Dataset Information

Crystal structure of the dimeric coiled-coil domain of the cytosolic nucleic acid sensor LRRFIP1.

ABSTRACT: LRRFIP1 binds cytoplasmic double-stranded DNA and RNA and interacts with FLI, the mammalian homolog of Drosophila flightless I, through a highly conserved 87-amino acid domain. Upon binding nucleic acid ligands, LRRFIP1 recruits and activates ?-catenin, leading to the IRF3-dependent production of type I interferon. However, the molecular mechanism of LRRFIP1 signaling is not well understood. Here we show that the FLI-interacting domain of LRRFIP1 forms a classic parallel, homodimeric coiled coil with 10 heptad repeats and 22 helical turns. The coiled coil domain is also a dimer in solution. However, a longer LRRFIP1 construct spanning the coiled coil and DNA binding domains assembles into higher order oligomers in solution. The structure of LRRFIP1-CC constitutes a valuable tool for probing the mechanism of LRRFIP1 signaling and for structural studies of larger LRRFIP1 constructs.

SUBMITTER: Nguyen JB

PROVIDER: S-EPMC3525766 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Crystal structure of the dimeric coiled-coil domain of the cytosolic nucleic acid sensor LRRFIP1.

Nguyen Jennifer B JB Modis Yorgo Y

Journal of structural biology 20121023 1

LRRFIP1 binds cytoplasmic double-stranded DNA and RNA and interacts with FLI, the mammalian homolog of Drosophila flightless I, through a highly conserved 87-amino acid domain. Upon binding nucleic acid ligands, LRRFIP1 recruits and activates β-catenin, leading to the IRF3-dependent production of type I interferon. However, the molecular mechanism of LRRFIP1 signaling is not well understood. Here we show that the FLI-interacting domain of LRRFIP1 forms a classic parallel, homodimeric coiled coil ...[more]

PMID: 23099021

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Project description:Bordetella pertussis controls the expression of its virulence regulon through the two-component system BvgAS. BvgS is a prototype for a family of multidomain sensor kinases. In BvgS, helical linkers connect periplasmic Venus flytrap (VFT) perception domains to a cytoplasmic Per-Arnt-Sim (PAS) domain and the PAS domain to the dimerization/histidine phosphotransfer (DHp) domain of the kinase. The two linkers can adopt coiled-coil structures but cannot do so simultaneously. The first linker forms a coiled coil in the kinase mode and the second in the phosphatase mode, with the other linker in both cases showing an increase in dynamic behavior. The intervening PAS domain changes its quaternary structure between the two modes. In BvgS homologues without a PAS domain, a helical "X" linker directly connects the VFT and DHp domains. Here, we used BvgS as a platform to characterize regulation in members of the PAS-less subfamily. BvgS chimeras of homologues with natural X linkers displayed various regulation phenotypes. We identified two distinct coiled-coil registers in the N- and C-terminal portions of the X linkers. Stable coil formation in the C-terminal moiety determines the phosphatase mode, similarly to BvgS; in contrast, coil formation in the N-terminal moiety along the other register leads to the kinase mode. Thus, antagonism between two registers in the VFT-DHp linker forms the basis for activity regulation in the absence of the PAS domain. The N and C moieties of the X linker play roles similar to those played by the two independent linkers in sensor kinases with a PAS domain, providing a unified mechanism of regulation for the entire family.IMPORTANCE The whooping cough agent Bordetella pertussis uses the BvgAS sensory transduction two-component system to regulate production of its virulence factors. BvgS serves as a model for a large family of multidomain bacterial sensor kinases. B. pertussis is virulent when BvgS functions as a kinase and avirulent when it switches to phosphatase activity in response to modulating signals. Understanding the molecular regulation of those proteins might lead to new antibacterial strategies. Here, we show that the linker regions between the perception and the enzymatic domains shift between distinct states of conformation in an alternating manner in response to signals and that their antagonistic changes control sensor kinase activity. These linker regions and mechanistic principles appear to be conserved among BvgS homologues, irrespective of the presence or absence of an intervening domain between the perception and the enzymatic domains. This work has thus uncovered general molecular mechanisms that regulate activity of sensor kinases in the BvgS family.

Project description:Two types of viruses are produced during the baculovirus life cycle: budded virus (BV) and occlusion-derived virus (ODV). A particular baculovirus protein, FP25K, is involved in the switch from BV to ODV production. Previously, FP25K from the model alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was shown to traffic ODV envelope proteins. However, FP25K localization and the domains involved are inconclusive. Here we used a quantitative approach to study FP25K subcellular localization during infection using an AcMNPV bacmid virus that produces a functional AcMNPV FP25K-green fluorescent protein (GFP) fusion protein. During cell infection, FP25K-GFP localized primarily to the cytoplasm, particularly amorphous structures, with a small fraction being localized in the nucleus. To investigate the sequences involved in FP25K localization, an alignment of baculovirus FP25K sequences revealed that the N-terminal putative coiled-coil domain is present in all alphabaculoviruses but absent in betabaculoviruses. Structural prediction indicated a strong relatedness of AcMNPV FP25K to long interspersed element 1 (LINE-1) open reading frame 1 protein (ORF1p), which contains an N-terminal coiled-coil domain responsible for cytoplasmic retention. Point mutations and deletions of this domain lead to a change in AcMNPV FP25K localization from cytoplasmic to nuclear. The coiled-coil and C-terminal deletion viruses increased BV production. Furthermore, a betabaculovirus FP25K protein lacking this N-terminal coiled-coil domain localized predominantly to the nucleus and exhibited increased BV production. These data suggest that the acquisition of this N-terminal coiled-coil domain in FP25K is important for the evolution of alphabaculoviruses. Moreover, with the divergence of preocclusion nuclear membrane breakdown in betabaculoviruses and membrane integrity in alphabaculoviruses, this domain represents an alphabaculovirus adaptation for nuclear trafficking of occlusion-associated proteins. IMPORTANCE:Baculovirus infection produces two forms of viruses: BV and ODV. Manufacturing of ODV involves trafficking of envelope proteins to the inner nuclear membrane, mediated partly through the FP25K protein. Since FP25K is present in alpha-, beta-, and gammabaculoviruses, it is uncertain if this trafficking function is conserved. In this study, we looked at alpha- and betabaculovirus FP25K trafficking by its localization. Alphabaculovirus FP25K localized primarily to the cytoplasm, whereas betabaculovirus FP25K localized to the nucleus. We found that an N-terminal coiled-coil domain present in all alphabaculovirus FP25K proteins, but absent in betabaculovirus FP25K, was critical for alphabaculovirus FP25K cytoplasmic localization. We believe that this represents an evolutionary process that partly led to the gain of function of this N-terminal coiled-coil domain in alphabaculovirus FP25K to aid in nuclear trafficking of occlusion-associated proteins. Due to betabaculovirus breakdown of the nuclear membrane before occlusion, this function is not needed, and the domain was lost or never acquired.

Dataset Information

Crystal structure of the dimeric coiled-coil domain of the cytosolic nucleic acid sensor LRRFIP1.

Publications

Crystal structure of the dimeric coiled-coil domain of the cytosolic nucleic acid sensor LRRFIP1.

Similar Datasets

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