Genome-wide functional analyses of plant coiled-coil NLR-type pathogen receptors reveal essential roles of their N-terminal domain in oligomerization, networking, and immunity.
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ABSTRACT:
SUBMITTER: Wroblewski T
PROVIDER: S-EPMC6312357 | biostudies-literature | 2018 Dec
REPOSITORIES: biostudies-literature
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Project description:The highly abundant ?-helical coiled-coil motif not only mediates crucial protein-protein interactions in the cell but is also an attractive scaffold in synthetic biology and material science and a potential target for disease intervention. Therefore a systematic understanding of the coiled-coil interactions (CCIs) at the organismal level would help unravel the full spectrum of the biological function of this interaction motif and facilitate its application in therapeutics. We report the first identified genome-wide CCI network in Saccharomyces cerevisiae, which consists of 3495 pair-wise interactions among 598 predicted coiled-coil regions. Computational analysis revealed that the CCI network is specifically and functionally organized and extensively involved in the organization of cell machinery. We further show that CCIs play a critical role in the assembly of the kinetochore, and disruption of the CCI network leads to defects in kinetochore assembly and cell division. The CCI network identified in this study is a valuable resource for systematic characterization of coiled coils in the shaping and regulation of a host of cellular machineries and provides a basis for the utilization of coiled coils as domain-based probes for network perturbation and pharmacological applications.
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Project description:Hantaviruses constitute a genus in the family Bunyaviridae. They are enveloped negative-strand RNA viruses with a tripartite genome encoding the nucleocapsid (N) protein, the two surface glycoproteins Gn and Gc, and an RNA-dependent RNA polymerase. The N protein is the most abundant component of the virion; it encapsidates genomic RNA segments forming ribonucleoproteins and participates in genome transcription and replication as well as virus assembly. In the course of RNA encapsidation, N protein forms intermediate trimers via head-to-head and tail-to-tail interactions. We analyzed the amino-terminal trimerization domain (amino acid residues 1 to 77) of Tula hantavirus using computer modeling, mammalian two-hybrid assay, and immunofluorescence assay. The results obtained were consistent with the existence of an antiparallel coiled-coil stabilized by interactions between hydrophobic residues. Residues L44, V51, and L58 were important for the N-N interaction; other residues, e.g., L25 and V32, also made a contribution, albeit a modest one. Our alignments of the N-terminal domain of the hantaviral N proteins suggest the coiled-coil structure, and hence the mode of N-protein oligomerization, is conserved among hantaviruses.
| S-EPMC1563903 | biostudies-literature
Project description:The Bcr oligomerization domain, from the Bcr-Abl oncoprotein, is an attractive therapeutic target for treating leukemias because it is required for cellular transformation. The domain homodimerizes via an antiparallel coiled coil with an adjacent short, helical swap domain. Inspection of the coiled-coil sequence does not reveal obvious determinants of helix-orientation specificity, raising the possibility that the antiparallel orientation preference and/or the dimeric oligomerization state are due to interactions of the swap domains. To better understand how structural specificity is encoded in Bcr, coiled-coil constructs containing either an N- or C-terminal cysteine were synthesized without the swap domain. When cross-linked to adopt exclusively parallel or antiparallel orientations, these showed similar circular dichroism spectra. Both constructs formed coiled-coil dimers, but the antiparallel construct was approximately 16 degrees C more stable than the parallel to thermal denaturation. Equilibrium disulfide-exchange studies confirmed that the isolated coiled-coil homodimer shows a very strong preference for the antiparallel orientation. We conclude that the orientation and oligomerization preferences of Bcr are not caused by the presence of the swap domains, but rather are directly encoded in the coiled-coil sequence. We further explored possible determinants of structural specificity by mutating residues in the d position of the coiled-coil core. Some of the mutations caused a change in orientation specificity, and all of the mutations led to the formation of higher-order oligomers. In the absence of the swap domain, these residues play an important role in disfavoring alternate states and are especially important for encoding dimeric oligomerization specificity.
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Project description:Coiled coils are extensively and successfully used nowadays to rationally design multistranded structures for applications, including basic research, biotechnology, nanotechnology, materials science, and medicine. The wide range of applications as well as the important functions these structures play in almost all biological processes highlight the need for a detailed understanding of the factors that control coiled-coil folding and oligomerization. Here, we address the important and unresolved question why the presence of particular oligomerization-state determinants within a coiled coil does frequently not correlate with its topology. We found an unexpected, general link between coiled-coil oligomerization-state specificity and trigger sequences, elements that are indispensable for coiled-coil formation. By using the archetype coiled-coil domain of the yeast transcriptional activator GCN4 as a model system, we show that well-established trimer-specific oligomerization-state determinants switch the peptide's topology from a dimer to a trimer only when inserted into the trigger sequence. We successfully confirmed our results in two other, unrelated coiled-coil dimers, ATF1 and cortexillin-1. We furthermore show that multiple topology determinants can coexist in the same trigger sequence, revealing a delicate balance of the resulting oligomerization state by position-dependent forces. Our experimental results should significantly improve the prediction of the oligomerization state of coiled coils. They therefore should have major implications for the rational design of coiled coils and consequently many applications using these popular oligomerization domains.
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Project description:Oligomerization is an important regulatory mechanism for many proteins, including oncoproteins and other pathogenic proteins. The oncoprotein Bcr-Abl relies on oligomerization via its coiled coil domain for its kinase activity, suggesting that a designed coiled coil domain with enhanced binding to Bcr-Abl and reduced self-oligomerization would be therapeutically useful. Key mutations in the coiled coil domain of Bcr-Abl were identified that reduce homo-oligomerization through intermolecular charge-charge repulsion yet increase interaction with the Bcr-Abl coiled coil through additional salt bridges, resulting in an enhanced ability to disrupt the oligomeric state of Bcr-Abl. The mutations were modeled computationally to optimize the design. Assays performed in vitro confirmed the validity and functionality of the optimal mutations, which were found to exhibit reduced homo-oligomerization and increased binding to the Bcr-Abl coiled coil domain. Introduction of the mutant coiled coil into K562 cells resulted in decreased phosphorylation of Bcr-Abl, reduced cell proliferation, and increased caspase-3/7 activity and DNA segmentation. Importantly, the mutant coiled coil domain was more efficacious than the wild type in all experiments performed. The improved inhibition of Bcr-Abl through oligomeric disruption resulting from this modified coiled coil domain represents a viable alternative to small molecule inhibitors for therapeutic intervention.
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Project description: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.
| S-EPMC7840751 | biostudies-literature
Project description:Minichromosome maintenance protein 10 (Mcm10) is an essential eukaryotic DNA-binding replication factor thought to serve as a scaffold to coordinate enzymatic activities within the replisome. Mcm10 appears to function as an oligomer rather than in its monomeric form (or rather than as a monomer). However, various orthologs have been found to contain 1, 2, 3, 4, or 6 subunits and thus, this issue has remained controversial. Here, we show that self-association of Xenopus laevis Mcm10 is mediated by a conserved coiled-coil (CC) motif within the N-terminal domain (NTD). Crystallographic analysis of the CC at 2.4 Å resolution revealed a three-helix bundle, consistent with the formation of both dimeric and trimeric Mcm10 CCs in solution. Mutation of the side chains at the subunit interface disrupted in vitro dimerization of both the CC and the NTD as monitored by analytical ultracentrifugation. In addition, the same mutations also impeded self-interaction of the full-length protein in vivo, as measured by yeast-two hybrid assays. We conclude that Mcm10 likely forms dimers or trimers to promote its diverse functions during DNA replication.
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Project description:Ubiquitin ligase TRAF6, together with ubiquitin-conjugating enzyme Ubc13/Uev1, catalyzes processive assembly of unanchored K63-linked polyubiquitin chains for TAK1 activation in the IL-1R/TLR pathways. However, what domain and how it functions to enable TRAF6's processivity are largely uncharacterized. Here, we find TRAF6 coiled-coil (CC) domain is crucial to enable its processivity. The CC domain mediates TRAF6 oligomerization to ensure efficient long polyubiquitin chain assembly. Mutating or deleting the CC domain impairs TRAF6 oligomerization and processive polyubiquitin chain assembly. Fusion of the CC domain to the E3 ubiquitin ligase CHIP/STUB1 renders the latter capable of NF-κB activation. Moreover, the CC domain, after oligomerization, interacts with Ubc13/Ub~Ubc13, which further contributes to TRAF6 processivity. Point mutations within the CC domain that weaken TRAF6 interaction with Ubc13/Ub~Ubc13 diminish TRAF6 processivity. Our results reveal that the CC oligomerization primes its interaction with Ubc13/Ub~Ubc13 to confer processivity to TRAF6 ubiquitin ligase activity.Ubiquitin ligase TRAF6 catalyzes assembly of free polyubiquitin chains for TAK1 activation in the IL-1R/TLR pathways, but the mechanism underlying its processivity is unclear. Here, the authors show that TRAF6 coiled-coil oligomerization domain primes its interaction with Ubc13/Ub~Ubc13 to confer processivity.
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Project description:The plant immune system is fundamental for plant survival in natural ecosystems and for productivity in crop fields. Substantial evidence supports the prevailing notion that plants possess a two-tiered innate immune system, called pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI is triggered by microbial patterns via cell surface-localized pattern-recognition receptors (PRRs), whereas ETI is activated by pathogen effector proteins via predominantly intracellularly localized receptors called nucleotide-binding, leucine-rich repeat receptors (NLRs)1-4. PTI and ETI are initiated by distinct activation mechanisms and involve different early signalling cascades5,6. Here we show that Arabidopsis PRR and PRR co-receptor mutants-fls2 efr cerk1 and bak1 bkk1 cerk1 triple mutants-are markedly impaired in ETI responses when challenged with incompatible Pseudomonas syrinage bacteria. We further show that the production of reactive oxygen species by the NADPH oxidase RBOHD is a critical early signalling event connecting PRR- and NLR-mediated immunity, and that the receptor-like cytoplasmic kinase BIK1 is necessary for full activation of RBOHD, gene expression and bacterial resistance during ETI. Moreover, NLR signalling rapidly augments the transcript and/or protein levels of key PTI components. Our study supports a revised model in which potentiation of PTI is an indispensable component of ETI during bacterial infection. This revised model conceptually unites two major immune signalling cascades in plants and mechanistically explains some of the long-observed similarities in downstream defence outputs between PTI and ETI.
| S-EPMC8016741 | biostudies-literature
Project description:Detailed mechanistic understanding of L1 retrotransposition is sparse, particularly with respect to ORF1p, a coiled coil-mediated homotrimeric nucleic acid chaperone that can form tightly packed oligomers on nucleic acids. Although the coiled coil motif is highly conserved, it is uniquely susceptible to evolutionary change. Here we studied three ORF1 proteins: a modern human one (111p), its resuscitated primate ancestor (555p) and a mosaic modern protein (151p) wherein 9 of the 30 coiled coil substitutions retain their ancestral state. While 111p and 555p equally supported retrotransposition, 151p was inactive. Nonetheless, they were fully active in bulk assays of nucleic acid interactions including chaperone activity. However, single molecule assays showed that 151p trimers form stably bound oligomers on ssDNA at <1/10th the rate of the active proteins, revealing that oligomerization rate is a novel critical parameter of ORF1p activity in retrotransposition conserved for at least the last 25 Myr of primate evolution.
| S-EPMC4705668 | biostudies-literature