KASH6 and SUN1-KASH5 (PDB accession 6R2I), showing χ² values of 2.22 and 1.32, respectively (for other combinations, χ² > 24). Source data are available in Supplementary Data 1. (d) SAXS ab initio models of SUN1-KASH6 core 9:6 and 6:6 species. Filtered averaged models from 20 independent DAMMIF runs are shown with the crystal structures of SUN1-KASH6 and SUN1-KASH5 docked into the respective SAXS envelopes. e Cryo-EM reference-free 2D class averages of SUN1-KASH6, corresponding to the 9:6 complex (top) and 6:6 complex (bottom). Scale bars, 50 Å. f SUN1-KASH6 forms a symmetric head-to-head 6:6 complex in solution that undergoes pH-triggered conformational change into an asymmetric trimer-of-trimers 9:6 complex.

Fig. 6. Molecular determinants of SUN1-KASH6 trimer-of-trimers assembly.

a–c Structural details of interactions formed by KASH6α amino-acids W534, H539 and H549. a W534 mediates inter-trimer interactions of KASH6α (red) by forming hydrophobic interactions with F671, I673 and L675 of KASH-lidβ from the opposing trimer (purple). It also co-ordinates a chloride ion along with E538 of KASH6α and W676 of KASH-lidα from the same trimer (yellow). b H539 interacts with a backbone carbonyl of KASH-lidγ from the same trimer (blue), which may constitute a salt bridge upon histidine protonation. This interface also includes interactions between H539, I540, P534 and F544 of KASH6α (red) and W676, M668 and L670 of KASH-lidγ from the same trimer (blue). c Superposition of KASH6α (red) and its associated SUN1 protomer with previous crystal structures (KASH1, green; KASH4, brown; KASH5, purple). KASH6α amino-acid H549 differs from other KASH proteins that have a tyrosine at this position (for clarity, only Y397 of KASH4 is shown). d–g Size-exclusion chromatography coupled to multi-angle light scattering of point mutants of the SUN1-KASH6 core complex in which samples were analysed in native conditions (yellow) and following treatment at pH 5.0 (blue). (d) W534A mutation largely blocked assembly, remaining in a 6:6 complex (143 kDa) following acidic treatment. e H539K mutation promoted assembly in the absence of acidic treatment, with untreated material forming 9:6 and 6:6 complexes (218 kDa and 141 kDa) at approximately 60% and 40% (by mass), respectively. f H539Q mutation largely blocked assembly, remaining in a 6:6 complex (149 kDa) following acidic treatment. g H549Y mutation partially blocked assembly, with treated material forming 9:6 and 6:6 complexes (205 kDa and 138 kDa) at approximately 45% and 55% (by mass), respectively. For comparison, the treated wild-type complex (Fig. 5a) formed 9:6 and 6:6 complexes at approximately 85% and 15% (by mass), respectively.

Fig. 7. Model of a SUN1-KASH6 9:6 LINC complex.

Schematic model of how a SUN1-KASH6 asymmetric trimer-of-trimers LINC complex may be positioned with the nuclear lumen. The six bound KASH6 domains emerge from the 9:6 complex on the top surface of the molecule, favouring insertion of their immediately upstream transmembrane helices into the outer nuclear membrane (ONM). The SUN1 trimers are tilted downwards within the trimer-of-trimers structure, facilitating the angled passage of SUN1’s upstream trimeric coiled-coil across the nuclear lumen, towards the inner nuclear membrane (INM).