C-terminal Coiled-coil Research Articles

Temperature Dependent Activity of the Voltage-Gated Proton Channel.

Voltage-gated proton channel (Hv) has activity of proton transport following electrochemical gradient of proton. Hv is expressed in neutrophils and macrophages of which functions are physiologically temperature-sensitive. Hv is also expressed in human sperm cells and regulates their locomotion. H+ transport through Hv is both regulated by membrane potential and pH difference across biological membrane. It is also reported that properties of Hv such as proton conductance and gating are highly temperature-dependent. Hv consists of the N-terminal cytoplasmic domain, the voltage sensor domain (VSD), and the C-terminal coiled-coil domain, and H+ permeates through VSD voltage-dependently. The functional unit of Hv is a dimer via the interaction between C-terminal coiled-coils assembly domain. We have reported that the coiled-coil domain of Hv has the nature of dissociation around our bodily temperature and mutational change of the coiled-coil affected temperature-sensitive gating, especially its temperature threshold. The temperature-sensitive gating is assessed from two separate points: temperature threshold and temperature dependence. In this chapter, I describe physiological roles and molecular structure mechanisms of Hv by mainly focusing on thermosensitive properties.

The intrinsically disordered region of coronins fine-tunes oligomerization and actin polymerization

Coronins play critical roles in actin network formation. The diverse functions of coronins are regulated by the structured N-terminal β propeller and the C-terminal coiled coil (CC). However, less is known about a middle "unique region" (UR), which is an intrinsically disordered region (IDR). The UR/IDR is an evolutionarily conserved signature in the coronin family. By integrating biochemical and cell biology experiments, coarse-grained simulations, and protein engineering, we find that the IDR optimizes the biochemical activities of coronins invivo and invitro. The budding yeast coronin IDR plays essential roles in regulating Crn1 activity by fine-tuning CC oligomerization and maintaining Crn1 as a tetramer. The IDR-guided optimization of Crn1 oligomerization is critical for F-actin cross-linking and regulation of Arp2/3-mediated actin polymerization. The final oligomerization status and homogeneity of Crn1 are contributed by three examined factors: helix packing, the energy landscape of the CC, and the length and molecular grammar of the IDR.

Structural basis of human PDZD8\u2013Rab7 interaction for the ER-late endosome tethering

The membrane contact sites (MCSs) between the ER and late endosomes (LEs) are essential for the regulation of endosomal protein sorting, dynamics, and motility. PDZD8 is an ER transmembrane protein containing a Synaptotagmin-like Mitochondrial lipid-binding Proteins (SMP) domain. PDZD8 tethers the ER to late endosomes and lysosomes by associating its C-terminal coiled-coil (CC) with the LE Rab7. To identify the structural determinants for the PDZD8–Rab7 interaction, we determined the crystal structure of the human PDZD8 CC domain in complex with the GTP-bound form of Rab7. The PDZD8 CC contains one short helix and the two helices forming an antiparallel coiled-coil. Two Rab7 molecules bind to the opposite sides of the PDZD8 CC in a 2:1 ratio. The switch I/II and interswitch regions of the GTP-loaded Rab7 form the binding interfaces, which correlates with the GTP-dependent interaction of PDZD8 and Rab7. Analysis of the protein interaction by isothermal titration calorimetry confirms that two Rab7 molecules bind the PDZD8 CC in a GTP-dependent manner. The structural model of the PDZD8 CC–Rab7 complex correlates with the recruitment of PDZD8 at the LE–ER interface and its role in lipid transport and regulation.

The carboxy terminal coiled-coil modulates Orai1 internalization during meiosis

Regulation of Ca2+ signaling is critical for the progression of cell division, especially during meiosis to prepare the egg for fertilization. The primary Ca2+ influx pathway in oocytes is Store-Operated Ca2+ Entry (SOCE). SOCE is tightly regulated during meiosis, including internalization of the SOCE channel, Orai1. Orai1 is a four-pass membrane protein with cytosolic N- and C-termini. Orai1 internalization requires a caveolin binding motif (CBM) in the N-terminus as well as the C-terminal cytosolic domain. However, the molecular determinant for Orai1 endocytosis in the C-terminus are not known. Here we show that the Orai1 C-terminus modulates Orai1 endocytosis during meiosis through a structural motif that is based on the strength of the C-terminal intersubunit coiled coil (CC) domains. Deletion mutants show that a minimal C-terminal sequence after transmembrane domain 4 (residues 260–275) supports Orai1 internalization. We refer to this region as the C-terminus Internalization Handle (CIH). Access to CIH however is dependent on the strength of the intersubunit CC. Mutants that increase the stability of the coiled coil prevent internalization independent of specific mutation. We further used human and Xenopus Orai isoforms with different propensity to form C-terminal CC and show a strong correlation between the strength of the CC and Orai internalization. Furthermore, Orai1 internalization does not depend on clathrin, flotillin or PIP2. Collectively these results argue that Orai1 internalization requires both the N-terminal CBM and C-terminal CIH where access to CIH is controlled by the strength of intersubunit C-terminal CC.

Dimerization of MORC2 through its C-terminal coiled-coil domain enhances chromatin dynamics and promotes DNA repair

Decondesation of the highly compacted chromatin architecture is essential for efficient DNA repair, but how this is achieved remains largely unknown. Here, we report that microrchidia family CW-type zinc finger protein 2 (MORC2), a newly identified ATPase-dependent chromatin remodeling enzyme, is required for nucleosome destabilization after DNA damage through loosening the histone-DNA interaction. Depletion of MORC2 attenuates phosphorylated histone H2AX (γH2AX) focal formation, compromises the recruitment of DNA repair proteins, BRCA1, 53BP1, and Rad51, to sites of DNA damage, and consequently reduces cell survival following treatment with DNA-damaging chemotherapeutic drug camptothecin (CPT). Furthermore, we demonstrate that MORC2 can form a homodimer through its C-terminal coiled-coil (CC) domain, a process that is enhanced in response to CPT-induced DNA damage. Deletion of the C-terminal CC domain in MORC2 disrupts its homodimer formation and impairs its ability to destabilize histone-DNA interaction after DNA damage. Consistently, expression of dimerization-defective MORC2 mutant results in impaired the recruitment of DNA repair proteins to damaged chromatin and decreased cell survival after CPT treatment. Together, these findings uncover a new mechanism for MORC2 in modulating chromatin dynamics and DDR signaling through its c-terminal dimerization.

Molecular and structural analysis of a mechanical transition of helices in the L. donovani coronin coiled-coil domain

Protein-protein interactions of cellular importance are mediated by coiled coils (CCs), the ubiquitous structural motif formed by the association of two or more α-helices in a knobs into holes manner. Coronins, actin-associated multi-functional proteins that possess distinct cytoskeleton-dependent and independent functions, oligomerize through their C-terminal CC domain. The structure of the L. donovani coronin CC domain (LdCoroCC; PDB ID 5CX2) revealed, in addition to a novel topology and architecture, an inherent asymmetry, with one of the helices of the 4-helix bundle axially shifted (~2 turns). The structural analysis identified that steric hindrance by Ile 486, Leu 493 and Met 500 as the cause for this asymmetry. To experimentally validate this hypothesis and to better understand the sequence-structure relationship in CCs, these amino acids have been mutated (I486A, L493A, M500V and the double mutant I486A-L493A) and characterized. Thermal CD studies suggest that the I486A and M500V mutants have comparable Tm values to LdCoroCC, while the other mutants have lower melting temperatures. The mutant crystal structures (I486A, M500V and the double mutant) retain the ‘ade’ core packing as LdcoroCC. While the M500V structure is similar to LdCoroCC, the I486A and the I486A-L493A structures show an asymmetry to symmetry transition. This study reveals crucial role of residues at position ‘a’ in coiled-coil domain play an important role in stabilizing the asymmetry in LdCoroCC, which might be necessary pursue specific biological function(s) inside the Leishmania.

Structure-based assessment of protein-protein interactions and accessibility of protein IX in adenoviruses with implications for antigen display

The exterior minor protein IX of adenoviruses (AdVs) is a frequent target of attachment of antigens and the modified AdVs are being used as potent vaccine platforms. The organization of protein IX is disticntly different between human adenoviruses (HAdVs) and non-HAdVs. The analysis of solvent accessibility, based on the near atomic resolution structures, suggests that the C-terminal residues of IX are more accessible in non-HAdVs (e.g., bovine adenovirus) than in HAdVs. Although the C-terminal fusions of IX are displayed on the capsid surface, they could disrupt the formation of tetrameric coiled-coils (4-HLXB) in HAdVs due to steric hinderance, thereby potentially affecting the capsid stability. Importantly, the parallel-antiparallel arrangement of helices seen in the 4-HLXB is not condusive for IX C-terminal fusions in HAdVs. In contrast, the parallel trimeric C-terminal coiled-coils in non-HAdVs are unlikely to be affected by the attachment of antigens and more efficiently displayed on the AdV surface.

TBK1 from orange-spotted grouper exerts antiviral activity against fish viruses and regulates interferon response

TANK-binding kinase-1 (TBK1) has been well studied in mammals because of its importance in type I interferon induction in antiviral immunity. However, the roles of fish TBK1 in virus infection still remained largely uncertain. In the current study, a TBK1 homolog from orange-spotted grouper (Epinephelus coioides) (EcTBK1) was cloned and its roles in fish viral infections were investigated. Sequence analysis showed that EcTBK1 encoded a 723-amino acid peptide which shared 98% and 73% identity to large yellow croaker (Larimichthys crocea) and human (homo sapiens), respectively. Multiple sequence alignments indicated that EcTBK1 contained conserved domains, including N-terminal kinase domain (KD), the middle ubiquitin-like domain (ULD) and C-terminal coiled-coil (CC) domains. The tissue distribution profiles demonstrated that EcTBK1 gene was constitutively expressed in all examined tissues, with predominant expression in intestine. Temporal expression analysis in vitro showed that the expression levels of EcTBK1 were significantly up-regulated in response to both red-spotted grouper nervous necrosis virus (RGNNV) and Singapore grouper iridovirus (SGIV) infection, suggested that EcTBK1 might exert crucial roles in fish virus infection. Subcellular localization indicated that EcTBK1 expression was primarily in the cytoplasm in GS cells. The ectopic expression of EcTBK1 significantly inhibited both SGIV and RGNNV replication. Furthermore, EcTBK1 overexpression significantly increased the expression levels of interferon related cytokines and pro-inflammatory factors. In addition, the overexpression of EcTBK1 increased the IRF3- and IRF7-regulated interferon promoter ISRE and IFN activity, and the regulatory effect on interferon immune response were dependent on its kinase domain. Together, we speculated that grouper TBK1 exerted antiviral activity against iridovirus and nodavirus via regulating the interferon immune and inflammatory response.

TBK1 of black carp plays an important role in host innate immune response against SVCV and GCRV

Tank-binding kinase 1 (TBK1) plays a pivotal role in the induction of type I IFNs in higher vertebrates. To explore the function of TBK1 in teleost, TBK1 of black carp (Mylopharyngodon Piceus) was cloned and characterized in this paper. The full-length cDNA of black carp TBK1 (bcTBK1) consists of 2857 nucleotides and the predicted bcTBK1 protein contains 727 amino acids, which includes an N-terminal kinase domain (KD), an ubiquitin-like domain (ULD) and two C-terminal coiled-coils. The transcription of bcTBK1 was constitutively detected in all the selected tissues and bcTBK1 mRNA level was increased in all selected tissues in response to SVCV or GCRV infection except that in muscle post GCRV invasion. The transcription of bcTBK1 in Mylopharyngodon Piceus fin (MPF) cells was up-regulated by the stimulation of SVCV, GCRV or poly (I:C) but not by LPS treatment. bcTBK1 migrated around 80 kDa in immunoblot assay and was identified as a cytosolic protein by immunofluorescence staining. bcTBK1 showed strong IFN-inducing ability in reporter assay and presented strong antiviral activity against both GCRV and SVCV in EPC cells. The reporter assay demonstrated that TRAF6 of black carp (bcTRAF6) up-regulated bcTBK1-induced IFN expression and the subcellular distribution of bcTBK1 overlapped with that of bcTRAF6 when these two proteins were co-expressed in EPC cells. Taken together, our study support the conclusion that bcTBK1 plays an important role in the antiviral innate immune response of black carp against SVCV and GCRV, in which its activity was positively regulated by bcTRAF6.

IFT54 regulates IFT20 stability but is not essential for tubulin transport during ciliogenesis.

Intraflagellar transport (IFT) is required for ciliogenesis by ferrying ciliary components using IFT complexes as cargo adaptors. IFT54 is a component of the IFT-B complex and is also associated with cytoplasmic microtubules (MTs). Loss of IFT54 impairs cilia assembly as well as cytoplasmic MT dynamics. The N-terminal calponin homology (CH) domain of IFT54 interacts with tubulins/MTs and has been proposed to transport tubulin during ciliogenesis, whereas the C-terminal coiled-coil (CC) domain binds IFT20. However, the precise function of these domains in vivo is not well understood. We showed that in Chlamydomonas, loss of IFT54 completely blocks ciliogenesis but does not affect spindle formation and proper cell cycle progression, even though IFT54 interacts with mitotic MTs. Interestingly, IFT54 lacking the CH domain allows proper flagellar assembly. The CH domain is required for the association of IFT54 with the axoneme but not with mitotic MTs, and also regulates the flagellar import of IFT54 but not IFT81 and IFT46. The C-terminal CC domain is essential for IFT54 to bind IFT20, and for its recruitment to the basal body and incorporation into IFT complexes. Complete loss of IFT54 or the CC domain destabilizes IFT20. ift54 mutant cells expressing the CC domain alone rescue the stability of IFT20 and form stunted flagella with accumulation of both IFT-A component IFT43 and IFT-B component IFT46, indicating that IFT54 also functions in IFT turn-around at the flagellar tip.

Plant 4/1 protein: potential player in intracellular, cell-to-cell and long-distance signaling

Originally isolated as a result of its ability to interact with the movement protein of Tomato spotted wilt virus in a yeast two-hybrid system, the 4/1 protein is proving to be an excellent tool for studying intracellular protein trafficking and intercellular communication. Expression of 4/1 in vivo is tightly regulated, first appearing in the veins of the cotyledon and later in the vasculature of the leaf and stem in association with the xylem parenchyma and phloem parenchyma. Structural studies indicate that 4/1 proteins contain as many as five coiled–coil (CC) domains; indeed, the highest level of sequence identity among 4/1 proteins involves their C-terminal CC domains, suggesting that protein–protein interaction is important for biological function. Recent data predict that the tertiary structure of this C-terminal CC domain is strikingly similar to that of yeast protein She2p; furthermore, like She2p, 4/1 protein exhibits RNA-binding activity, and mutational analysis has shown that the C-terminal CC domain is responsible for RNA binding. The 4/1 protein contains a nuclear export signal. Additional microscopy studies involving leptomycin and computer prediction suggest the presence of a nuclear localization signal as well.

Structure-Function Analysis of DipA, a Francisella tularensis Virulence Factor Required for Intracellular Replication

Francisella tularensis is a highly infectious bacterium whose virulence relies on its ability to rapidly reach the macrophage cytosol and extensively replicate in this compartment. We previously identified a novel Francisella virulence factor, DipA (FTT0369c), which is required for intramacrophage proliferation and survival, and virulence in mice. DipA is a 353 amino acid protein with a Sec-dependent signal peptide, four Sel1-like repeats (SLR), and a C-terminal coiled-coil (CC) domain. Here, we determined through biochemical and localization studies that DipA is a membrane-associated protein exposed on the surface of the prototypical F. tularensis subsp. tularensis strain SchuS4 during macrophage infection. Deletion and substitution mutagenesis showed that the CC domain, but not the SLR motifs, of DipA is required for surface exposure on SchuS4. Complementation of the dipA mutant with either DipA CC or SLR domain mutants did not restore intracellular growth of Francisella , indicating that proper localization and the SLR domains are required for DipA function. Co-immunoprecipitation studies revealed interactions with the Francisella outer membrane protein FopA, suggesting that DipA is part of a membrane-associated complex. Altogether, our findings indicate that DipA is positioned at the host–pathogen interface to influence the intracellular fate of this pathogen.

Molecular Determinants of TRPC Channels Gating by STIM1

We investigated if STIM1 SOAR, which has a coiled‐coil (CC) domain, interacts with the TRPC channel N‐ (NT) and/or C‐terminal (CT) CCs by determining how mutations in the TRPC CCs affect their interaction with SOAR and their gating by STIM1. We found that SOAR binds directly to TRPC1, TRPC4 and TRPC5, but not to TRPC3 and TRPC6. Mutations of 3 residues in the NT CCs of all TRPCs eliminate their surface expression and reduce binding of TRPC1 and TRPC5 to STIM1, while increasing binding of TRPC3 and TRPC6 to STIM1. In contrast, most single mutations in the NT CC of TRPC3 enhance binding to STIM1 with no appreciable effect on surface expression. Notably, I807S in the CT CC of TRPC3 has no effect on its interaction with STIM1, but eliminates the increased interaction observed by mutations of the NT CC. Accordingly, sensitivity of TRPC3 and TRPC3(L245S) to STIM1 knockdown by siRNA and scavenging STIM1 by the channel dead Orai1(R91W) show that a greater inhibition of STIM1 function is required to inhibit the function of TRPC3(L245S) compared to WT TRPC3. Lastly, TRPC3(I807S) eliminates regulation of TRPC3 and TRPC3(L245S) by STIM1. These findings suggest a model in which the TRPC3 NT and CT CCs interact to shield the CT CC from binding to STIM1. Dissociation of this interaction allows STIM1 SOAR to access and bind to the CT CC, to present the STIM1 polybasic domain to the conserved negative charges on TRPCs, and, ultimately, to open the channel.

Host-dependent differential expression of factor H binding proteins, their affinity to factor H and complement evasion by Lyme and relapsing fever borreliae

Binding of complement factor H is crucial for the resistance of Borrelia to complement-mediated lysis. This study was aimed to assess the correlation between the expression of fH binding proteins (FHBPs) during the early phase of infection (48 h after the entry of Borrelia into the blood circulation) and complement resistance of the Borrelia genus. As expected, B. afzelii, B. burgdorferi sensu stricto and B. garinii (Serotype 4, PBi) showed resistance to complement mediated lysis when incubated with human and dog complement, which coincided with the significantly higher expression ( P < 0.05) of the FHBPs. Similarly, B. coriaceae showed resistance to cattle complement. In non-reservoir hosts borreliae failed to induce expression of FHBPs within 48 h of complement challenge, and did not survive. It is important to note that not only the expression of FHBP but also their binding to fH is required for borrelial resistance to the complement. fH binding may depend on the coiled-coil (CC) motifs observed in the FHBPs, especially at the C terminus. A loss of the C-terminal CC motif in BgCRASP-1 of SKT-1 strain was found in in-silico CC prediction, and may be coupled with SKT-1's inability to bind factor H and evade complement-mediated attack. In contrast, the C-terminal CC motif was observed ( P – 1.0) in BgCRASP-1 of PBi that may contributed to its factor H binding and human complement resistance.

Ubiquitin-like Sequence in ASK1 Plays Critical Roles in the Recognition and Stabilization by USP9X and Oxidative Stress-Induced Cell Death

Ubiquitination is an important posttranslational modification that regulates various cellular processes, including signal transduction. However, physiological roles of ubiquitination in the regulation of MAPK pathways are poorly understood. Here, we identified the deubiquitinating enzyme USP9X as a binding partner of ASK1 that mediates oxidative stress-induced cell death through activation of the JNK and p38 MAPK pathways. In the recognition of ubiquitin by deubiquitinating enzymes, the importance of a tandem glycine-glycine sequence in the ubiquitin C terminus has been suggested. Interestingly, ASK1 contains six amino acids identical to the ubiquitin C terminus (LRLRGG), and the GG sequence of ASK1 was required for the USP9X-ASK1 interaction. We also found that USP9X interacted with oxidative stress-activated ASK1 and prevented it from undergoing ubiquitin-dependent degradation. In USP9X-deficient cells, oxidative stress-induced JNK activation and subsequent cell death were reduced. These results demonstrate that USP9X-dependent stabilization of activated ASK1 plays a crucial role in oxidative stress-induced cell death.

Probing the Interaction between the Coiled Coil Leucine Zipper of cGMP-dependent Protein Kinase Iα and the C Terminus of the Myosin Binding Subunit of the Myosin Light Chain Phosphatase

Nitric oxide and nitrovasodilators induce vascular smooth muscle cell relaxation in part by cGMP-dependent protein kinase I (PKG-Ialpha)-mediated activation of myosin phosphatase (MLCP). Mechanistically it has been proposed that protein-protein interactions between the N-terminal leucine zipper (LZ) domain of PKG-Ialpha ((PKG-Ialpha(1-59)) and the LZ and/or coiled coil (CC) domain of the myosin binding subunit (MBS) of MLCP are localized in the C terminus of MBS. Although recent studies have supported these interactions, the critical amino acids responsible for these interactions have not been identified. Here we present structural and biophysical data identifying that the LZ domain of PKG-Ialpha(1-59) interacts with a well defined 42-residue CC motif (MBS(CT42)) within the C terminus of MBS. Using glutathione S-transferase pulldown experiments, chemical cross-linking, size exclusion chromatography, circular dichroism, and isothermal titration calorimetry we identified a weak dimer-dimer interaction between PKG-Ialpha(1-59) and this C-terminal CC domain of MBS. The K(d) of this non-covalent complex is 178.0+/-1.5 microm. Furthermore our (1)H-(15)N heteronuclear single quantum correlation NMR data illustrate that this interaction is mediated by several PKG-Ialpha residues that are on the a, d, e, and g hydrophobic and electrostatic interface of the C-terminal heptad layers 2, 4, and 5 of PKG-Ialpha. Taken together these data support a role for the LZ domain of PKG-Ialpha and the CC domain of MBS in this requisite contractile complex.

Ubiquitin binding and conjugation regulate the recruitment of Rabex-5 to early endosomes

Rab GTPases and ubiquitination are critical regulators of transmembrane cargo sorting in endocytic and lysosomal targeting pathways. The endosomal protein Rabex-5 intersects these two layers of regulation by being both a guanine nucleotide exchange factor (GEF) for Rab5 and a substrate for ubiquitin (Ub) binding and conjugation. The ability of trafficking machinery components to bind ubiquitinated proteins is known to have a function in cargo sorting. Here, we demonstrate that Ub binding is essential for the recruitment of Rabex-5 from the cytosol to endosomes, independently of its GEF activity and of Rab5. We also show that monoubiquitinated Rabex-5 is enriched in the cytosol. These observations are consistent with a model whereby a cycle of Ub binding and monoubiquitination regulates the association of Rabex-5 with endosomes.

Cardiolipin Controls the Osmotic Stress Response and the Subcellular Location of Transporter ProP in Escherichia coli

The phospholipid composition of the membrane and transporter structure control the subcellular location and function of osmosensory transporter ProP in Escherichia coli. Growth in media of increasing osmolality increases, and entry to stationary phase decreases, the proportion of phosphatidate in anionic lipids (phosphatidylglycerol (PG) plus cardiolipin (CL)). Both treatments increase the CL:PG ratio. Transporters ProP and LacY are concentrated with CL (and not PG) near cell poles and septa. The polar concentration of ProP is CL-dependent. Here we show that the polar concentration of LacY is CL-independent. The osmotic activation threshold of ProP was directly proportional to the CL content of wild type bacteria, the PG content of CL-deficient bacteria, and the anionic lipid content of cells and proteoliposomes. CL was effective at a lower concentration in cells than in proteoliposomes, and at a much lower concentration than PG in either system. Thus, in wild type bacteria, osmotic induction of CL synthesis and concentration of ProP with CL at the cell poles adjust the osmotic activation threshold of ProP to match ambient conditions. ProP proteins linked by homodimeric, C-terminal coiled-coils are known to activate at lower osmolalities than those without such structures and coiled-coil disrupting mutations raise the osmotic activation threshold. Here we show that these mutations also prevent polar concentration of ProP. Stabilization of the C-terminal coiled-coil by covalent cross-linking of introduced Cys reverses the impact of increasing CL on the osmotic activation of ProP. Association of ProP C termini with the CL-rich membrane at cell poles may raise the osmotic activation threshold by blocking coiled-coil formation. Mutations that block coiled-coil formation may also block association of the C termini with the CL-rich membrane.

Identification of the Coiled-coil Domains of Enterococcus faecalis DivIVA that Mediate Oligomerization and their Importance for Biological Function

Bacillus subtilis (Bs) DivIVA comprises coiled-coil structures and self-associates forming a 10-12 mer complex in vitro. Using bioinformatic approaches, we determined that Enterococcus faecalis (Ef) DivIVA comprises four coiled-coil domains, one at the N-terminus, the second and the third in the central region of the protein and the fourth at the C-terminus. We determined that DivIVA(Ef) self-interacts and forms a 10-12 multimeric complex. Point mutations or deletions of the central regions predicted bioinformatically to disrupt the coiled-coil structures either eliminated or weakened DivIVA(Ef) self-interaction and reduced oligomerization. Mutations disrupting the N- and C-terminal coiled-coils of DivIVA(Ef) did not affect DivIVA(Ef) oligomerization. The introduction of DivIVA(Ef) mutations to both the N-terminal and the central coiled-coil domains were lethal unless rescued by expressing wild-type DivIVA(Ef) in trans. E. faecalis cells expressing these mutations displayed aberrant cell morphology, indicating disruption of the normal cell division phenotype. The results in E. faecalis also indicate that both the N-terminal and the central coiled-coil structures of DivIVA(Ef) are indispensable for proper biological function. Overexpression of wild-type DivIVA(Ef) in both rod-shaped and round Escherichia coli cells resulted in morphological changes, while the overexpression of DivIVA(Ef) mutations failed to induce such alterations.

On the self-association potential of transmembrane tight junction proteins.

Tight junctions seal intercellular clefts via membrane-related strands, hence, maintaining important organ functions. We investigated the self-association of strand-forming transmembrane tight junction proteins. The regulatory tight junction protein occludin was differently tagged and cotransfected in eucaryotic cells. These occludins colocalized within the plasma membrane of the same cell, coprecipitated and exhibited fluorescence resonance energy transfer. Differently tagged strand-forming claudin-5 also colocalized in the plasma membrane of the same cell and showed fluorescence resonance energy transfer. This demonstrates self-association in intact cells both of occludin and claudin-5 in one plasma membrane. In search of dimerizing regions of occludin, dimerization of its cytosolic C-terminal coiledcoil domain was identified. In claudin-5, the second extracellular loop was detected as a dimer. Since the transmembrane junctional adhesion molecule also is known to dimerize, the assumption that homodimerization of transmembrane tight junction proteins may serve as a common structural feature in tight junction assembly is supported.