The tetrameric L27 domain complex as an organization platform for supramolecular assemblies

L27 domain, initially identified in the Caenorhabditis elegans Lin-2 and Lin-7 proteins, is a protein interaction module that exists in a large family of scaffold proteins. The domain can function as an organization center of large protein assemblies required for establishment and maintenance of cell polarity. We have solved the high-resolution NMR structure of a tetrameric complex of L27 domains containing two SAP97-mLin-2 L27 domain heterodimers. Each L27 domain contains three a-helices. The first two helices of each domain are packed together to form a four-helical bundle in the heterodimer. The third helix of each L27 domain forms another four-helical bundle that assembles the two heterodimers into a tetramer. The structure of the complex provides a mechanistic explanation for L27 domain-mediated polymerization of scaffold proteins, a process that is crucial for the assembly of supramolecular complexes in asymmetric cells.     

Drosophila Varicose, a member of a new subgroup of basolateral MAGUKs, is required for septate junctions and tracheal morphogenesis

Epithelial tubes are the functional units of many organs, but little is known about how tube sizes are established. Using the Drosophila tracheal system as a model, we previously showed that mutations in varicose (vari) cause tubes to become elongated without increasing cell number. Here we show vari is required for accumulation of the tracheal size-control proteins Vermiform and Serpentine in the tracheal lumen. We also show that vari is an essential septate junction (SJ) gene encoding a membrane associated guanylate kinase (MAGUK). In vivo analyses of domains important for MAGUK scaffolding functions demonstrate that while the Vari HOOK domain is essential, the L27 domain is dispensable. Phylogenetic analyses reveal that Vari helps define a new MAGUK subgroup that includes mammalian PALS2. Importantly, both Vari and PALS2 are basolateral, and the interaction of Vari with the cell-adhesion protein Neurexin IV parallels the interaction of PALS2 and another cell-adhesion protein, Necl-2. Vari therefore bolsters the similarity between Drosophila and vertebrate epithelial basolateral regions, which had previously been limited to the common basolateral localization of Scrib, Dlg and Lgl, proteins required for epithelial polarization at the beginning of embryogenesis. However, by contrast to Scrib, Dlg and Lgl, Vari is not required for cell polarity but rather is part of a cell-adhesion complex. Thus, Vari fundamentally extends the similarity of Drosophila and vertebrate basolateral regions from sharing only polarity complexes to sharing both polarity and cell-adhesion complexes.     

A general model for preferential hetero-oligomerization of LIN-2/7 domains: mechanism underlying directed assembly of supramolecular signaling complexes

LIN-2/7 (L27) domains are protein interaction modules that preferentially hetero-oligomerize, a property critical for their function in directing specific assembly of supramolecular signaling complexes at synapses and other polarized cell-cell junctions. We have solved the solution structure of the heterodimer composed of the L27 domains from LIN-2 and LIN-7. Comparison of this structure with other L27 domain structures has allowed us to formulate a general model for why most L27 domains form an obligate heterodimer complex. L27 domains can be divided in two types (A and B), with each heterodimer comprising an A/B pair. We have identified two keystone positions that play a central role in discrimination. The residues at these positions are energetically acceptable in the context of an A/B heterodimer, but would lead to packing defects or electrostatic repulsion in the context of A/A and B/B homodimers. As predicted by the model, mutations of keystone residues stabilize normally strongly disfavored homodimers. Thus, L27 domains are specifically optimized to avoid homodimeric interactions.     

Identification of multiple binding partners for the amino-terminal domain of synapse-associated protein 97

Multiprotein complexes mediate static and dynamic functions to establish and maintain cell polarity in both epithelial cells and neurons. Membrane-associated guanylate kinase (MAGUK) proteins are thought to be scaffolding molecules in these processes and bind multiple proteins via their obligate postsynaptic density (PSD)-95/Disc Large/Zona Occludens-1, Src homology 3, and guanylate kinase-like domains. Subsets of MAGUK proteins have additional protein-protein interaction domains. An additional domain we identified in SAP97 called the MAGUK recruitment (MRE) domain binds the LIN-2,7 amino-terminal (L27N) domain of mLIN-2/CASK, a MAGUK known to bind mLIN-7. Here we show that SAP97 binds two other mLIN-7 binding MAGUK proteins. One of these MAGUK proteins, DLG3, coimmunoprecipitates with SAP97 in lysates from rat brain and transfected Madin-Darby canine kidney cells. This interaction requires the MRE domain of SAP97 and surprisingly, both the L27N and L27 carboxyl-terminal (L27C) domains of DLG3. We also demonstrate that SAP97 can interact with the MAGUK protein, DLG2, but not the highly related protein, PALS2. The ability of SAP97 to interact with multiple MAGUK proteins is likely to be important for the targeting of specific protein complexes in polarized cells.     

The structural basis for partitioning of the XRCC1/DNA ligase III-α BRCT-mediated dimer complexes

The ultimate step common to almost all DNA repair pathways is the ligation of the nicked intermediate to form contiguous double-stranded DNA. In the mammalian nucleotide and base excision repair pathways, the ligation step is carried out by ligase III-α. For efficient ligation, ligase III-α is constitutively bound to the scaffolding protein XRCC1 through interactions between the C-terminal BRCT domains of each protein. Although structural data for the individual domains has been available, no structure of the complex has been determined and several alternative proposals for this interaction have been advanced. Interpretation of the models is complicated by the formation of homodimers that, depending on the model, may either contribute to, or compete with heterodimer formation. We report here the structures of both homodimer complexes as well as the heterodimer complex. Structural characterization of the heterodimer formed from a longer XRCC1 BRCT domain construct, including residues comprising the interdomain linker region, revealed an expanded heterodimer interface with the ligase III-α BRCT domain. This enhanced linker-mediated binding interface plays a significant role in the determination of heterodimer/homodimer selectivity. These data provide fundamental insights into the structural basis of BRCT-mediated dimerization, and resolve questions related to the organization of this important repair complex.     

PATJ regulates tight junction formation and polarity in mammalian epithelial cells

Recent studies have revealed an important role for tight junction protein complexes in epithelial cell polarity. One of these complexes contains the apical transmembrane protein, Crumbs, and two PSD95/discs large/zonula occludens domain proteins, protein associated with Lin seven 1 (PALS1)/Stardust and PALS1-associated tight junction protein (PATJ). Although Crumbs and PALS1/Stardust are known to be important for cell polarization, recent studies have suggested that Drosophila PATJ is not essential and its function is unclear. Here, we find that PATJ is targeted to the apical region and tight junctions once cell polarization is initiated. We show using RNAi techniques that reduction in PATJ expression leads to delayed tight junction formation as well as defects in cell polarization. These effects are reversed by reintroduction of PATJ into these RNAi cells. This study provides new functional information on PATJ as a polarity protein and increases our understanding of the Crumbs-PALS1-PATJ complex function in epithelial polarity.     

FERM protein EPB41L5 is a novel member of the mammalian CRB-MPP5 polarity complex

Cell polarity is induced and maintained by separation of the apical and basolateral domains through specialized cell-cell junctions. The Crumbs protein and its binding partners are involved in formation and stabilization of adherens junctions. In this study, we describe a novel component of the mammalian Crumbs complex, the FERM domain protein EPB41L5, which associates with the intracellular domains of all three Crumbs homologs through its FERM domain. Surprisingly, the same FERM domain is involved in binding to the HOOK domain of MPP5/PALS1, a previously identified interactor of Crumbs. Co-expression and co-localization studies suggested that in several epithelial derived tissues Epb4.1l5 interacts with at least one Crumbs homolog, and with Mpp5. Although at early embryonic stages Epb4.1l5 is found at the basolateral membrane compartment, in adult tissues it co-localizes at the apical domain with Crumbs proteins and Mpp5. Overexpression of Epb4.1l5 in polarized MDCK cells affects tightness of cell junctions and results in disorganization of the tight junction markers ZO-1 and PATJ. Our results emphasize the importance of a conserved Crumbs-MPP5-EPB41L5 polarity complex in mammals.     

Direct interaction of two polarity complexes implicated in epithelial tight junction assembly

Tight junctions help establish polarity in mammalian epithelia by forming a physical barrier that separates apical and basolateral membranes. Two evolutionarily conserved multi-protein complexes, Crumbs (Crb)-PALS1 (Stardust)-PATJ (DiscsLost) and Cdc42-Par6-Par3-atypical protein kinase C (aPKC), have been implicated in the assembly of tight junctions and in polarization of Drosophila melanogaster epithelia. Here we identify a biochemical and functional link between these two complexes that is mediated by Par6 and PALS1 (proteins associated with Lin7). The interaction between Par6 and PALS1 is direct, requires the amino terminus of PALS1 and the PDZ domain of Par6, and is regulated by Cdc42-GTP. The transmembrane protein Crb can recruit wild-type Par6, but not Par6 with a mutated PDZ domain, to the cell surface. Expression of dominant-negative PALS1-associated tight junction protein (PATJ) in MDCK cells results in mis-localization of PALS1, members of the Par3-Par6-aPKC complex and the tight junction marker, ZO-1. Similarly, overexpression of Par6 in MDCK cells inhibits localization of PALS1 to the tight junction. Our data highlight a previously unrecognized link between protein complexes that are essential for epithelial polarity and formation of tight junctions.     

Mammalian lin-7 stabilizes polarity protein complexes

Mammalian Lin-7 forms a complex with several proteins, including PALS1, that have a role in polarity determination in epithelial cells. In this study we have found that loss of Lin-7 protein from the polarized epithelial cell line Madin-Darby canine kidney II by small hairpin RNA results in defects in tight junction formation as indicated by lowered transepithelial electrical resistance and mislocalization of the tight junction protein ZO-1 after calcium switch. The knock down of Lin-7 also resulted in the loss of expression of several Lin-7 binding partners, including PALS1 and the polarity protein PATJ. The effects of Lin-7 knock down were rescued by the exogenous expression of murine Lin-7 constructs that contained the L27 domain, but not the PDZ domain alone. Furthermore, exogenously expressed PALS1, but not other Lin-7 binding partners, also rescued the effects of Lin-7 knock down, including the restoration of PATJ protein in rescued cell lines. Finally, the effects of Lin-7 knock down appeared to be due to instability of PALS1 protein in the absence of Lin-7, as indicated by an increased rate of PALS1 protein degradation. Taken together, these results indicate that Lin-7 functions in tight junction formation by stabilizing its membrane-associated guanylate kinase binding partner PALS1.     

Localization and Function of Pals1-associated Tight Junction Protein in Drosophila Is Regulated by Two Distinct Apical Complexes

The transmembrane protein Crumbs (Crb) and its intracellular adaptor protein Pals1 (Stardust, Sdt in Drosophila) play a crucial role in the establishment and maintenance of apical-basal polarity in epithelial cells in various organisms. In contrast, the multiple PDZ domain-containing protein Pals1-associated tight junction protein (PATJ), which has been described to form a complex with Crb/Sdt, is not essential for apical basal polarity or for the stability of the Crb/Sdt complex in the Drosophila epidermis. Here we show that, in the embryonic epidermis, Sdt is essential for the correct subcellular localization of PATJ in differentiated epithelial cells but not during cellularization. Consistently, the L27 domain of PATJ is crucial for the correct localization and function of the protein. Our data further indicate that the four PDZ domains of PATJ function, to a large extent, in redundancy, regulating the function of the protein. Interestingly, the PATJ-Sdt heterodimer is not only recruited to the apical cell-cell contacts by binding to Crb but depends on functional Bazooka (Baz). However, biochemical experiments show that PATJ associates with both complexes, the Baz-Sdt and the Crb-Sdt complex, in the mature epithelium of the embryonic epidermis, suggesting a role of these two complexes for the function of PATJ during the development of Drosophila.     

Integrated activity of PDZ protein complexes regulates epithelial polarity

Polarized cells contain numerous membrane domains, but it is unclear how the formation of these domains is coordinated to create a single integrated cell architecture. Genetic screens of Drosophila melanogaster embryos have identified three complexes, each containing one of the PDZ domain proteins--Stardust (Sdt), Bazooka (Baz) and Scribble (Scrib)--that control epithelial polarity and formation of zonula adherens. We find that these complexes can be ordered into a single regulatory hierarchy that is initiated by cell adhesion-dependent recruitment of the Baz complex to the zonula adherens. The Scrib complex represses apical identity along basolateral surfaces by antagonizing Baz-initiated apical polarity. The Sdt-containing Crb complex is recruited apically by the Baz complex to counter antagonistic Scrib activity. Thus, a finely tuned balance between Scrib and Crb complex activity sets the limits of the apical and basolateral membrane domains and positions cell junctions. Our data suggest a model in which the maturation of epithelial cell polarity is driven by integration of the sequential activities of PDZ-based protein complexes.     

Structure of an L27 domain heterotrimer from cell polarity complex Patj/Pals1/Mals2 reveals mutually independent L27 domain assembly mode

The assembly of supramolecular complexes in multidomain scaffold proteins is crucial for the control of cell polarity. The scaffold protein of protein associated with Lin-7 1 (Pals1) forms a complex with two other scaffold proteins, Pals-associated tight junction protein (Patj) and mammalian homolog-2 of Lin-7 (Mals2), through its tandem Lin-2 and Lin-7 (L27) domains to regulate apical-basal polarity. Here, we report the crystal structure of a 4-L27 domain-containing heterotrimer derived from the tripartite complex Patj/Pals1/Mals2. The heterotrimer consists of two cognate pairs of heterodimeric L27 domains with similar conformations. Structural analysis and biochemical data further show that the dimers assemble mutually independently. Additionally, such mutually independent assembly of the two heterodimers can be observed in another tripartite complex, Disks large homolog 1 (DLG1)/calcium-calmodulin-dependent serine protein kinase (CASK)/Mals2. Our results reveal a novel mechanism for tandem L27 domain-mediated, supramolecular complex assembly with a mutually independent mode.     

Polarity complex proteins

The formation of functional epithelial tissues involves the coordinated action of several protein complexes, which together produce a cell polarity axis and develop cell-cell junctions. During the last decade, the notion of polarity complexes emerged as the result of genetic studies in which a set of genes was discovered first in Caenorhabditis elegans and then in Drosophila melanogaster. In epithelial cells, these complexes are responsible for the development of the apico-basal axis and for the construction and maintenance of apical junctions. In this review, we focus on apical polarity complexes, namely the PAR3/PAR6/aPKC complex and the CRUMBS/PALS1/PATJ complex, which are conserved between species and along with a lateral complex, the SCRIBBLE/DLG/LGL complex, are crucial to the formation of apical junctions such as tight junctions in mammalian epithelial cells. The exact mechanisms underlying their tight junction construction and maintenance activities are poorly understood, and it is proposed to focus in this review on establishing how these apical polarity complexes might regulate epithelial cell morphogenesis and functions. In particular, we will present the latest findings on how these complexes regulate epithelial homeostasis.     

The SARS Coronavirus E Protein Interacts with PALS1 and Alters Tight Junction Formation and Epithelial Morphogenesis

Intercellular tight junctions define epithelial apicobasal polarity and form a physical fence which protects underlying tissues from pathogen invasions. PALS1, a tight junction-associated protein, is a member of the CRUMBS3-PALS1-PATJ polarity complex, which is crucial for the establishment and maintenance of epithelial polarity in mammals. Here we report that the carboxy-terminal domain of the SARS-CoV E small envelope protein (E) binds to human PALS1. Using coimmunoprecipitation and pull-down assays, we show that E interacts with PALS1 in mammalian cells and further demonstrate that the last four carboxy-terminal amino acids of E form a novel PDZ-binding motif that binds to PALS1 PDZ domain. PALS1 redistributes to the ERGIC/Golgi region, where E accumulates, in SARS-CoV–infected Vero E6 cells. Ectopic expression of E in MDCKII epithelial cells significantly alters cyst morphogenesis and, furthermore, delays formation of tight junctions, affects polarity, and modifies the subcellular distribution of PALS1, in a PDZ-binding motif-dependent manner. We speculate that hijacking of PALS1 by SARS-CoV E plays a determinant role in the disruption of the lung epithelium in SARS patients.     

Structural basis for Arl1-dependent targeting of homodimeric GRIP domains to the Golgi apparatus

Golgins are large coiled-coil proteins that play a role in Golgi structure and vesicle traffic. The Arf-like GTPase Arl1 regulates the translocation of GRIP domain-containing golgins to Golgi membranes. We report here the 1.7 A resolution structure of human Arl1-GTP in a complex with the GRIP domain of golgin-245. The structure reveals that the GRIP domain consists of an S-shaped arrangement of three helices. The domain forms a homodimer that binds two Arl1-GTPs using two helices from each monomer. The structure is consistent with golgin-245 forming parallel coiled-coils and suggests how Arl1-GTP/GRIP complexes interact with Golgi membranes via the N termini of Arl1-GTP and the C-terminal tails of the GRIP domains. In cells, bivalent association with Arl1-GTP would increase residence time of the golgins on Golgi membranes. Despite no conservation of sequence, topology, or even helical direction, several other effectors form similar interactions with small GTPases via a pair of alpha helices, suggesting a common structural basis for effector recognition.     

Polarity complex proteins

The formation of functional epithelial tissues involves the coordinated action of several protein complexes, which together produce a cell polarity axis and develop cell-cell junctions. During the last decade, the notion of polarity complexes emerged as the result of genetic studies in which a set of genes was discovered first in Caenorhabditis elegans and then in Drosophila melanogaster. In epithelial cells, these complexes are responsible for the development of the apico-basal axis and for the construction and maintenance of apical junctions. In this review, we focus on apical polarity complexes, namely the PAR3/PAR6/aPKC complex and the CRUMBS/PALS1/PATJ complex, which are conserved between species and along with a lateral complex, the SCRIBBLE/DLG/LGL complex, are crucial to the formation of apical junctions such as tight junctions in mammalian epithelial cells. The exact mechanisms underlying their tight junction construction and maintenance activities are poorly understood, and it is proposed to focus in this review on establishing how these apical polarity complexes might regulate epithelial cell morphogenesis and functions. In particular, we will present the latest findings on how these complexes regulate epithelial homeostasis.     

Energetic determinants of animal cell polarity regulator Par-3 interaction with the Par complex

The animal cell polarity regulator Par-3 recruits the Par complex (consisting of Par-6 and atypical PKC, aPKC) to specific sites on the cell membrane. Although numerous physical interactions have been reported between Par-3 and the Par complex, it is unclear how each of these interactions contributes to the overall binding. Using a purified, intact Par complex and a quantitative binding assay, here, we found that the energy required for this interaction is provided by the second and third PDZ protein interaction domains of Par-3. We show that both Par-3 PDZ domains bind to the PDZ-binding motif of aPKC in the Par complex, with additional binding energy contributed from the adjacent catalytic domain of aPKC. In addition to highlighting the role of Par-3 PDZ domain interactions with the aPKC kinase domain and PDZ-binding motif in stabilizing Par-3–Par complex assembly, our results indicate that each Par-3 molecule can potentially recruit two Par complexes to the membrane during cell polarization. These results provide new insights into the energetic determinants and structural stoichiometry of the Par-3–Par complex assembly.     

大鼠MUPP1蛋白PDZ8结构域的生化及晶体结构特性

多重PDZ结构域蛋白1型（MUPP1）是一种存在于上皮细胞和神经细胞内含有13个PDZ结构域的重要支架蛋白.在上皮细胞中,MUPP1蛋白在紧密连接结构的形成和上皮细胞的极化过程中发挥重要作用.而在中枢神经系统中,MUPP1基因的1个提前终止突变导致了其最后12个PDZ结构域的缺失,以及严重的先天性脑积水.此外,MUPP1蛋白的表达水平与酒精依赖性和药物戒断的严重性也具有显著的相关性.因此,对MUPP1蛋白所含的PDZ结构域进行纯化和性质鉴定,将有助于深入研究MUPP1蛋白的功能和分子机制.在本文研究中,利用亲和纯化和分子筛技术,对大鼠来源的MUPP1蛋白的第8个PDZ结构域进行了表达和纯化.多角度激光光散射的数据表明: MUPP1-PDZ8结构域在溶液中为单体,分子量为16.4 kD.圆二色谱结果表明,MUPP1-PDZ8结构域具有较好的二级结构折叠,测得其熔解温度为71.6摄氏度,暗示该PDZ结构域在溶液中非常稳定.最后,MUPP1-PDZ8结构域的晶体结构显示,该结构域属于I 型PDZ 结构域,包含3个α螺旋和6个β折叠.其中GLGL模块、β折叠B上的1 351位亮氨酸,以及α螺旋B上的1 405位异亮氨酸/1 398位组氨酸形成的PDZ结合口袋,可以特异性地与其目标蛋白质的羧基末端相结合.综上所述,本文的研究提供了MUPP1-PDZ8结构域的生化特性,以及该结构域与其目标蛋白质相互作用的分子机制,这将为MUPP1蛋白的功能研究提供生物化学与结构生物学的理论基础.     

The conformationally dynamic C helix of the RIalpha subunit of protein kinase A mediates isoform-specific domain reorganization upon C subunit binding

Different isoforms of the full-length protein kinase A (PKA) regulatory subunit homodimer (R2) and the catalytic (C) subunit-bound holoenzyme (R2C2) have very different global structures despite similar molecular weights and domain organization within their primary sequences. To date, it has been the linker sequence between the R subunit dimerization/docking domain and cAMP-binding domain A that has been implicated in modulating domain interactions to give rise to these differences in global structure. The small angle solution scattering data presented here for three different isoforms of PKA heterodimer (deltaR-C) complexes reveal a role for another conformationally dynamic sequence in modulating inter-subunit and domain interactions, the C helix that connects the cAMP-binding domains A and B of the R subunit. The deltaR-C heterodimer complexes studied here were each formed with a monomeric N-terminal deletion mutant of the R subunit (deltaR) that contains the inhibitor sequence and both cAMP-binding domains. The scattering data show that type IIalpha and type IIbeta deltaR-C heterodimers are relatively compact and globular, with the C subunit contacting the inhibitor sequence and both cAMP-binding domains. In contrast, the type Ialpha heterodimer is significantly more extended, with the C subunit interacting with the inhibitor sequence and cAMP-binding domain A, whereas domain B extends out such that its surface is almost completely solvent exposed. These data implicate the C helix of RIalpha in modulating isoform-specific interdomain communication in the PKA holoenzyme, adding another layer of structural complexity to our understanding of signaling dynamics in this multisubunit, multidomain protein kinase.