scholarly journals Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistance

eLife ◽  
2022 ◽  
Vol 11 ◽  
Author(s):  
Kaitlyn Tsai ◽  
Vanja Stojković ◽  
Lianet Noda-Garcia ◽  
Iris D Young ◽  
Alexander G Myasnikov ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Ribosomal Rna ◽  
Binding Sites ◽  
Molecular Basis ◽  
Direct Evidence ◽  
Resistance Mechanism ◽  
Cryo Electron Microscopy ◽  
New Antibiotics ◽  
Natural Isolates ◽  
Adenosine Nucleotide

Alteration of antibiotic binding sites through modification of ribosomal RNA (rRNA) is a common form of resistance to ribosome-targeting antibiotics. The rRNA-modifying enzyme Cfr methylates an adenosine nucleotide within the peptidyl transferase center, resulting in the C-8 methylation of A2503 (m8A2503). Acquisition of cfr results in resistance to eight classes of ribosome-targeting antibiotics. Despite the prevalence of this resistance mechanism, it is poorly understood whether and how bacteria modulate Cfr methylation to adapt to antibiotic pressure. Moreover, direct evidence for how m8A2503 alters antibiotic binding sites within the ribosome is lacking. In this study, we performed directed evolution of Cfr under antibiotic selection to generate Cfr variants that confer increased resistance by enhancing methylation of A2503 in cells. Increased rRNA methylation is achieved by improved expression and stability of Cfr through transcriptional and post-transcriptional mechanisms, which may be exploited by pathogens under antibiotic stress as suggested by natural isolates. Using a variant that achieves near-stoichiometric methylation of rRNA, we determined a 2.2 Å cryo-electron microscopy structure of the Cfr-modified ribosome. Our structure reveals the molecular basis for broad resistance to antibiotics and will inform the design of new antibiotics that overcome resistance mediated by Cfr.

scholarly journals Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistance

2021 ◽  
Author(s):  
Kaitlyn Tsai ◽  
Vanja Stojkovic ◽  
Lianet Noda-Garcia ◽  
Iris D. Young ◽  
Alexander G. Myasnikov ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Binding Sites ◽  
Molecular Basis ◽  
Direct Evidence ◽  
Resistance Mechanisms ◽  
Cross Resistance ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Bacterial Ribosome ◽  
Mechanisms Of Adaptation

Many clinically useful antibiotics inhibit the bacterial ribosome. The ribosomal RNA-modifying enzyme Cfr methylates an adenosine (m8A2503) in the peptidyl transferase center and causes cross-resistance to several classes of antibiotics. Despite the prevalence of this mode of resistance, mechanisms of adaptation to antibiotic pressure that exploit ribosome modification by Cfr are poorly understood. Moreover, direct evidence for how m8A2503 alters antibiotic binding sites within the ribosome is lacking. To address these questions, we evolved Cfr under antibiotic selection to generate variants that confer increased resistance and methylation of rRNA, provided by enhanced Cfr expression and stability. Using a variant which achieves near-stoichiometric methylation, we determined a 2.2Å cryo-EM structure of the Cfr-modified ribosome, revealing the molecular basis for resistance and informing design of antibiotics that overcome Cfr resistance.

scholarly journals Structures of Rhodopseudomonas palustris RC-LH1 complexes with open or closed quinone channels

2021 ◽  
Vol 7 (3) ◽  
pp. eabe2631
Author(s):  
David J. K. Swainsbury ◽  
Pu Qian ◽  
Philip J. Jackson ◽  
Kaitlyn M. Faries ◽  
Dariusz M. Niedzwiedzki ◽  
...  
Keyword(s):  
Binding Sites ◽  
Rhodopseudomonas Palustris ◽  
Ring Closure ◽  
Light Harvesting Complex ◽  
The Core ◽  
Quinone Binding ◽  
Cryo Electron Microscopy ◽  
Unique Protein ◽  
Center Light ◽  
Resolution Structure

The reaction-center light-harvesting complex 1 (RC-LH1) is the core photosynthetic component in purple phototrophic bacteria. We present two cryo–electron microscopy structures of RC-LH1 complexes from Rhodopseudomonas palustris. A 2.65-Å resolution structure of the RC-LH114-W complex consists of an open 14-subunit LH1 ring surrounding the RC interrupted by protein-W, whereas the complex without protein-W at 2.80-Å resolution comprises an RC completely encircled by a closed, 16-subunit LH1 ring. Comparison of these structures provides insights into quinone dynamics within RC-LH1 complexes, including a previously unidentified conformational change upon quinone binding at the RC QB site, and the locations of accessory quinone binding sites that aid their delivery to the RC. The structurally unique protein-W prevents LH1 ring closure, creating a channel for accelerated quinone/quinol exchange.

scholarly journals A Modified Autonomous En Transposon in Maize (Zea mays L.) Elicits a Differential Response of Reporter Alleles

Genetics ◽  
1997 ◽  
Vol 147 (3) ◽  
pp. 1329-1338
Author(s):  
Peter A Peterson
Keyword(s):  
Molecular Structure ◽  
Binding Sites ◽  
Molecular Basis ◽  
Zea Mays L ◽  
Phenotypic Expression ◽  
Terminal Inverted Repeat ◽  
Coding Sequences ◽  
Defective Element ◽  
Related Element ◽  
Unstable Genes

Transposable elements in maize are composed of a defined molecular structure that includes coding sequences, determiners of functionality and ordered terminal motifs that provide binding sites for transposase proteins. Alterations in these components change the phenotypic expression of unstable genes with transposon inserts. The molecular basis for the altered timing and frequency of transposition as determined by the size and number of spots on kernels or stripes on leaves has generally been described for defective inserts in genes. Most differential patterns can be ascribed to alterations in the terminal motifs of the reporter allele structure that supplies a substrate (terminal inverted repeat motifs) for transposase activity. For autonomously functioning alleles, the explanations for changes in phenotype are not so clear. In this report, an En-related element identified as F-En is described that shares with En the recognition of a specific defective element c1(mr)888104 but differs from En in that this F-En element does not recognize the canonical c1(mr) elements that are recognized by En. Evidence is provided suggesting that F-En does not recognize other En/Spm-related defective elements, some of whose sequences are known. This modified En arose from a c1-m autonomously mutating En allele.

scholarly journals Dynamic oligopeptide acquisition by the RagAB transporter from Porphyromonas gingivalis

2019 ◽  
Author(s):  
Mariusz Madej ◽  
Joshua B. R. White ◽  
Zuzanna Nowakowska ◽  
Shaun Rawson ◽  
Carsten Scavenius ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Direct Evidence ◽  
Inflammatory Diseases ◽  
Extracellular Proteins ◽  
Substrate Selectivity ◽  
Binding Studies ◽  
Uptake Mechanism ◽  
X Ray ◽  
Cryo Electron Microscopy ◽  
Binding Surface

AbstractPorphyromonas gingivalis, an asaccharolytic Bacteroidetes, is a keystone pathogen in human periodontitis that may also contribute to the development of other chronic inflammatory diseases, such as rheumatoid arthritis, cardiovascular disease and Alzheimer’s disease. P. gingivalis utilizes protease-generated peptides derived from extracellular proteins for growth, but how those peptides enter the cell is not clear. Here we identify RagAB as the outer membrane importer for peptides. X-ray crystal structures show that the transporter forms a dimeric RagA2B2 complex with the RagB substrate binding surface-anchored lipoprotein forming a closed lid on the TonB-dependent transporter RagA. Cryo-electron microscopy structures reveal the opening of the RagB lid and thus provide direct evidence for a “pedal bin” nutrient uptake mechanism. Together with mutagenesis, peptide binding studies and RagAB peptidomics, our work identifies RagAB as a dynamic OM oligopeptide acquisition machine with considerable substrate selectivity that is essential for the efficient utilisation of proteinaceous nutrients by P. gingivalis.

scholarly journals The molecular mechanism of the inherited phosphofructokinase deficiency associated with hemolysis and myopathy

Blood ◽  
1980 ◽  
Vol 55 (4) ◽  
pp. 629-635
Author(s):  
S Vora ◽  
L Corash ◽  
WK Engel ◽  
S Durham ◽  
C Seaman ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Direct Evidence ◽  
The Other ◽  
Heterogeneous Mixture ◽  
Muscle Type ◽  
Crossover Point ◽  
Normal Human ◽  
Phosphofructokinase Deficiency ◽  
2,3 Dpg ◽  
Chromatographic Profiles

Normal human erythrocyte phosphofructokinase (ATP:c D-fructose-6, P-1- phosphotransferase, EC 2.7.1.11; PFK) has recently been shown to consist of a heterogeneous mixture of five tetrameric isozymes: M4, M3L, M2L2, ML3, and L4 (M, muscle type; L, liver type). In the light of these findings, we have investigated the molecular basis of the inherited erythrocyte PFK deficiency associated with myopathy and hemolysis (Tarui disease). The propositus, a 31-yr-old male, suffered from muscle weakness and myoglobinuria on exertion. He showed mild erythrocytosis despite laboratory evidence of hemolysis. In his erythrocytes a metabolic crossover point was found at the level of PFK; 2,3-diphosphoglycerate (2,3-DPG) was also significantly reduced. The PFK from the patient's erythrocytes consisted exclusively of the L4 isozyme, and there was a complete absence of the other four. The leukocyte and platelet PFKs from the patient showed normal activities, chromatographic profiles, and precipitation with anti-M4 antibody. These studies provide direct evidence that in Tarui disease the M-type subunits are absent; but the liver- and platelet-type subunits of PFK are unaffected. The paradox of mild erythrocytosis despite hemolysis reflects the decreased production of 2,3-DPG.

Eukaryotic Ribosome Assembly

2019 ◽  
Vol 88 (1) ◽  
pp. 281-306 ◽  
Author(s):  
Jochen Baßler ◽  
Ed Hurt
Keyword(s):  
Ribosomal Rna ◽  
Nuclear Export ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Cryo Electron Microscopy ◽  
A Cell ◽  
Cellular Pathways ◽  
Nucleolar Rnas ◽  
Shed Light

Ribosomes, which synthesize the proteins of a cell, comprise ribosomal RNA and ribosomal proteins, which coassemble hierarchically during a process termed ribosome biogenesis. Historically, biochemical and molecular biology approaches have revealed how preribosomal particles form and mature in consecutive steps, starting in the nucleolus and terminating after nuclear export into the cytoplasm. However, only recently, due to the revolution in cryo–electron microscopy, could pseudoatomic structures of different preribosomal particles be obtained. Together with in vitro maturation assays, these findings shed light on how nascent ribosomes progress stepwise along a dynamic biogenesis pathway. Preribosomes assemble gradually, chaperoned by a myriad of assembly factors and small nucleolar RNAs, before they reach maturity and enter translation. This information will lead to a better understanding of how ribosome synthesis is linked to other cellular pathways in humans and how it can cause diseases, including cancer, if disturbed.

Aldosterone binding sites along nephron of Xenopus and rabbit

1989 ◽  
Vol 257 (1) ◽  
pp. R87-R95 ◽  
Author(s):  
A. Gnionsahe ◽  
M. Claire ◽  
N. Koechlin ◽  
J. P. Bonvalet ◽  
N. Farman
Keyword(s):  
Binding Sites ◽  
Direct Evidence ◽  
Distal Tubule ◽  
Distal Segment ◽  
The Other ◽  
Type I ◽  
Thick Ascending Limb ◽  
Type Ii ◽  
Straight Segment ◽  
Dry Film

Distal segment of several amphibians exhibits aldosterone-modulated ion transport properties. On the other hand, A6 cells, derived from Xenopus laevis (XL) kidney, are aldosterone sensitive. We examined the distribution of aldosterone binding sites in isolated tubules of XL compared with rabbit. After incubation with 2 nM [3H]aldosterone, microdissected tubular segments from proximal (PT), distal straight segment (DST), and flask cell collecting (CT) tubules from XL and from rabbit cortical thick ascending limb (CTAL), connecting (CNT), and collecting (CCD) tubules were processed for dry film autoradiography. In XL, specific nuclear labeling of type I (mineralocorticoid) sites was restricted to DST. Labeling of type II (glucocorticoid) sites was present all along the tubule. No specific cytoplasmic labeling was observed, except for type II sites in PT. In the rabbit, aldosterone binds to both type I and type II sites in the three tubular segments studied. In these segments, the binding was about fourfold higher than in DST of XL. These results bring direct evidence in designating the distal tubule of amphibians as a target epithelium for aldosterone. In addition, they suggest that A6 cell line may derive from DST of the Xenopus nephron.

BIOBOARD

2018 ◽  
Vol 22 (04) ◽  
pp. 49-56
Keyword(s):  
Resistance Mechanism ◽  
Drug Carriers ◽  
Resistant Bacteria ◽  
Antibiotics Resistance ◽  
Drug Resistant ◽  
Chiral Molecules ◽  
Liver Cancer Cells ◽  
Drug Resistant Bacteria ◽  
New Findings ◽  
New Antibiotics

ASIA – Natural products identified as potential new antibiotic against various drug-resistant bacteria. ASIA – HKUST discovers new antibiotics resistance mechanism. ASIA – Green tea-based drug carriers improve cancer treatment. ASIA – New molecule can kill five types of deadly drug-resistant superbugs. ASIA – Scientists grow liver cancer cells in lab. ASIA – New findings reveal sex-based and age-based differences in how the gut microbiome affects brain immunity. ASIA – HKUST scientists find new way to produce chiral molecules.

Molecular basis for pore blockade of human Na+ channel Nav1.2 by the μ-conotoxin KIIIA

Science ◽  
2019 ◽  
Vol 363 (6433) ◽  
pp. 1309-1313 ◽  
Author(s):  
Xiaojing Pan ◽  
Zhangqiang Li ◽  
Xiaoshuang Huang ◽  
Gaoxingyu Huang ◽  
Shuai Gao ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Rational Design ◽  
Na Channel ◽  
Auxiliary Subunit ◽  
Cryo Electron Microscopy ◽  
Initiation And Propagation ◽  
The Central Nervous System ◽  
Immunoglobulin Domain ◽  
Pore Domain ◽  
Pore Blocker

The voltage-gated sodium channel Nav1.2 is responsible for the initiation and propagation of action potentials in the central nervous system. We report the cryo–electron microscopy structure of human Nav1.2 bound to a peptidic pore blocker, the μ-conotoxin KIIIA, in the presence of an auxiliary subunit, β2, to an overall resolution of 3.0 angstroms. The immunoglobulin domain of β2 interacts with the shoulder of the pore domain through a disulfide bond. The 16-residue KIIIA interacts with the extracellular segments in repeats I to III, placing Lys7 at the entrance to the selectivity filter. Many interacting residues are specific to Nav1.2, revealing a molecular basis for KIIIA specificity. The structure establishes a framework for the rational design of subtype-specific blockers for Nav channels.

Sign in / Sign up

Export Citation Format

Share Document