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Structural Insights Into SUN-KASH Complexes Across the Nuclear Envelope

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Structural Insights Into SUN-KASH Complexes Across the Nuclear Envelope npg Crystal structure of the SUN-KASH complex Cell Research (2012) 22:1440-1452. 1440 © 2012 IBCB, SIBS, CAS All rights reserved 1001-0602/12 $ 32.00 npg ORIGINAL ARTICLE www.nature.com/cr Structural insights into SUN-KASH complexes across the nuclear envelope Wenjia Wang1, *, Zhubing Shi1, *, Shi Jiao1, *, Cuicui Chen1, Huizhen Wang1, Guoguang Liu1, Qiang Wang2, Yun Zhao1, Mark I Greene3, Zhaocai Zhou1 1State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; 2Women’s Cancer Program at the Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA; 3Department of Pathology and Laboratory Medicine, Uni- versity of Pennsylvania, Philadelphia, PA 19104, USA Linker of the nucleoskeleton and the cytoskeleton (LINC) complexes are composed of SUN and KASH domain- containing proteins and bridge the inner and outer membranes of the nuclear envelope. LINC complexes play criti- cal roles in nuclear positioning, cell polarization and cellular stiffness. Previously, we reported the homotrimeric structure of human SUN2. We have now determined the crystal structure of the human SUN2-KASH complex. In the complex structure, the SUN domain homotrimer binds to three independent “hook”-like KASH peptides. The overall conformation of the SUN domain in the complex closely resembles the SUN domain in its apo state. A major conformational change involves the AA’-loop of KASH-bound SUN domain, which rearranges to form a mini β-sheet that interacts with the KASH peptide. The PPPT motif of the KASH domain fits tightly into a hydrophobic pocket on the homotrimeric interface of the SUN domain, which we termed the BI-pocket. Moreover, two adjacent protomers of the SUN domain homotrimer sandwich the KASH domain by hydrophobic interaction and hydrogen bonding. Mutations of these binding sites disrupt or reduce the association between the SUN and KASH domains in vitro. In addition, transfection of wild-type, but not mutant, SUN2 promotes cell migration in Ovcar-3 cells. These results pro- vide a structural model of the LINC complex, which is essential for additional study of the physical and functional coupling between the cytoplasm and the nucleoplasm. Keywords: crystal structure; SUN2; KASH; LINC complexes; mechanical force transduction; nuclear envelope Cell Research (2012) 22:1440-1452. doi:10.1038/cr.2012.126; published online 4 September 2012 Introduction complex has been well studied, and only minimal knowl- edge about the remaining NE exists. Only recently have In eukaryotic cells, the nuclear envelope (NE) sepa- molecular characterization and studies of the functional rates the nucleoplasm from the cytoplasm, which is im- importance of the nuclear double membrane architecture portant for maintaining the integrity of genetic material emerged. For example, the linker of the nucleoskeleton and the complex regulation of gene expression and func- and the cytoskeleton (LINC) complex has been identified tion. The NE consists of two lipid bilayers, the inner and as a molecular bridge across the NE for the transduction the outer nuclear membrane (INM and ONM), which are of signaling and mechanical force [1, 2]. LINC complex- connected at nuclear pores. The ONM is contiguous with es primarily consist of proteins containing a SUN (Sad1 the endoplasmic reticulum membrane, and the INM ad- and UNC84) domain or a KASH (Klarsicht, ANC-1 and heres to the nuclear lamina matrix. Only the nuclear pore Syne homology) domain, which function as a part of the mechano/signaling cascade from the cellular periphery to the genome. *These three authors contribute equally to this work. As core components of the LINC complexes, both the Correspondence: Zhaocai Zhou SUN and KASH domains are evolutionarily conserved, E-mail: [email protected] Received 8 June 2012; revised 23 July 2012; accepted 24 July 2012; pub- supporting a fundamental and conserved role of LINC lished online 4 September 2012 complexes. Indeed, mounting evidence indicates that Cell Research | Vol 22 No 10 | October 2012 Wenjia Wang et al. npg 1441 LINC complexes function in multiple biological pro- membrane for interaction with the SUN domain of INM cesses, including nuclear migration and repositioning, proteins (Figure 1A). In the lumen of the NE, the SUN meiotic telomere tethering and chromatin organization, domain interacts with the KASH domain, ensuring that centrosome migration and attachment, nucleokinesis KASH domain proteins are targeted to the ONM. During and cellular rigidity homeostasis [1-3]. Recently, LINC actin-dependent nuclear movement, the LINC complex complexes have been implicated in the regulation of components Nesprin 2G and SUN2 were found to pro- apoptosis [4, 5], the maturation and survival of germlines vide a direct linkage between the nucleus and the actin [6], and in the pathology of human diseases linked to network [18, 19]. Recently, mutations of KASH domain mutations of A-type lamins, such as laminopathies and proteins have been linked to Emery-Dreifuss muscular Emery-Dreifuss muscular dystrophy [7-9]. dystrophy [20, 21]. During a study of centrosome-related proteins that The NE-spanning LINC complexes that are formed were affected by erbB transformation, we cloned and through interactions between the SUN domain and the identified the SUN domain protein SUN2 [10]. Human KASH domain exemplify a physical linkage that may SUN2 is a ubiquitously expressed type II transmembrane affect many aspects of cellular functions. However, the protein that localizes at the INM. A transmembrane do- assembly of the SUN-KASH core complex has not been main separates the N-terminal nucleoplasmic region, fully defined at the atomic level, hindering further ex- which interacts with lamina and/or chromatin binding ploration of its functions. Based on our previous crystal- proteins, from the C-terminal luminal region, which pro- lographic study of the SUN domain [12], we performed trudes into the perinuclear space. The luminal region of structural, biochemical and cell-based studies of the SUN2 contains two coiled-coil domains and a conserved SUN-KASH complex. We observed a 3:3 ratio in the SUN domain at the C-terminus [11] (Figure 1A). Accord- hexameric SUN-KASH complex in which the SUN do- ing to our recent structural study of SUN2, the conserved main trimer binds three separate KASH peptides. Our SUN domain is essential and sufficient for KASH bind- results also indicate that the two regions corresponding ing [12]. Endogenous SUN2 is localized to the NE and to the first and second coiled-coil motifs preceding the is partly dependent on lamin A, but not lamin C, for NE SUN domain may function as activation and inhibitory retention [13]. SUN2 is targeted to the INM through clas- domains, respectively, for its KASH-binding activity. sical nuclear localization signal motifs at the N-terminus and the perinuclear SUN domain at the C-terminus [14]. Results SUN2 appears to associate with the centrosome in a cell cycle-dependent manner. Previously, we showed that Overall structure of the SUN-KASH complex SUN2 may form homo-oligomers, which is regulated We previously reported the crystal structure of the by its two coiled-coil regions and the SUN domain [10]. SUN domain of human SUN2 as a homotrimer and SUN2 can also form hetero-oligomers with SUN1, an- mapped the essential domains required for SUN-KASH other SUN domain protein that is associated with the association [12]. We demonstrated that a synthesized nuclear pore complex [10, 15]. Both SUN1 and SUN2 peptide (termed the KASH peptide), corresponding to the are widely expressed KASH-binding partners and may conserved C-terminus of the human Nesprin 2G KASH have partially redundant functions. Double knockout of domain, can bind to the SUN domain of human SUN2 SUN1 and SUN2 is lethal in mice, similar to the disrup- and that disruption of the SUN domain homotrimer tion of KASH domain-containing proteins, highlighting abolished the binding of the KASH peptide. To further the functional importance of the LINC complex [16]. understand the structural basis of the LINC complex as- Upon formation of the LINC complex, the INM sembly and its functional mechanism at an atomic level, proteins with SUN domains recruit the proteins with a we crystallized the SUN2 SUN domain (G522-H717) KASH domain to the OMN via interactions between in complex with the conserved KASH domain peptide their SUN and KASH surfaces [17]. Nesprins are rep- (SFYPMLRYTNGPPPT). The crystal structure of the resentative KASH domain proteins that contain a single SUN-KASH complex was determined to 3.05 Å resolu- transmembrane anchor at the C-terminus for ONM local- tion by molecular replacement using the SUN domain ization. The N-terminal domains of KASH proteins are monomer as a search model (PDB code 3UNP). highly diversified and are associated with the cytoskel- In the SUN-KASH complex structure, the SUN do- eton by connecting to actin filaments, microtubules and main forms a cloverleaf-like homotrimer with an overall intermediate filaments. The conserved C-terminal KASH conformation that is highly similar to that observed for domain consists of approximately 35 amino acids that the apo-SUN domain homotrimer (Figure 1B). Briefly, are exposed to the luminal space of the nuclear double the SUN domain monomer is composed of an N-terminal www.cell-research.com | Cell Research npg Crystal structure of the SUN-KASH complex 1442 Figure 1 Overall structure of the SUN-KASH complex. (A) A schematic illustration of the domain organization for human SUN2 and Nesprin 2G. (B) The top and side views of the SUN domain homotrimer shown in cartoon representation. The three protomers of the SUN homotrimer are colored in cyan, light blue and pale green. (C) The top and side views of the SUN-KASH complex. The three KASH domain peptides are colored in yellow, magenta and red and bind to three separate trimeric interfaces of the SUN domain homotrimer.
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