Faculty Opinions recommendation of Identification of structural and molecular determinants of the tyrosine-kinase Wzc and implications in capsular polysaccharide export.

Many pathogenic bacteria are encased in a layer of capsular polysaccharide (CPS). This layer is important for virulence by masking surface antigens, preventing opsonophagocytosis, and avoiding mucus entrapment. The bacterial tyrosine kinase (BY-kinase) regulates capsule synthesis and helps bacterial pathogens to survive different host niches. BY-kinases autophosphorylate at the C-terminal tyrosine residues upon external stimuli, but the role of phosphorylation is still unclear. Here, we report that the BY-kinase CpsCD is required for growth in Streptococcus pneumoniae. Cells lacking a functional cpsC or cpsD accumulated low molecular weight CPS and lysed because of the lethal sequestration of the lipid carrier undecaprenyl phosphate, resulting in inhibition of peptidoglycan (PG) synthesis. CpsC interacts with CpsD and the polymerase CpsH. CpsD phosphorylation reduces the length of CPS polymers presumably by controlling the activity of CpsC. Finally, pulse–chase experiments reveal the spatiotemporal coordination between CPS and PG synthesis. This coordination is dependent on CpsC and CpsD. Together, our study provides evidence that BY-kinases regulate capsule polymer length by fine-tuning CpsC activity through autophosphorylation.

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Mutational Analysis of the Carboxy-Terminal (YGX)4 Repeat Domain of CpsD, an Autophosphorylating Tyrosine Kinase Required for Capsule Biosynthesis in Streptococcus pneumoniae

Journal of Bacteriology ◽

10.1128/jb.185.10.3009-3019.2003 ◽

2003 ◽

Vol 185 (10) ◽

pp. 3009-3019 ◽

Cited By ~ 68

Author(s):

Judy K. Morona ◽

Renato Morona ◽

David C. Miller ◽

James C. Paton

Keyword(s):

Streptococcus Pneumoniae ◽

Tyrosine Kinase ◽

Type Strain ◽

Wild Type Strain ◽

Capsular Polysaccharide ◽

Wild Type ◽

Protein Tyrosine ◽

Tyrosine Residues ◽

Repeat Domain ◽

Carboxy Terminal

ABSTRACT In Streptococcus pneumoniae, CpsB, CpsC, and CpsD are essential for encapsulation, and mutants containing deletions of cpsB, cpsC, or cpsD exhibit rough colony morphologies. CpsD is an autophosphorylating protein-tyrosine kinase, CpsC is required for CpsD tyrosine phosphorylation, and CpsB is a phosphotyrosine-protein phosphatase. We have previously shown that autophosphorylation of CpsD at tyrosine attenuates its activity and consequently reduces the level of encapsulation and negatively regulates CPS production. In this study, we further investigated the role of the carboxy-terminal (YGX)4 repeat domain of CpsD in encapsulation. A CpsD truncation mutant in which the entire (YGX)4 repeat domain was removed was indistinguishable from a strain in which the entire cpsD gene had been deleted, indicating that the carboxy-terminal (YGX)4 tail is required for CpsD activity in capsular polysaccharide production. Double mutants having a single tyrosine residue at position 2, 3, or 4 in the (YGX)4 repeat domain and lacking CpsB exhibited a rough colony morphology, indicating that in the absence of an active protein-tyrosine phosphatase, phosphorylation of just one of the tyrosine residues in the (YGX)4 repeat was sufficient to inactivate CpsD. When various mutants in which CpsD had either one or combinations of two or three tyrosine residues in the (YGX)4 repeat domain were examined, only those with three tyrosine residues in the (YGX)4 repeat domain were indistinguishable from the wild-type strain. The mutants with either one or two tyrosine residues exhibited mucoid colony morphologies. Further analysis of the mucoid strains indicated that the mucoid phenotype was not due to overproduction of capsular polysaccharide, as these strains actually produced less capsular polysaccharide than the wild-type strain. Thus, the tyrosine residues in the (YGX)4 repeat domain are essential for normal functioning of CpsD.

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Synthesis of capsular polysaccharide at the division septum of Streptococcus pneumoniae is dependent on a bacterial tyrosine kinase

Molecular Microbiology ◽

10.1111/j.1365-2958.2011.07828.x ◽

2011 ◽

Vol 82 (2) ◽

pp. 515-534 ◽

Cited By ~ 30

Author(s):

Mafalda X. Henriques ◽

Tatiana Rodrigues ◽

Madalena Carido ◽

Luís Ferreira ◽

Sérgio R. Filipe

Keyword(s):

Streptococcus Pneumoniae ◽

Tyrosine Kinase ◽

Capsular Polysaccharide

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Detection and mapping of interactions between chromatin and the nuclear envelope in drosophila embryos

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140191 ◽

1995 ◽

Vol 53 ◽

pp. 764-765

Author(s):

W.F. Marshall ◽

A.F. Dernburg ◽

B. Harmon ◽

J.W. Sedat

Keyword(s):

Nuclear Envelope ◽

Chromatin Condensation ◽

Three Dimensional ◽

Physiological Role ◽

Nuclear Surface ◽

Intact Cells ◽

Molecular Determinants ◽

Surface Harmonic ◽

Drosophila Embryos

Interactions between chromatin and nuclear envelope (NE) have been implicated in chromatin condensation, gene regulation, nuclear reassembly, and organization of chromosomes within the nucleus. To further investigate the physiological role played by such interactions, it will be necessary to determine which loci specifically interact with the nuclear envelope. This will not only facilitate identification of the molecular determinants of this interaction, but will also allow manipulation of the pattern of chromatin-NE interactions to probe possible functions. We have developed a microscopic approach to detect and map chromatin-NE interactions inside intact cells.Fluorescence in situ hybridization (FISH) is used to localize specific chromosomal regions within the nucleus of Drosophila embryos and anti-lamin immunofluorescence is used to detect the nuclear envelope. Widefield deconvolution microscopy is then used to obtain a three-dimensional image of the sample (Fig. 1). The nuclear surface is represented by a surface-harmonic expansion (Fig 2). A statistical test for association of the FISH spot with the surface is then performed.

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SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana

Biochemical Journal ◽

10.1042/bcj20190674 ◽

2020 ◽

Vol 477 (1) ◽

pp. 173-189 ◽

Cited By ~ 1

Author(s):

Marco Pedretti ◽

Carolina Conter ◽

Paola Dominici ◽

Alessandra Astegno

Keyword(s):

Excision Repair ◽

Complex Structure ◽

Binding Motif ◽

C Terminus ◽

Repair Protein ◽

Nucleotide Excision ◽

Ef Hand ◽

Molecular Determinants ◽

Structure In Solution

Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I8W4W1). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp–CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca2+-saturated CML19, but also with apo-CML19 to form a protein–peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca2+-CML19. Importantly, the peptide dramatically increases the affinity for Ca2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca2+-saturated and bound to SAC3B even at resting Ca2+-levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export.

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