scholarly journals Involvement of FtsE ATPase and FtsX Extracellular Loops 1 and 2 in FtsEX-PcsB Complex Function in Cell Division of Streptococcus pneumoniae D39

mBio ◽  
2013 ◽  
Vol 4 (4) ◽  
Author(s):  
Lok-To Sham ◽  
Katelyn R. Jensen ◽  
Kevin E. Bruce ◽  
Malcolm E. Winkler
Keyword(s):  
Signal Transduction ◽  
Amino Acids ◽  
Cell Division ◽  
Streptococcus Pneumoniae ◽  
Amino Acid ◽  
Stationary Phase ◽  
Coiled Coil ◽  
Extracellular Loop ◽  
Coiled Coil Domain ◽  
Loop Domain

ABSTRACT The FtsEX protein complex has recently been proposed to play a major role in coordinating peptidoglycan (PG) remodeling by hydrolases with the division of bacterial cells. According to this model, cytoplasmic FtsE ATPase interacts with the FtsZ divisome and FtsX integral membrane protein and powers allosteric activation of an extracellular hydrolase interacting with FtsX. In the major human respiratory pathogen Streptococcus pneumoniae (pneumococcus), a large extracellular-loop domain of FtsX (ECL1FtsX) is thought to interact with the coiled-coil domain of the PcsB protein, which likely functions as a PG amidase or endopeptidase required for normal cell division. This paper provides evidence for two key tenets of this model. First, we show that FtsE protein is essential, that depletion of FtsE phenocopies cell defects caused by depletion of FtsX or PcsB, and that changes of conserved amino acids in the FtsE ATPase active site are not tolerated. Second, we show that temperature-sensitive (Ts) pcsB mutations resulting in amino acid changes in the PcsB coiled-coil domain (CCPcsB) are suppressed by ftsX mutations resulting in amino acid changes in the distal part of ECL1FtsX or in a second, small extracellular-loop domain (ECL2FtsX). Some FtsX suppressors are allele specific for changes in CCPcsB, and no FtsX suppressors were found for amino acid changes in the catalytic PcsB CHAP domain (CHAPPcsB). These results strongly support roles for both ECL1FtsX and ECL2FtsX in signal transduction to the coiled-coil domain of PcsB. Finally, we found that pcsB CC(Ts) mutants (Ts mutants carrying mutations in the region of pcsB corresponding to the coiled-coil domain) unexpectedly exhibit delayed stationary-phase autolysis at a permissive growth temperature. IMPORTANCE Little is known about how FtsX interacts with cognate PG hydrolases in any bacterium, besides that ECL1FtsX domains somehow interact with coiled-coil domains. This work used powerful genetic approaches to implicate a specific region of pneumococcal ECL1FtsX and the small ECL2FtsX in the interaction with CCPcsB. These findings identify amino acids important for in vivo signal transduction between FtsX and PcsB for the first time. This paper also supports the central hypothesis that signal transduction between pneumococcal FtsX and PcsB is linked to ATP hydrolysis by essential FtsE, which couples PG hydrolysis to cell division. The classical genetic approaches used here can be applied to dissect interactions of other integral membrane proteins involved in PG biosynthesis. Finally, delayed autolysis of the pcsB CC(Ts) mutants suggests that the FtsEX-PcsB PG hydrolase may generate a signal in the PG necessary for activation of the major LytA autolysin as pneumococcal cells enter stationary phase.

Three differentially expressed survivin cDNA variants encode proteins with distinct antiapoptotic functions

Blood ◽  
2000 ◽  
Vol 95 (4) ◽  
pp. 1435-1442 ◽  
Author(s):  
Edward M. Conway ◽  
Saskia Pollefeyt ◽  
Jan Cornelissen ◽  
Inky DeBaere ◽  
Marta Steiner-Mosonyi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Messenger Rna ◽  
Coiled Coil ◽  
Cdna Clones ◽  
Exon 2 ◽  
Reading Frame ◽  
Coiled Coil Domain ◽  
Acid Protein ◽  
Apoptosis Protein

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is believed to play a role in oncogenesis. To elucidate further its physiologic role(s), we have characterized the murinesurvivin gene and complementary DNA (cDNA). The structural organization of the survivin gene, located on chromosome 11E2, is similar to that of its human counterpart, both containing 4 exons. Surprisingly, 3 full-length murine survivin cDNA clones were isolated, predicting the existence of 3 distinct survivin proteins. The longest open reading frame, derived from all 4 exons, predicts a 140-amino acid residue protein, survivin140, similar to human survivin, which contains a single IAP repeat and a COOH-terminal coiled-coil domain that links its function to the cell cycle. A second cDNA, which retains intron 3, predicts the existence of a 121-amino acid protein, survivin121 that lacks the coiled-coil domain. Removal of exon 2-derived sequences by alternative pre-messenger RNA (mRNA) splicing results in a third 40-amino acid residue protein, survivin40, lacking the IAP repeat and coiled-coil structure. Predictably, only recombinant survivin140 and survivin121 inhibited caspase-3 activity. All 3 mRNA species were variably expressed during development from 7.5 days postcoitum. Of the adult tissues surveyed, thymus and testis accumulated high levels of survivin140 mRNA, whereas survivin121-specific transcripts were detected in all tissues, while those representing survivin40 were absent. Human counterparts to the 3 survivin mRNA transcripts were identified in a study of human cells and tissues. The presence of distinct isoforms of survivin that are expressed differentially suggests that survivin plays a complex role in regulating apoptosis.

scholarly journals Structural studies on lamin. Similarities and differences between lamin and intermediate-filament proteins

1986 ◽  
Vol 238 (1) ◽  
pp. 305-308 ◽  
Author(s):  
D A D Parry ◽  
J F Conway ◽  
P M Steinert
Keyword(s):  
Amino Acid ◽  
Coiled Coil ◽  
Amino Acid Sequences ◽  
Ionic Interactions ◽  
Structural Studies ◽  
Intermediate Filament Proteins ◽  
Coiled Coil Domain ◽  
Similarities And Differences ◽  
Almost Continuous

Analysis of the amino acid sequences of lamins A and C has revealed that each chain has an almost continuous heptad-containing coiled-coil domain containing structural regularities in the linear disposition of the acidic and the basic residues. The data suggest that the lamin molecules are two-stranded ropes, that the two chains are parallel to one another and in axial register, and that the molecules aggregate in vivo through periodic ionic interactions. These results indicate that significant changes in stability of the nuclear envelope may be achieved between interphase and mitosis through changes in the degree of phosphorylation of the lamin proteins.

scholarly journals Sec2 protein contains a coiled-coil domain essential for vesicular transport and a dispensable carboxy terminal domain.

1990 ◽  
Vol 110 (6) ◽  
pp. 1897-1909 ◽  
Author(s):  
J Nair ◽  
H Müller ◽  
M Peterson ◽  
P Novick
Keyword(s):  
Amino Acids ◽  
Secretory Pathway ◽  
Synthetic Lethality ◽  
Coiled Coil ◽  
Protein Product ◽  
Temperature Sensitive ◽  
Vesicular Traffic ◽  
Amino Terminal ◽  
Native Molecular Mass ◽  
Coiled Coil Domain

SEC2 function is required at the post-Golgi apparatus stage of the yeast secretory pathway. The SEC2 sequence encodes a protein product of 759 amino acids containing an amino terminal region that is predicted to be in an alpha-helical, coiled-coil conformation. Two temperature-sensitive alleles, sec2-41 and sec2-59, encode proteins truncated by opal stop codons and are suppressible by an opal tRNA suppressor. Deletion analysis indicates that removal of the carboxyl terminal 251 amino acids has no apparent phenotype, while truncation of 368 amino acids causes temperature sensitivity. The amino terminal half of the protein, containing the putative coiled-coil domain, is essential at all temperatures. Sec2 protein is found predominantly in the soluble fraction and displays a native molecular mass of greater than 500 kD. All phenotypes of the temperature-sensitive sec2 alleles are partially suppressed by duplication of the SEC4 gene, but the lethality of a sec2 disruption is not suppressed. The sec2-41 mutation exhibits synthetic lethality with the same subset of the late acting sec mutants as does sec4-8 and sec15-1. The Sec2 protein may function in conjunction with the Sec4 and Sec15 proteins to control vesicular traffic.

Characterization of Two New Imatinib-Responsive Fusion Genes Generated by Disruption of PDGFRB in Eosinophilia-Associated Chronic Myeloproliferative Disorders.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 667-667
Author(s):  
Christoph Walz ◽  
Georgia Metzgeroth ◽  
Claudia Schoch ◽  
Torsten Haferlach ◽  
Rudiger Hehlmann ◽  
...  
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Tyrosine Kinase ◽  
Coiled Coil ◽  
Myeloproliferative Disorders ◽  
Fusion Genes ◽  
Protein Protein Interaction ◽  
Coiled Coil Domain ◽  
Exon 11

Abstract Fusion genes involving PDGFRA, PDGFRB, FGFR1 and JAK2 are seen in a substantial number of patients with BCR-ABL negative myeloproliferative disorders (MPD) and result in constitutive activation of the corresponding tyrosine kinase moiety. The vast majority of tyrosine kinase fusion partners contain coiled-coil domains or other dimerization motifs properties that are essential for malignant transformation. We have identified two patients presenting with eosinophilia-associated MPD and a t(5;12)(q31;q24) or a complex translocation t(1;5;11) with involvement of 5q31, respectively, suggesting a possible involvement of the PDGFRB gene which is located at chromosome band 5q31–33. 5′-rapid amplification of cDNA ends (5′-RACE) for the t(5;12) identified an in-frame mRNA fusion between ’G protein-coupled receptor kinase interactor 2′ (GIT2) exon 12 at 12q24 and PDGFRB exon 11. GIT2 is a member of the GIT protein family that is extensively alternative spliced in many distinct forms causing its functional diversity. A reciprocal transcript was amplified by RT-PCR with a fusion between PDGFRB exon 10 and GIT2 exon 13. GIT2-PDGFRB is predicted to be translated into a 742 amino acid fusion protein that retains the GIT2 N-terminal protein-protein interaction motif Ankyrin and an Arf GTPase activating protein (ArfGAP) domain fused to the transmembrane and catalytic domain of PDGFRB. The truncated GIT2 protein lacks coiled-coil domains as they are lost in the fusion protein due to the breakpoint within GIT2 intron 12. We therefore speculate that the Ankyrin repeat, which is one of the most common protein-protein interaction motifs in nature, may have replaced the function of a coiled-coil domain offering dimerization properties to the fusion protein. 5′-RACE for the complex t(1;5;11) identified an in-frame mRNA fusion between ’GPI-anchored membrane protein 1′ (GPIAP1) exon 7 at 11p13 and PDGFRB exon 11. Normal GPIAP1 is a cytoplasmic phosphoprotein which plays a mainly uncharacterized role in cellular activation or proliferation. The chimeric mRNA is predicted to encode an 803 amino acid fusion protein retaining the coiled-coil domain of GPIAP1 fused to the transmembrane and catalytic domains of PDGFRB. Both patients have been treated with 400 mg/day imatinib, which is a selective inhibitor of PDGFRB, and achieved rapid complete clinical and hematological remission. Residual GIT2-PDGFRB transcripts could be detected repeatedly during a 17 months follow up in case 1 whereas no follow-up samples have been available for case 2. These data give further evidence that numerous partner genes fuse to PDGFRB in BCR-ABL negative MPDs. In addition, the data demonstrate that cytogenetic analysis is a mandatory technique for the identification of tyrosine kinase fusion genes. In cases with abnormalities of chromosome 5q, a possible involvement of PDGFRB should be screened by adequate FISH and PCR-based techniques. Although their occurrence is rare in general, the identification of these fusion genes is essential for the successful treatment with tyrosine kinase inhibitors.

scholarly journals Mutations in the Drosophila hook gene inhibit endocytosis of the boss transmembrane ligand into multivesicular bodies.

1996 ◽  
Vol 133 (6) ◽  
pp. 1205-1215 ◽  
Author(s):  
H Krämer ◽  
M Phistry
Keyword(s):  
Amino Acids ◽  
Transmembrane Protein ◽  
Coiled Coil ◽  
Specific Pattern ◽  
Multivesicular Bodies ◽  
Endocytic Compartment ◽  
Coiled Coil Domain ◽  
Type Pattern ◽  
Cell Type Specific ◽  
Endocytic Vesicles

Transmembrane ligands can be internalized across cell boundaries into receptor-expressing cells. In the developing Drosophila eye imaginal disc, the bride of sevenless transmembrane protein (boss) is expressed on the surface of R8 cells. After internalization into neighboring R7 cells, the boss protein accumulates in multivesicular bodies. In a search for genes that affect this cell-type-specific pattern of boss endocytosis, we found that mutations in the hook gene inhibit the accumulation of boss in multivesicular bodies of R7 cells. In addition, hook flies exhibit pleiotropic phenotypes including abnormal bristle morphology and eye degeneration. The wild-type-pattern of boss endocytosis was restored in hook mutants by a genomic rescue fragment containing the hook gene or by a hook cDNA expressed in R7 cells under control of a sevenless (sev) enhancer. The hook gene encodes a novel cytoplasmic protein of 679 amino acids with a central coiled-coil domain of some 200 amino acids. Truncated, epitope-tagged hook proteins coimmunoprecipitated the full-length protein, indicating dimerization mediated by the coiled-coil domain. The hook protein localizes to vesicular structures that are part of the endocytic compartment. The requirement of the hook protein in R7 cells for the accumulation of boss protein in multivesicular bodies, and the localization of the hook protein to endocytic vesicles indicate that the hook gene encodes a novel component of the endocytic compartment that plays an important role in the endocytosis of transmembrane ligands or their transport to multivesicular bodies.

scholarly journals Amino acid sensing in dietary-restriction-mediated longevity: roles of signal-transducing kinases GCN2 and TOR

2012 ◽  
Vol 449 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Jordan Gallinetti ◽  
Eylul Harputlugil ◽  
James R. Mitchell
Keyword(s):  
Signal Transduction ◽  
Amino Acids ◽  
Amino Acid ◽  
Dietary Restriction ◽  
Stress Resistance ◽  
Essential Amino Acid ◽  
Signal Transduction Pathways ◽  
Evolutionarily Conserved ◽  
Amino Acid Intake ◽  
Metabolic Fitness

DR (dietary restriction), or reduced food intake without malnutrition, is associated with extended longevity, improved metabolic fitness and increased stress resistance in a wide range of organisms. DR is often referred to as calorie restriction, implying that reduced energy intake is responsible for its widespread and evolutionarily conserved benefits. However, recent data indicate dietary amino acid restriction as a key mediator of DR benefits. In fruitflies, an imbalance in essential amino acid intake is thought to underlie longevity benefits of DR. In mammals, reduced dietary protein or essential amino acid intake can extend longevity, improve metabolic fitness and increase stress resistance. In the present paper we review two evolutionarily conserved signal transduction pathways responsible for sensing amino acid levels. The eIF2α (eukaryotic initiation factor 2α) kinase GCN2 (general amino acid control non-derepressible 2) senses the absence of one or more amino acids by virtue of direct binding to uncharged cognate tRNAs. The presence of certain amino acids, such as leucine, permits activation of the master growth regulating kinase TOR (target of rapamycin). These two signal transduction pathways react to amino acid deprivation by inhibiting general protein translation while at the same time increasing translation of specific mRNAs involved in restoring homoeostasis. Together, these pathways may contribute to the regulation of longevity, metabolic fitness and stress resistance.

scholarly journals Nonideal polar residues in the core of the coiled coil of FtsLB are critical for modulating its stability and activation

2021 ◽  
Author(s):  
Samuel J Craven ◽  
Samson G.F. Condon ◽  
Gladys Diaz-Vazquez ◽  
Qiang Cui ◽  
Alessandro Senes
Keyword(s):  
Amino Acids ◽  
Cell Wall ◽  
Cell Division ◽  
Conformational Changes ◽  
Structural Model ◽  
Coiled Coil ◽  
Fine Tuning ◽  
Marginal Stability ◽  
Structural Element ◽  
The Core

The FtsLB complex is a critical regulator of bacterial cell division, acting as a switch that modulates cell wall reconstruction. Evidence indicates that FtsLB exists in either an off or on state which supports the corresponding activation state of the peptidoglycan synthase complex FtsWI. In Escherichia coli, residues within FtsLB that are critical for this activation are located in a region near the C-terminal end of the periplasmic coiled coil, raising questions about the precise role of this conserved domain in the mechanism. Here, we investigate an unusual cluster of polar amino acids occurring within the core of the coiled coil. These amino acids likely reduce the structural stability of the domain and thus may be important for governing conformational changes. We found that mutating these positions to hydrophobic residues increased the thermal stability of FtsLB but caused cell division defects, suggesting that the coiled-coil domain is an intentionally "detuned" structural element. In addition, suppressor mutations were identified within the polar cluster, indicating that the precise identity of the polar amino acids is important for fine-tuning the structural balance between the off and on states. Based on energetic and sequence propensity considerations, we propose a revised structural model of the tetrameric FtsLB (named the "Y-model") in which the periplasmic domain splits into a pair of coiled-coil branches. In this configuration, the polar amino acids participate in packing within the core, but their hydrophilic terminal moieties remain more favorably exposed to water than in the original four-helix bundle model ("I-model"). The Y-model remains well structured during molecular dynamics simulations, unlike the I-model, and satisfies all known experimental constraints. For this reason, we propose the Y-model as the configuration of the coiled coil of FtsLB and that a shift in this architecture, dependent on its marginal stability, is involved in activating the complex during the process that triggers septal cell wall reconstruction.

scholarly journals Mutations Enhancing Amino Acid Catabolism Confer a Growth Advantage in Stationary Phase

1999 ◽  
Vol 181 (18) ◽  
pp. 5800-5807 ◽  
Author(s):  
Erik R. Zinser ◽  
Roberto Kolter
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Stationary Phase ◽  
Carbon Starvation ◽  
Amino Acid Catabolism ◽  
Rich Media ◽  
Dying Cells

ABSTRACT Starved cultures of Escherichia coli undergo successive rounds of population takeovers by mutants of increasing fitness. These mutants express the growth advantage in stationary phase (GASP) phenotype. Previous work identified the rpoS819 allele as a GASP mutation allowing cells to take over stationary-phase cultures after growth in rich media (M. M. Zambrano, D. A. Siegele, M. A. Almirón, A. Tormo, and R. Kolter, Science 259:1757–1760, 1993). Here we have identified three new GASP loci from an aged rpoS819 strain: sgaA, sgaB, and sgaC. Each locus is capable of conferring GASP on therpoS819 parent, and they can provide successively higher fitnesses for the bacteria in the starved cultures. All four GASP mutations isolated thus far allow for faster growth on both individual and mixtures of amino acids. Each mutation confers a growth advantage on a different subset of amino acids, and these mutations act in concert to increase the overall catabolic capacity of the cell. We present a model whereby this enhanced ability to catabolize amino acids is responsible for the fitness gain during carbon starvation, as it may allow GASP mutants to outcompete the parental cells when growing on the amino acids released by dying cells.

scholarly journals Identification of functional regions on the tail of Acanthamoeba myosin-II using recombinant fusion proteins. II. Assembly properties of tails with NH2- and COOH-terminal deletions.

1990 ◽  
Vol 111 (6) ◽  
pp. 2417-2426 ◽  
Author(s):  
J H Sinard ◽  
D L Rimm ◽  
T D Pollard
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Residue ◽  
Fusion Proteins ◽  
Divalent Cations ◽  
Myosin Ii ◽  
Coiled Coil ◽  
Second Step ◽  
Recombinant Fusion Proteins ◽  
Functional Regions

We used purified fusion proteins containing parts of the Acanthamoeba myosin-II tail to localize those regions of the tail responsible for each of the three steps in the successive dimerization mechanism (Sinard, J. H., W. F. Stafford, and T. D. Pollard. 1989. J. Cell Biol. 107:1537-1547) for Acanthamoeba myosin-II minifiliment assembly. Fusion proteins containing the terminal approximately 90% of the myosin-II tail assemble normally, but deletions within the last 100 amino acids of the tail sequence alter or prevent assembly. The first step in minifilament assembly, formation of antiparallel dimers, requires the COOH-terminal approximately 30 amino acids that are thought to form a nonhelical domain at the end of the coiled-coil. The second step, formation of antiparallel tetramers, requires the last approximately 40 residues in the coiled-coil. The final step, the association of two antiparallel tetramers to form the completed octameric minifilament, requires residues approximately 40-70 from the end of the coiled-coil. A region of the tail near the junction with the heads is important for tight packing of the tails in the minifilaments. Divalent cations induce the lateral aggregation of minifilaments formed from native myosin-II or fusion proteins containing a nonmyosin "head," but under the same conditions fusion proteins composed essentially only of myosin tail sequences with very little nonmyosin sequences form paracrystals. The region of the tail necessary for this paracrystal formation lies NH2-terminal to amino acid residue 1,468 in the native myosin-II sequence.

scholarly journals Amino Acid Residues in the Fusion Peptide Pocket Regulate the pH of Activation of the H5N1 Influenza Virus Hemagglutinin Protein

2009 ◽  
Vol 83 (8) ◽  
pp. 3568-3580 ◽  
Author(s):  
Mark L. Reed ◽  
Hui-Ling Yen ◽  
Rebecca M. DuBois ◽  
Olga A. Bridges ◽  
Rachelle Salomon ◽  
...  
Keyword(s):  
Amino Acid ◽  
Influenza A Virus ◽  
Influenza A ◽  
Coiled Coil ◽  
Fusion Peptide ◽  
Influenza Viruses ◽  
Amino Acid Residues ◽  
H5n1 Influenza ◽  
Coiled Coil Domain ◽  
Ha Protein

ABSTRACT The receptor specificity and cleavability of the hemagglutinin (HA) protein have been shown to regulate influenza A virus transmissibility and pathogenicity, but little is known about how its pH of activation contributes to these important biological properties. To identify amino acid residues that regulate the acid stability of the HA protein of H5N1 influenza viruses, we performed a mutational analysis of the HA protein of the moderately pathogenic A/chicken/Vietnam/C58/04 (H5N1) virus. Nineteen HA proteins containing point mutations in the HA2 coiled-coil domain or in an HA1 histidine or basic patch were generated. Wild-type and mutant HA plasmids were transiently transfected in cell culture and analyzed for total protein expression, surface expression, cleavage efficiency, pH of fusion, and pH of conformational change. Four mutations to residues in the fusion peptide pocket, Y23H and H24Q in the HA1 subunit and E105K and N114K in the HA2 subunit, and a K58I mutation in the HA2 coiled-coil domain significantly altered the pH of activation of the H5 HA protein. In some cases, the magnitude and direction of changes of individual mutations in the H5 HA protein differed considerably from similar mutations in other influenza A virus HA subtypes. Introduction of Y23H, H24Q, K58I, and N114K mutations into recombinant viruses resulted in virus-expressed HA proteins with similar shifts in the pH of fusion. Overall, the data show that residues comprising the fusion peptide pocket are important in triggering pH-dependent activation of the H5 HA protein.

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