scholarly journals Crystallographic study of the phosphoethanolamine transferase EptC required for polymyxin resistance and motility inCampylobacter jejuni

2014 ◽  
Vol 70 (10) ◽  
pp. 2730-2739 ◽  
Author(s):  
Christopher D. Fage ◽  
Dusty B. Brown ◽  
Joseph M. Boll ◽  
Adrian T. Keatinge-Clay ◽  
M. Stephen Trent
Keyword(s):  
Cell Surface ◽  
Lipid A ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Cationic Antimicrobial Peptide ◽  
Cell Surface Structures ◽  
Zinc Binding Site ◽  
Flagellar Assembly ◽  
Antimicrobial Peptide Resistance

The foodborne enteric pathogenCampylobacter jejunidecorates a variety of its cell-surface structures with phosphoethanolamine (pEtN). Modifying lipid A with pEtN promotes cationic antimicrobial peptide resistance, whereas post-translationally modifying the flagellar rod protein FlgG with pEtN promotes flagellar assembly and motility, which are processes that are important for intestinal colonization. EptC, the pEtN transferase required for all known pEtN cell-surface modifications inC. jejuni, is a predicted inner-membrane metalloenzyme with a five-helix N-terminal transmembrane domain followed by a soluble sulfatase-like catalytic domain in the periplasm. The atomic structure of the catalytic domain of EptC (cEptC) was crystallized and solved to a resolution of 2.40 Å. cEptC adopts the α/β/α fold of the sulfatase protein family and harbors a zinc-binding site. A phosphorylated Thr266 residue was observed that was hypothesized to mimic a covalent pEtN–enzyme intermediate. The requirement for Thr266 as well as the nearby residues Asn308, Ser309, His358 and His440 was ascertainedvia in vivoactivity assays on mutant strains. The results establish a basis for the design of pEtN transferase inhibitors.

Interaction of the endoplasmic reticulum α1,2-mannosidase Mns1p with Rer1p using the split-ubiquitin system

2001 ◽  
Vol 114 (24) ◽  
pp. 4629-4635
Author(s):  
Michel J. Massaad ◽  
Annette Herscovics
Keyword(s):  
Endoplasmic Reticulum ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Protein A ◽  
Yeast Cells ◽  
Type Ii ◽  
Ubiquitin System ◽  
Endoplasmic Reticulum Protein ◽  
Split Ubiquitin

The α1,2-mannosidase Mns1p involved in the N-glycosidic pathway in Saccharomyces cerevisiae is a type II membrane protein of the endoplasmic reticulum. The localization of Mns1p depends on retrieval from the Golgi through a mechanism that involves Rer1p. A chimera consisting of the transmembrane domain of Mns1p fused to the catalytic domain of the Golgi α1,2-mannosyltransferase Kre2p was localized in the endoplasmic reticulum of Δpep4 cells and in the vacuoles of rer1/Δpep4 by indirect immunofluorescence. The split-ubiquitin system was used to determine if there is an interaction between Mns1p and Rer1p in vivo. Co-expression of NubG-Mns1p and Rer1p-Cub-protein A-lexA-VP16 in L40 yeast cells resulted in cleavage of the reporter molecule, protein A-lexA-VP16, detected by western blot analysis and by expression of β-galactosidase activity. Sec12p, another endoplasmic reticulum protein that depends on Rer1p for its localization, also interacted with Rer1p using the split-ubiquitin assay, whereas the endoplasmic reticulum protein Ost1p showed no interaction. A weak interaction was observed between Alg5p and Rer1p. These results demonstrate that the transmembrane domain of Mns1p is sufficient for Rer1p-dependent endoplasmic reticulum localization and that Mns1p and Rer1p interact. Furthermore, the split-ubiquitin system demonstrates that the C-terminal of Rer1p is in the cytosol.

scholarly journals Posttranslational Modifications Required for Cell Surface Localization and Function of the Fungal Adhesin Aga1p

2003 ◽  
Vol 2 (5) ◽  
pp. 1099-1114 ◽  
Author(s):  
Guohong Huang ◽  
Mingliang Zhang ◽  
Scott E. Erdman
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Posttranslational Modifications ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Cell Types ◽  
Fungal Species ◽  
Surface Proteins ◽  
Functional Requirements

ABSTRACT Adherence of fungal cells to host substrates and each other affects their access to nutrients, sexual conjugation, and survival in hosts. Adhesins are cell surface proteins that mediate these different cell adhesion interactions. In this study, we examine the in vivo functional requirements for specific posttranslational modifications to these proteins, including glycophosphatidylinositol (GPI) anchor addition and O-linked glycosylation. The processing of some fungal GPI anchors, creating links to cell wall β-1,6 glucans, is postulated to facilitate postsecretory traffic of proteins to cell wall domains conducive to their functions. By studying the yeast sexual adhesin subunit Aga1p, we found that deletion of its signal sequence for GPI addition eliminated its activity, while deletions of different internal domains had various effects on function. Substitution of the Aga1p GPI signal domain with those of other GPI-anchored proteins, a single transmembrane domain, or a cysteine capable of forming a disulfide all produced functional adhesins. A portion of the cellular pool of Aga1p was determined to be cell wall resident. Aga1p and the α-agglutinin Agα1p were shown to be under glycosylated in cells lacking the protein mannosyltransferase genes PMT1 and PMT2, with phenotypes manifested only in MATα cells for single mutants but in both cell types when both genes are absent. We conclude that posttranslational modifications to Aga1p are necessary for its biogenesis and activity. Our studies also suggest that in addition to GPI-glucan linkages, other cell surface anchorage mechanisms, such as transmembrane domains or disulfides, may be employed by fungal species to localize adhesins.

scholarly journals An amphipathic and cationic antimicrobial peptide kills colistin resistant Gram-negative pathogens in vivo

2021 ◽  
Author(s):  
Thomas T. Thomsen ◽  
Mette Kolpen ◽  
Vinoth Wigneswaran ◽  
Ulrik Kromann ◽  
Anna Ebbensgaard ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lipid A ◽  
Multidrug Resistant ◽  
Colistin Resistance ◽  
Cationic Antimicrobial Peptide ◽  
Gram Negative ◽  
Excellent Activity ◽  
New Antibiotics ◽  
Charge Change

New antibiotics are needed against multidrug resistant Gram-negative pathogens that have compromised global health systems. Antimicrobial peptides are generally considered promising lead candidates for the next generation of antibiotics but have not fulfilled this expectation. Here we demonstrate activity of a cationic amphipathic undecapeptide (ChIP; Charge change Independent Peptide) against a wide panel of multidrug resistant Gram-negative pathogens. Importantly, the antimicrobial activity of ChIP is independent of the surface charge changes that confer colistin resistance through modification of Lipid A, while decreased activity of ChIP correlates with GlcN1 tri-acylation of Lipid A. In an in vivo peritonitis mouse model ChIP displays excellent activity against both colistin sensitive and resistant Escherichia coli and Acinetobacter baumannii strains.

scholarly journals Receptor-Like Protein Tyrosine Phosphatase α Homodimerizes on the Cell Surface

2000 ◽  
Vol 20 (16) ◽  
pp. 5917-5929 ◽  
Author(s):  
Guoqiang Jiang ◽  
Jeroen den Hertog ◽  
Tony Hunter
Keyword(s):  
Biological Activity ◽  
Cell Surface ◽  
Protein Tyrosine Phosphatase ◽  
Intracellular Signaling ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Tyrosine Phosphatase ◽  
Receptor Protein ◽  
Dependent Manner ◽  
Protein Tyrosine

ABSTRACT We reported previously that the N-terminal D1 catalytic domain of receptor protein-tyrosine phosphatase α (RPTPα) forms a symmetrical, inhibited dimer in a crystal structure, in which a helix-turn-helix wedge element from one monomer is inserted into the catalytic cleft of the other monomer. Previous functional studies also suggested that dimerization inhibits the biological activity of a CD45 chimeric RPTP and the catalytic activity of an isolated RPTPς D1 catalytic domain. Most recently, we have also shown that enforced dimerization inhibits the biological activity of full-length RPTPα in a wedge-dependent manner. The physiological significance of such inhibition is unknown, due to a lack of understanding of how RPTPα dimerization is regulated in vivo. In this study, we show that transiently expressed cell surface RPTPα exists predominantly as homodimers, suggesting that dimerization-mediated inhibition of RPTPα biological activity is likely to be physiologically relevant. Consistent with our published and unpublished crystallographic data, we show that mutations in the wedge region of D1 catalytic domain and deletion of the entire D2 catalytic domain independently reduced but did not abolish RPTPα homodimerization, suggesting that both domains are critically involved but that neither is essential for homodimerization. Finally, we also provide evidence that both the RPTPα extracellular domain and the transmembrane domain were independently able to homodimerize. These results lead us to propose a zipper model in which inactive RPTPα dimers are stabilized by multiple, relatively weak dimerization interfaces. Dimerization in this manner would provide a potential mechanism for negative regulation of RPTPα. Such RPTPα dimers could be activated by extracellular ligands or intracellular binding proteins that induce monomerization or by intracellular signaling events that induce an open conformation of the dimer.

scholarly journals Proteolytic processing of the serine protease matriptase-2: identification of the cleavage sites required for its autocatalytic release from the cell surface

2010 ◽  
Vol 430 (1) ◽  
pp. 87-95 ◽  
Author(s):  
Marit Stirnberg ◽  
Eva Maurer ◽  
Angelika Horstmeyer ◽  
Sonja Kolp ◽  
Stefan Frank ◽  
...  
Keyword(s):  
Cell Surface ◽  
Disulfide Bonds ◽  
Serine Proteases ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Bond Cleavage ◽  
Proteolytic Processing ◽  
Cleavage Sites ◽  
Single Chain

Matriptase-2 is a member of the TTSPs (type II transmembrane serine proteases), an emerging class of cell surface proteases involved in tissue homoeostasis and several human disorders. Matriptase-2 exhibits a domain organization similar to other TTSPs, with a cytoplasmic N-terminus, a transmembrane domain and an extracellular C-terminus containing the non-catalytic stem region and the protease domain. To gain further insight into the biochemical functions of matriptase-2, we characterized the subcellular localization of the monomeric and multimeric form and identified cell surface shedding as a defining point in its proteolytic processing. Using HEK (human embryonic kidney)-293 cells, stably transfected with cDNA encoding human matriptase-2, we demonstrate a cell membrane localization for the inactive single-chain zymogen. Membrane-associated matriptase-2 is highly N-glycosylated and occurs in monomeric, as well as multimeric, forms covalently linked by disulfide bonds. Furthermore, matriptase-2 undergoes shedding into the conditioned medium as an activated two-chain form containing the catalytic domain, which is cleaved at the canonical activation motif, but is linked to a released portion of the stem region via a conserved disulfide bond. Cleavage sites were identified by MS, sequencing and mutational analysis. Interestingly, cell surface shedding and activation of a matriptase-2 variant bearing a mutation at the active-site serine residue is dependent on the catalytic activity of co-expressed or co-incubated wild-type matriptase-2, indicating a transactivation and trans-shedding mechanism.

scholarly journals Identification of cptA, a PmrA-Regulated Locus Required for Phosphoethanolamine Modification of the Salmonella enterica Serovar Typhimurium Lipopolysaccharide Core

2005 ◽  
Vol 187 (10) ◽  
pp. 3391-3399 ◽  
Author(s):  
R. Tamayo ◽  
B. Choudhury ◽  
A. Septer ◽  
M. Merighi ◽  
R. Carlson ◽  
...  
Keyword(s):  
Surface Charge ◽  
Salmonella Enterica ◽  
Lipid A ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Regulatory System ◽  
Polymyxin B ◽  
Serovar Typhimurium ◽  
Antimicrobial Peptide Resistance

ABSTRACT In response to the in vivo environment, the Salmonella enterica serovar Typhimurium lipopolysaccharide (LPS) is modified. These modifications are controlled in part by the two-component regulatory system PmrA-PmrB, with the addition of 4-aminoarabinose (Ara4N) to the lipid A and phosphoethanolamine (pEtN) to the lipid A and core. Here we demonstrate that the PmrA-regulated STM4118 (cptA) gene is necessary for the addition of pEtN to the LPS core. pmrC, a PmrA-regulated gene necessary for the addition of pEtN to lipid A, did not affect core pEtN addition. Although imparting a similar surface charge modification as Ara4N, which greatly affects polymyxin B resistance and murine virulence, neither pmrC nor cptA plays a dramatic role in antimicrobial peptide resistance in vitro or virulence in the mouse model. Therefore, factors other than surface charge/electrostatic interaction contribute to resistance to antimicrobial peptides such as polymyxin B.

scholarly journals Continuous, Topologically Guided Protein Crystallization Controls Bacterial Surface Layer Self-Assembly

2019 ◽  
Author(s):  
Colin J. Comerci ◽  
Jonathan Herrmann ◽  
Joshua Yoon ◽  
Fatemeh Jabbarpour ◽  
Xiaofeng Zhou ◽  
...  
Keyword(s):  
Surface Layer ◽  
Cell Surface ◽  
Self Assembly ◽  
Cell Envelope ◽  
Protein Crystallization ◽  
Bacterial Surface ◽  
Cell Surface Structures ◽  
Complicated Topology ◽  
Layer Assembly

AbstractBacteria assemble the cell envelope using localized enzymes to account for growth and division of a topologically complicated surface1–3. However, a regulatory pathway has not been identified for assembly and maintenance of the surface layer (S-layer), a 2D crystalline protein coat surrounding the curved 3D surface of a variety of bacteria4,5. By specifically labeling, imaging, and tracking native and purified RsaA, the S-layer protein (SLP) fromC. crescentus, we show that protein self-assembly alone is sufficient to assemble and maintain the S-layerin vivo. By monitoring the location of newly produced S-layer on the surface of living bacteria, we find that S-layer assembly occurs independently of the site of RsaA secretion and that localized production of new cell wall surface area alone is insufficient to explain S-layer assembly patterns. When the cell surface is devoid of a pre-existing S-layer, the location of S-layer assembly depends on the nucleation characteristics of SLP crystals, which grow by capturing RsaA molecules freely diffusing on the outer bacterial surface. Based on these observations, we propose a model of S-layer assembly whereby RsaA monomers are secreted randomly and diffuse on the lipopolysaccharide (LPS) outer membrane until incorporated into growing 2D S-layer crystals. The complicated topology of the cell surface enables formation of defects, gaps, and grain boundaries within the S-layer lattice, thereby guiding the location of S-layer assembly without enzymatic assistance. This unsupervised mechanism poses unique challenges and advantages for designing treatments targeting cell surface structures or utilizing S-layers as self-assembling macromolecular nanomaterials. As an evolutionary driver, 2D protein self-assembly rationalizes the exceptional S-layer subunit sequence and species diversity6.

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