The structure of ribonuclease P protein from Staphylococcus aureus reveals a unique binding site for single-stranded RNA

2000 ◽  
Vol 295 (1) ◽  
pp. 105-115 ◽  
Author(s):  
Claus Spitzfaden ◽  
Neville Nicholson ◽  
Jo J Jones ◽  
Sabine Guth ◽  
Ruth Lehr ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Binding Site ◽  
Ribonuclease P ◽  
P Protein

scholarly journals Crystal structure of the ribonuclease-P-protein subunit from Staphylococcus aureus

2018 ◽  
Vol 74 (10) ◽  
pp. 632-637
Author(s):  
Lisha Ha ◽  
Jennifer Colquhoun ◽  
Nicholas Noinaj ◽  
Chittaranjan Das ◽  
Paul M. Dunman ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Staphylococcus Aureus ◽  
Rna Processing ◽  
Bacterial Species ◽  
Rna Degradation ◽  
Ribonuclease P ◽  
Protein Subunit ◽  
Aromatic Residues ◽  
Cellular Processes ◽  
P Protein

Staphylococcus aureus ribonuclease-P-protein subunit (RnpA) is a promising antimicrobial target that is a key protein component for two essential cellular processes, RNA degradation and transfer-RNA (tRNA) maturation. The first crystal structure of RnpA from the pathogenic bacterial species, S. aureus, is reported at 2.0 Å resolution. The structure presented maintains key similarities with previously reported RnpA structures from bacteria and archaea, including the highly conserved RNR-box region and aromatic residues in the precursor-tRNA 5′-leader-binding domain. This structure will be instrumental in the pursuit of structure-based designed inhibitors targeting RnpA-mediated RNA processing as a novel therapeutic approach for treating S. aureus infections.

scholarly journals Inhibition of Ubiquitination and Stabilization of Human Ubiquitin E3 Ligase PIRH2 by Measles Virus Phosphoprotein

2005 ◽  
Vol 79 (18) ◽  
pp. 11824-11836 ◽  
Author(s):  
Mingzhou Chen ◽  
Jean-Claude Cortay ◽  
Ian R. Logan ◽  
Vasileia Sapountzi ◽  
Craig N. Robson ◽  
...  
Keyword(s):  
Binding Site ◽  
Measles Virus ◽  
Fluorescent Protein ◽  
E3 Ligase ◽  
Yeast Two Hybrid ◽  
Ubiquitin E3 Ligase ◽  
P Protein ◽  
Two Hybrid

ABSTRACT Using a C-terminal domain (PCT) of the measles virus (MV) phosphoprotein (P protein) as bait in a yeast two-hybrid screen, a cDNA identical to the recently described human p53-induced-RING-H2 (hPIRH2) cDNA was isolated. A glutathione S-transferase-hPIRH2 fusion protein expressed in bacteria was able to pull down P protein when mixed with an extract from P-expressing HeLa cells in vitro, and myc-tagged hPIRH2 could be reciprocally coimmunoprecipitated with MV P protein from human cells. Additionally, immunoprecipitation experiments demonstrated that hPIRH2-myc, MV P, and nucleocapsid (N) proteins form a ternary complex. The hPIRH2 binding site was mapped to the C-terminal X domain region of the P protein by using a yeast two-hybrid assay. The PCT binding site was mapped on hPIRH2 by using a novel yeast two-hybrid tagged PCR approach and by coimmunoprecipitation of hPIRH2 cysteine mutants and mouse/human PIRH2 chimeras. The hPIRH2 C terminus could mediate the interaction with MV P which was favored by the RING-H2 motif. When coexpressed with an enhanced green fluorescent protein-tagged hPIRH2 protein, MV P alone or in a complex with MV N was able to redistribute hPIRH2 to outside the nucleus, within intracellular aggregates. Finally, MV P efficiently stabilized hPIRH2-myc expression and prevented its ubiquitination in vivo but had no effect on the stability or ubiquitination of an alternative ubiquitin E3 ligase, Mdm2. Thus, MV P protein is the first protein from a pathogen that is able to specifically interact with and stabilize the ubiquitin E3 ligase hPIRH2 by preventing its ubiquitination.

scholarly journals Interplay between substrate recognition, 5’ end tRNA processing and methylation activity of human mitochondrial RNase P

2018 ◽  
Author(s):  
Agnes Karasik ◽  
Carol A. Fierke ◽  
Markos Koutmos
Keyword(s):  
Mitochondrial Diseases ◽  
Protein S ◽  
Rnase P ◽  
Ribonuclease P ◽  
Mitochondrial Rna ◽  
P Protein ◽  
Adenosyl Methionine ◽  
Methylation Activity

ABSTRACTHuman mitochondrial ribonuclease P (mtRNase P) is an essential three protein complex that catalyzes the 5’ end maturation of mitochondrial precursor tRNAs (pre-tRNAs). MRPP3 (Mitochondrial RNase P Protein 3), a protein-only RNase P (PRORP), is the nuclease component of the mtRNase P complex and requires a two-protein S-adenosyl methionine (SAM)-dependent methyltransferase MRPP1/2 sub-complex to function. Dysfunction of mtRNase P is linked to several human mitochondrial diseases, such as mitochondrial myopathies. Despite its central role in mitochondrial RNA processing, little is known about how the protein subunits of mtRNase P function synergistically. Here we use purified mtRNase P to demonstrate that mtRNase P recognizes, cleaves, and methylates some, but not all, mitochondrial pre-tRNAs in vitro. Additionally, mtRNase P does not process all mitochondrial pre-tRNAs uniformly, suggesting the possibility that some pre-tRNAs require additional factors to be cleaved in vivo. Consistent with this, we found that addition of the MRPP1 co-factor SAM enhances the ability of mtRNase P to bind and cleave some mitochondrial pre-tRNAs. Furthermore, the presence of MRPP3 can enhance the methylation activity of MRPP1/2. Taken together, our data demonstrate that the subunits of mtRNase P work together to efficiently recognize, process and methylate human mitochondrial pre-tRNAs.

scholarly journals A screening platform to monitor RNA processing and protein-RNA interactions in ribonuclease P uncovers a small molecule inhibitor

2019 ◽  
Vol 47 (12) ◽  
pp. 6425-6438 ◽  
Author(s):  
Ezequiel-Alejandro Madrigal-Carrillo ◽  
Carlos-Alejandro Díaz-Tufinio ◽  
Hugo-Aníbal Santamaría-Suárez ◽  
Marcelino Arciniega ◽  
Alfredo Torres-Larios
Keyword(s):  
Rna Processing ◽  
Rnase P ◽  
Ribonuclease P ◽  
Binding Assays ◽  
Antibiotic Development ◽  
Processing Enzymes ◽  
P Protein ◽  
Molecule Inhibitor ◽  
Screening Platform ◽  
Dynamics Simulations

AbstractRibonucleoprotein (RNP) complexes and RNA-processing enzymes are attractive targets for antibiotic development owing to their central roles in microbial physiology. For many of these complexes, comprehensive strategies to identify inhibitors are either lacking or suffer from substantial technical limitations. Here, we describe an activity-binding-structure platform for bacterial ribonuclease P (RNase P), an essential RNP ribozyme involved in 5′ tRNA processing. A novel, real-time fluorescence-based assay was used to monitor RNase P activity and rapidly identify inhibitors using a mini-helix and a pre-tRNA-like bipartite substrate. Using the mini-helix substrate, we screened a library comprising 2560 compounds. Initial hits were then validated using pre-tRNA and the pre-tRNA-like substrate, which ultimately verified four compounds as inhibitors. Biolayer interferometry-based binding assays and molecular dynamics simulations were then used to characterize the interactions between each validated inhibitor and the P protein, P RNA and pre-tRNA. X-ray crystallographic studies subsequently elucidated the structure of the P protein bound to the most promising hit, purpurin, and revealed how this inhibitor adversely affects tRNA 5′ leader binding. This integrated platform affords improved structure-function studies of RNA processing enzymes and facilitates the discovery of novel regulators or inhibitors.

scholarly journals Inhibition of Staphylococcus aureus Cell Wall Biosynthesis by Desleucyl-Oritavancin: a Quantitative Peptidoglycan Composition Analysis by Mass Spectrometry

2017 ◽  
Vol 199 (15) ◽  
Author(s):  
James D. Chang ◽  
Erin E. Foster ◽  
Aanchal N. Thadani ◽  
Alejandro J. Ramirez ◽  
Sung Joon Kim
Keyword(s):  
Mass Spectrometry ◽  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Binding Site ◽  
Mode Of Action ◽  
Composition Analysis ◽  
Edman Degradation ◽  
Glycopeptide Antibiotics ◽  
Content Type ◽  
The Cross

ABSTRACT Oritavancin is a lipoglycopeptide antibiotic that exhibits potent activities against vancomycin-resistant Gram-positive pathogens. Oritavancin differs from vancomycin by a hydrophobic side chain attached to the drug disaccharide, which forms a secondary binding site to enable oritavancin binding to the cross-linked peptidoglycan in the cell wall. The mode of action of secondary binding site was investigated by measuring the changes in the peptidoglycan composition of Staphylococcus aureus grown in the presence of desleucyl-oritavancin at subinhibitory concentration using liquid chromatography-mass spectrometry (LC-MS). Desleucyl-oritavancin is an Edman degradation product of oritavancin that exhibits potent antibacterial activities despite the damaged d-Ala–d-Ala binding site due to its functional secondary binding site. Accurate quantitative peptidoglycan composition analysis based on 83 muropeptide ions determined that cell walls of S. aureus grown in the presence of desleucyl-oritavancin showed a reduction of peptidoglycan cross-linking, increased muropeptides with a tetrapeptide-stem structure, decreased O-acetylation of MurNAc, and increased N-deacetylation of GlcNAc. The changes in peptidoglycan composition suggest that desleucyl-oritavancin targets the peptidoglycan template to induce cell wall disorder and interferes with cell wall maturation. IMPORTANCE Oritavancin is a lipoglycopeptide antibiotic with a secondary binding site that targets the cross-linked peptidoglycan bridge structure in the cell wall. Even after the loss of its primary d-Ala–d-Ala binding site through Edman degradation, desleucyl-oritavancin exhibits potent antimicrobial activities through its still-functioning secondary binding site. In this study, we characterized the mode of action for desleucyl-oritavancin's secondary binding site using LC-MS. Peptidoglycan composition analysis of desleucyl-oritavancin-treated S. aureus was performed by determining the relative abundances of 83 muropeptide ions matched from a precalculated library through integrating extracted ion chromatograms. Our work highlights the use of quantitative peptidoglycan composition analysis by LC-MS to provide insights into the mode of action of glycopeptide antibiotics.

scholarly journals NMR and XAS reveal an inner-sphere metal binding site in the P4 helix of the metallo-ribozyme ribonuclease P

2010 ◽  
Vol 107 (6) ◽  
pp. 2479-2484 ◽  
Author(s):  
K. S. Koutmou ◽  
A. Casiano-Negroni ◽  
M. M. Getz ◽  
S. Pazicni ◽  
A. J. Andrews ◽  
...  
Keyword(s):  
Binding Site ◽  
Metal Binding ◽  
Ribonuclease P ◽  
Metal Binding Site ◽  
Inner Sphere

Binding of staphylococcal cell surface polysaccharide to human fibrinogen

1990 ◽  
Vol 36 (3) ◽  
pp. 206-210
Author(s):  
Toshichika Ohtomo ◽  
Tsugiaki Kobayashi ◽  
Yukio Ohshima ◽  
Yukio Usui ◽  
Masaru Suganuma ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Surface ◽  
Binding Site ◽  
Key Words ◽  
Active Substance ◽  
Soft Agar ◽  
Alkaline Ph ◽  
Human Fibrinogen ◽  
Surface Polysaccharide ◽  
Alkaline Range

The interaction between the binding site of a polysaccharide (called compact colony forming active substance (CCFAS)), obtained from the cell surface of a strain of Staphylococcus, and human fibrinogen (HF) was investigated. The CCFAS was found to bind specifically to both the Bβ and γ chains of HF at pH 7.0 and 8.0, and the Aα chain at pH 5.0. The binding of CCFAS with fibrinogen fragments obtained by digestion with plasmin were also investigated. Fragments with Mr of 55 000, 24 000, and 19 000 were the major bands precipitated by CCFAS at pH 7.0 and 8.0. Fragments with Mr of 85 000 and 75 000 bound to CCFAS at pH 5.0. Binding of CCFAS (7 μg) with fibrinogen could be inhibited by 1.2 μg of Bβ chain and 1.5 μg γ chain at alkaline pH or 6.2 μg of the Aα chain at pH 5.0. CCFAS was, therefore, assumed to be specifically bonded with HF molecules, in the alkaline range at least, resulting in compact colony forming activity in serum soft agar and paracoagulation. Key words: cell surface, polysaccharide, Staphylococcus aureus, fibrinogen.

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