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 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 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 Yeast mitochondrial RNase P RNA synthesis is altered in an RNase P protein subunit mutant: insights into the biogenesis of a mitochondrial RNA-processing enzyme.

1996 ◽  
Vol 16 (7) ◽  
pp. 3429-3436 ◽  
Author(s):  
V Stribinskis ◽  
G J Gao ◽  
P Sulo ◽  
Y L Dang ◽  
N C Martin
Keyword(s):  
Rna Processing ◽  
Mitochondrial Protein ◽  
Rnase P ◽  
Mitochondrial Rna ◽  
Wild Type ◽  
Protein Subunit ◽  
Mitochondrial Trna ◽  
P Deficiency ◽  
F Gene ◽  
P Protein

Rpm2p is a protein subunit of Saccharomyces cerevisiae yeast mitochondrial RNase P, an enzyme which removes 5' leader sequences from mitochondrial tRNA precursors. Precursor tRNAs accumulate in strains carrying a disrupted allele of RPM2. The resulting defect in mitochondrial protein synthesis causes petite mutants to form. We report here that alteration in the biogenesis of Rpm1r, the RNase P RNA subunit, is another consequence of disrupting RPM2. High-molecular-weight transcripts accumulate, and no mature Rpm1r is produced. Transcript mapping reveals that the smallest RNA accumulated is extended on both the 5' and 3' ends relative to mature Rpm1r. This intermediate and other longer transcripts which accumulate are also found as low-abundance RNAs in wild-type cells, allowing identification of processing events necessary for conversion of the primary transcript to final products. Our data demonstrate directly that Rpm1r is transcribed with its substrates, tRNA met f and tRNAPro, from a promoter located upstream of the tRNA met f gene and suggest that a portion also originates from a second promoter, located between the tRNA met f gene and RPM1. We tested the possibility that precursors accumulate because the RNase P deficiency prevents the removal of the downstream tRNAPro. Large RPM1 transcripts still accumulate in strains missing this tRNA. Thus, an inability to process cotranscribed tRNAs does not explain the precursor accumulation phenotype. Furthermore, strains with mutant RPM1 genes also accumulate precursor Rpm1r, suggesting that mutations in either gene can lead to similar biogenesis defects. Several models to explain precursor accumulation are presented.

scholarly journals Crystal structures of the archaeal RNase P protein Rpp38 in complex with RNA fragments containing a K-turn motif

2018 ◽  
Vol 74 (1) ◽  
pp. 57-64 ◽  
Author(s):  
Kosuke Oshima ◽  
Xuzhu Gao ◽  
Seiichiro Hayashi ◽  
Toshifumi Ueda ◽  
Takashi Nakashima ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Rnase P ◽  
Ribonuclease P ◽  
Stem Loop ◽  
P Protein ◽  
Human Rnase ◽  
Human Proteins ◽  
Rna Fragments ◽  
Specificity Domain

A characteristic feature of archaeal ribonuclease P (RNase P) RNAs is that they have extended helices P12.1 and P12.2 containing kink-turn (K-turn) motifs to which the archaeal RNase P protein Rpp38, a homologue of the human RNase P protein Rpp38, specifically binds. PhoRpp38 from the hyperthermophilic archaeon Pyrococcus horikoshii is involved in the elevation of the optimum temperature of the reconstituted RNase P by binding the K-turns in P12.1 and P12.2. Previously, the crystal structure of PhoRpp38 in complex with the K-turn in P12.2 was determined at 3.4 Å resolution. In this study, the crystal structure of PhoRpp38 in complex with the K-turn in P12.2 was improved to 2.1 Å resolution and the structure of PhoRpp38 in complex with the K-turn in P12.1 was also determined at a resolution of 3.1 Å. Both structures revealed that Lys35, Asn38 and Glu39 in PhoRpp38 interact with characteristic G·A and A·G pairs in the K-turn, while Thr37, Asp59, Lys84, Glu94, Ala96 and Ala98 in PhoRpp38 interact with the three-nucleotide bulge in the K-turn. Moreover, an extended stem-loop containing P10–P12.2 in complex with PhoRpp38, as well as PhoRpp21 and PhoRpp29, which are the archaeal homologues of the human proteins Rpp21 and Rpp29, respectively, was affinity-purified and crystallized. The crystals thus grown diffracted to a resolution of 6.35 Å. Structure determination of the crystals will demonstrate the previously proposed secondary structure of stem-loops including helices P12.1 and P12.2 and will also provide insight into the structural organization of the specificity domain in P. horikoshii RNase P RNA.

scholarly journals GENETIC MAPPING AND SOME CHARACTERIZATION OF THE rnpA49 MUTATION OF ESCHERICHIA COLI THAT AFFECTS THE RNA-PROCESSING ENZYME RIBONUCLEASE P

Genetics ◽  
1980 ◽  
Vol 94 (2) ◽  
pp. 291-299
Author(s):  
David Apirion
Keyword(s):  
Escherichia Coli ◽  
Genetic Mapping ◽  
Temperature Sensitivity ◽  
Rna Processing ◽  
Rnase P ◽  
Ribonuclease P ◽  
E Coli ◽  
Processing Enzyme

ABSTRACT A mutant defective in the enzyme RNase P was isolated by P. SCHEDL and P. PRIMAKOFF (1973). The mutation rnpA49 found in this strain, which confers temperature sensitivity on carrier strains, was mapped by conjugation and transduction experiments and located around minute 82 of the E . coli map, with the suggested order rnpA bglB phoS rbsP ilv. As expected, the rnpA49 mutation is recessive. Even though this mutation is conditional, it is manifested at temperatures at which the carrier strains can grow.

scholarly journals Interactions between RNase P protein subunits in Archaea

Archaea ◽  
2004 ◽  
Vol 1 (4) ◽  
pp. 247-254 ◽  
Author(s):  
Thomas A. Hall ◽  
James W. Brown
Keyword(s):  
Protein Interactions ◽  
Rnase P ◽  
Ribonuclease P ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Protein Subunits ◽  
P Protein ◽  
Yeast Two Hybrid System ◽  
Methanothermobacter Thermoautotrophicus ◽  
Two Hybrid

A yeast two-hybrid system was used to identify protein–protein interactions between the ribonuclease P (RNase P) protein subunits Mth11p, Mth687p, Mth688p and Mth1618p from the archaeonMethanothermobacter thermoautotrophicus. Clear interactions between Mth688p and Mth687p, and between Mth1618p and Mth11p, were confirmed byHIS3andLacZreporter expression. Weaker interactions of Mth687p and Mth688p with Mth11p, and Mth11p with itself, are also suggested. These interactions resemble, and confirm, those previously seen among the homologs of these proteins in the more complex yeast RNase P holoenzyme.

scholarly journals Kinetic mechanism of human mitochondrial RNase P

2019 ◽  
Author(s):  
Xin Liu ◽  
Nancy Wu ◽  
Aranganathan Shanmuganathan ◽  
Bradley P. Klemm ◽  
Michael J. Howard ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Rnase P ◽  
Kinetic Mechanism ◽  
Ribonuclease P ◽  
Substrate Affinity ◽  
P Protein ◽  
Binding Data ◽  
Catalytically Active

ABSTRACTA first step in processing mitochondrial precursor tRNA (pre-tRNA) is cleavage of the 5’ leader catalyzed by ribonuclease P (RNase P). Human mitochondrial RNase P (mtRNase P) is composed of three protein subunits: mitochondrial RNase P protein (MRPP) 1, 2 and 3. Even though MRPP3 is the metallonuclease subunit responsible for catalysis, cleavage is observed only in the presence of the MRPP1/2 subcomplex. To understand the functional role of MRPP1/2, we reconstituted human mitochondrial RNase P in vitro and performed kinetic and thermodynamic analyses. MRPP1/2 significantly enhances both the catalytic activity and the apparent substrate affinity of mtRNase P. Additionally, pull-down and binding data demonstrate synergy between binding pre-tRNA and formation of a catalytically active MRPP1/2/3 complex. These data suggest that conformational changes in the MRPP1/2-pre-tRNA complex lead to protein-protein or protein-RNA interactions that increase both MRPP3 recognition and cleavage efficiency. This work presents the first kinetic model for human mtRNase P, providing a fundamental framework for the function of MRPP1/2 for recognition and processing of pre-tRNA.

scholarly journals Peptides presenting the binding site of human CD4 for the HIV-1 envelope glycoprotein gp120

2012 ◽  
Vol 8 ◽  
pp. 1858-1866 ◽  
Author(s):  
Julia Meier ◽  
Kristin Kassler ◽  
Heinrich Sticht ◽  
Jutta Eichler
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Envelope Glycoprotein ◽  
Conformational Stability ◽  
Cellular Receptor ◽  
Binding Assays ◽  
Glycoprotein Gp120 ◽  
Dynamics Simulations ◽  
Key Residues ◽  
Hiv 1

Based on the structure of the HIV-1 glycoprotein gp120 in complex with its cellular receptor CD4, we have designed and synthesized peptides that mimic the binding site of CD4 for gp120. The ability of these peptides to bind to gp120 can be strongly enhanced by increasing their conformational stability through cyclization, as evidenced by binding assays, as well as through molecular-dynamics simulations of peptide–gp120 complexes. The specificity of the peptide–gp120 interaction was demonstrated by using peptide variants, in which key residues for the interaction with gp120 were replaced by alanine or D-amino acids.

scholarly journals The substrate-binding cap of the UDP-diacylglucosamine pyrophosphatase LpxH is highly flexible, enabling facile substrate binding and product release

2018 ◽  
Vol 293 (21) ◽  
pp. 7969-7981 ◽  
Author(s):  
Thomas E. Bohl ◽  
Pek Ieong ◽  
John K. Lee ◽  
Thomas Lee ◽  
Jayakanth Kankanala ◽  
...  
Keyword(s):  
Rational Design ◽  
Substrate Binding ◽  
Flexible Substrate ◽  
Binding Mode ◽  
Antibiotic Development ◽  
Product Release ◽  
Outer Leaflet ◽  
Hydrogen Deuterium ◽  
Dynamics Simulations ◽  
Secondary Membrane

Gram-negative bacteria are surrounded by a secondary membrane of which the outer leaflet is composed of the glycolipid lipopolysaccharide (LPS), which guards against hydrophobic toxins, including many antibiotics. Therefore, LPS synthesis in bacteria is an attractive target for antibiotic development. LpxH is a pyrophosphatase involved in LPS synthesis, and previous structures revealed that LpxH has a helical cap that binds its lipid substrates. Here, crystallography and hydrogen–deuterium exchange MS provided evidence for a highly flexible substrate-binding cap in LpxH. Furthermore, molecular dynamics simulations disclosed how the helices of the cap may open to allow substrate entry. The predicted opening mechanism was supported by activity assays of LpxH variants. Finally, we confirmed biochemically that LpxH is inhibited by a previously identified antibacterial compound, determined the potency of this inhibitor, and modeled its binding mode in the LpxH active site. In summary, our work provides evidence that the substrate-binding cap of LpxH is highly dynamic, thus allowing for facile substrate binding and product release between the capping helices. Our results also pave the way for the rational design of more potent LpxH inhibitors.

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