30S subunit recognition and G1405 modification by the aminoglycoside-resistance 16S ribosomal RNA methyltransferase RmtC.

Acquired ribosomal RNA (rRNA) methylation has emerged as a significant mechanism of aminoglycoside resistance in pathogenic bacterial infections. Modification of a single nucleotide in the ribosome decoding center by the aminoglycoside-resistance 16S rRNA (m7G1405) methyltransferases effectively blocks the action of all 4,6-deoxystreptamine ring-containing aminoglycosides, including the latest generation of drugs. To define the molecular basis of 30S subunit recognition and G1405 modification by these enzymes, we used a S-adenosyl-L-methionine analog to trap the complex in a postcatalytic state to enable determination of a global 3.0 Å cryo-electron microscopy structure of the m7G1405 methyltransferase RmtC bound to the mature Escherichia coli 30S ribosomal subunit. This structure, together with functional analyses of RmtC variants, identifies the RmtC N-terminal domain as critical for recognition and docking of the enzyme on a conserved 16S rRNA tertiary surface adjacent to G1405 in 16S rRNA helix 44 (h44). To access the G1405 N7 position for modification, a collection of residues across one surface of RmtC, including a loop that undergoes a disorder-to order transition upon 30S subunit binding, induces significant distortion of h44. This distortion flips G1405 into the enzyme active site where it is positioned for modification by two almost universally conserved RmtC residues. These studies expand our understanding of ribosome recognition by rRNA modification enzymes and present a more complete structural basis for future development of strategies to inhibit m7G1405 modification to resensitize bacterial pathogens to aminoglycosides.

Structural Characterization of the Chlorophyllide a Oxygenase (CAO) Enzyme Through an In Silico Approach.

Chlorophyllide a oxygenase (CAO) is responsible for converting chlorophyll a to chlorophyll b in a two-step oxygenation reaction. CAO belongs to the family of Rieske-mononuclear iron oxygenases. Although the structure and reaction mechanism of other Rieske monooxygenases have been described, a member of plant Rieske non-heme iron-dependent monooxygenase has not been structurally characterized. The enzymes in this family usually form a trimeric structure and electrons are transferred between the non-heme iron site and the Rieske center of the adjoining subunits. CAO is supposed to form a similar structural arrangement. However, in Mamiellales such as Micromonas and Ostreococcus, CAO is encoded by two genes where non-heme iron site and Rieske cluster localize on the distinct polypeptides. It is not clear if they can form a similar structural organization to achieve the enzymatic activity. In this study, the tertiary structures of CAO from the model plant Arabidopsis thaliana and the Prasinophyte Micromonas pusilla were predicted by deep learning-based methods, followed by energy minimization and subsequent stereochemical quality assessment of the predicted models. Furthermore, the chlorophyll a binding cavity and the interaction of ferredoxin, which is the electron donor, on the surface of Micromonas CAO were predicted. The electron transfer pathway was predicted in Micromonas CAO and the overall structure of the CAO active site was conserved even though it forms a heterodimeric complex. The structures presented in this study will serve as a basis for understanding the reaction mechanism and regulation of the plant monooxygenase family to which CAO belongs.

30S subunit recognition and G1405 modification by the aminoglycoside-resistance 16S ribosomal RNA methyltransferase RmtC.

Acquired ribosomal RNA (rRNA) methylation has emerged as a significant mechanism of aminoglycoside resistance in pathogenic bacterial infections. Modification of a single nucleotide in the ribosome decoding center by the aminoglycoside-resistance 16S rRNA (m 7 G1405) methyltransferases effectively blocks the action of all 4,6-deoxystreptamine ring-containing aminoglycosides, including the latest generation of drugs. To define the molecular basis of 30S subunit recognition and G1405 modification by these enzymes, we used a S-adenosyl-L-methionine (SAM) analog to trap the complex in a post-catalytic state to enable determination of an overall 3.0 Å cryo-electron microscopy structure of the m 7 G1405 methyltransferase RmtC bound to the mature Escherichia coli 30S ribosomal subunit. This structure, together with functional analyses of RmtC variants, identifies the RmtC N-terminal domain as critical for recognition and docking of the enzyme on a conserved 16S rRNA tertiary surface adjacent to G1405 in 16S rRNA helix 44 (h44). To access the G1405 N7 position for modification, a collection of residues across one surface of RmtC, including a loop that undergoes a disorder to order transition upon 30S subunit binding, induces significant distortion of h44. This distortion flips G1405 into the enzyme active site where it is positioned for modification by two almost universally conserved RmtC residues. These studies expand our understanding of ribosome recognition by rRNA modification enzymes and present a more complete structural basis for future development of strategies to inhibit m 7 G1405 modification to re-sensitize bacterial pathogens to aminoglycosides.

Tied up in knots: Untangling substrate recognition by the SPOUT methyltransferases.

The SpoU-TrmD (SPOUT) methyltransferase superfamily was designated when structural similarity was identified between the transfer RNA-modifying enzymes TrmH (SpoU) and TrmD. SPOUT methyltransferases are found in all domains of life and predominantly modify transfer RNA or ribosomal RNA substrates, though one instance of an enzyme with a protein substrate has been reported. Modifications placed by SPOUT methyltransferases play diverse roles in regulating cellular processes such as ensuring translational fidelity, altering RNA stability, and conferring bacterial resistance to antibiotics. This large collection of S-adenosyl-L-methionine-dependent methyltransferases is defined by a unique α/ß fold with a deep trefoil knot in their catalytic (SPOUT) domain. Herein, we describe current knowledge of SPOUT enzyme structure, domain architecture, and key elements of catalytic function, including S-adenosyl-L-methionine co-substrate binding, beginning with a new sequence alignment that divides the SPOUT methyltransferase superfamily into four major clades. Finally, a major focus of this review will be on our growing understanding of how these diverse enzymes accomplish the molecular feat of specific substrate recognition and modification, as highlighted by recent advances in our knowledge of protein-RNA complex structures and the discovery of the dependence of one SPOUT methyltransferase on metal ion binding for catalysis. Considering the broad biological roles of RNA modifications, developing a deeper understanding of the process of substrate recognition by the SPOUT enzymes will be critical for defining many facets of fundamental RNA biology with implications for human disease.

50S subunit recognition and modification by the Mycobacterium tuberculosis ribosomal RNA methyltransferase TlyA.

Changes in bacterial ribosomal RNA (rRNA) methylation status can alter the activity of diverse groups of ribosome-targeting antibiotics. These modifications are typically incorporated by a single methyltransferase that acts on one nucleotide target and rRNA methylation directly prevents drug binding, thereby conferring drug resistance. Loss of intrinsic methylation can also result in antibiotic resistance. For example, Mycobacterium tuberculosis becomes sensitized to tuberactinomycin antibiotics, such as capreomycin and viomycin, due to the action of the intrinsic methyltransferase TlyA. TlyA is unique among antibiotic resistance-associated methyltransferases as it has dual 16S and 23S rRNA substrate specificity and can incorporate cytidine-2'-O-methylations within two structurally distinct contexts. Here, we report the structure of a mycobacterial 50S subunit-TlyA complex trapped in a postcatalytic state with a S-adenosyl-L-methionine analog using single-particle cryogenic electron microscopy. Together with complementary functional analyses, this structure reveals critical roles in 23S rRNA substrate recognition for conserved residues across an interaction surface that spans both TlyA domains. These interactions position the TlyA active site over the target nucleotide C2144, which is flipped from 23S Helix 69 in a process stabilized by stacking of TlyA residue Phe157 on the adjacent A2143. Base flipping may thus be a common strategy among rRNA methyltransferase enzymes, even in cases where the target site is accessible without such structural reorganization. Finally, functional studies with 30S subunit suggest that the same TlyA interaction surface is employed to recognize this second substrate, but with distinct dependencies on essential conserved residues.

Role of helical structure and dynamics in oligoadenylate synthetase 1 (OAS1) mismatch tolerance and activation by short dsRNAs.

The 2'-5'-oligoadenylate synthetases (OAS) are innate immune sensors of cytosolic double-stranded RNA (dsRNA) that play a critical role in limiting viral infection. How these proteins are able to avoid aberrant activation by cellular RNAs is not fully understood, but adenosine-to-inosine (A-to-I) editing has been proposed to limit accumulation of endogenous RNAs that might otherwise cause stimulation of the OAS/RNase L pathway. Here, we aim to uncover whether and how such sequence modifications can restrict the ability of short, defined dsRNAs to activate the single-domain form of OAS, OAS1. Unexpectedly, we find that all tested inosine-containing dsRNAs have an increased capacity to activate OAS1, whether in a destabilizing (I•U) or standard Watson-Crick-like base pairing (I-C) context. Additional variants with strongly destabilizing A•C mismatches or stabilizing G-C pairs also exhibit increased capacity to activate OAS1, eliminating helical stability as a factor in the relative ability of the dsRNAs to activate OAS1. Using thermal difference spectra and molecular dynamics simulations, we identify both increased helical dynamics and specific local changes in helical structure as important factors in the capacity of short dsRNAs to activate OAS1. These helical features may facilitate more ready adoption of the distorted OAS1-bound conformation or stabilize important structures to predispose the dsRNA for optimal binding and activation of OAS1. These studies thus reveal the molecular basis for the greater capacity of some short dsRNAs to activate OAS1 in a sequence-independent manner.

Role of Neutrophil to Lymphocyte Ratio in Predicting Severity of Obstructive Sleep Apnea.

Obstructive sleep apnea (OSA) has been linked to and is associated with increased cardiovascular and cerebrovascular morbidity. Ongoing inflammatory responses play an important role in this association. Systemic inflammation is important in pathophysiology of OSA and its comorbidity. In this study, we aimed to evaluate the role of neutrophil-to-lymphocyte ratio (NLR) in OSA patients and comparing with other well-known inflammatory marker, C-reactive protein (CRP) along with thyroid-stimulating hormone(TSH) and body-mass index(BMI). We conducted a retrospective analysis of 162 patients with OSA and divided them into 2 categories based on apnea-hypopnea index (AHI) (< 30 and > = 30), and recorded their leukocyte profiles, sex, age and body mass index. 80 matched healthy controls were taken. Patients were excluded if they had underlying cancer, chronic inflammatory disease, any systemic infection, uncontrolled hypertension and diabetes mellitus, a known acute coronary syndrome, valvular heart disease, renal or hepatic dysfunction. We found that N/L Ratio in severe OSA patients was significantly higher compared with mild and moderate OSA patients and healthy controls (p < 0.001). CRP levels were not different in all OSA stages (p = 0.595). We noted a significant difference in mean BMI of the two groups. In the wake of increase in prevalence of OSA in a developing country like India coupled with inadequate proportion of sleep labs, NLR is an inexpensive, easy to obtain, widely available marker of inflammation that might in combination with other markers assist in identifying patients with severe OSA.

Academic Crisis During COVID 19: Online Classes, a Panacea for Imminent Doctors.

Introduction: COVID 19 made a serious impact on many aspects of everyday life. The world saw a paradigm shift in the education system favouring online learning during the constrains of pandemic. Methodology: To assess the attitude of the students towards online learning in subject of ENT, we conducted an observational study among 170 third year MBBS undergraduate students of our institute attending online classes through the student portal of our university website. Results: Our survey revealed students favoured online learning to sustain their academic interest and development during this pandemic. Yet, they perceived many challenges during online learning like lack of face-to-face interactions, lack of socialization, distraction by social media, technology related issues etc. Students also opted for a combined approach of learning in the post pandemic period. Conclusion: This article reflects the challenges faced during online learning and added the innovative methods that can be included to overcome the obstacles of online learning. During this period of COVID, one must embrace the alternative to classroom learning to keep up with one's academic development and can consider an integrated approach of learning after the pandemic.

Heterozygous OAS1 gain-of-function variants cause an autoinflammatory immunodeficiency.

Analysis of autoinflammatory and immunodeficiency disorders elucidates human immunity and fosters the development of targeted therapies. Oligoadenylate synthetase 1 is a type I interferon-induced, intracellular double-stranded RNA (dsRNA) sensor that generates 2'-5'-oligoadenylate to activate ribonuclease L (RNase L) as a means of antiviral defense. We identified four de novo heterozygous OAS1 gain-of-function variants in six patients with a polymorphic autoinflammatory immunodeficiency characterized by recurrent fever, dermatitis, inflammatory bowel disease, pulmonary alveolar proteinosis, and hypogammaglobulinemia. To establish causality, we applied genetic, molecular dynamics simulation, biochemical, and cellular functional analyses in heterologous, autologous, and inducible pluripotent stem cell-derived macrophages and/or monocytes and B cells. We found that upon interferon-induced expression, OAS1 variant proteins displayed dsRNA-independent activity, which resulted in RNase L-mediated RNA cleavage, transcriptomic alteration, translational arrest, and dysfunction and apoptosis of monocytes, macrophages, and B cells. RNase L inhibition with curcumin modulated and allogeneic hematopoietic cell transplantation cured the disorder. Together, these data suggest that human OAS1 is a regulator of interferon-induced hyperinflammatory monocyte, macrophage, and B cell pathophysiology.

Substrate recognition by the Pseudomonas aeruginosa EF-Tu-modifying methyltransferase EftM.

The opportunistic bacterial pathogen Pseudomonas aeruginosa is a leading cause of serious infections in individuals with cystic fibrosis, compromised immune systems, or severe burns. P. aeruginosa adhesion to host epithelial cells is enhanced by surface-exposed translation elongation factor EF-Tu carrying a Lys-5 trimethylation, incorporated by the methyltransferase EftM. Thus, the EF-Tu modification by EftM may represent a target to prevent P. aeruginosa infections in vulnerable individuals. Here, we extend our understanding of EftM activity by defining the molecular mechanism by which it recognizes EF-Tu. Acting on the observation that EftM can bind to EF-Tu lacking its N-terminal peptide (encompassing the Lys-5 target site), we generated an EftM homology model and used it in protein/protein docking studies to predict EftM/EF-Tu interactions. Using site-directed mutagenesis of residues in both proteins, coupled with binding and methyltransferase activity assays, we experimentally validated the predicted protein/protein interface. We also show that EftM cannot methylate the isolated N-terminal EF-Tu peptide and that binding-induced conformational changes in EftM are likely needed to enable placement of the first 5-6 amino acids of EF-Tu into a conserved peptide-binding channel in EftM. In this channel, a group of residues that are highly conserved in EftM proteins position the N-terminal sequence to facilitate Lys-5 modification. Our findings reveal that EftM employs molecular strategies for substrate recognition common among both class I (Rossmann fold) and class II (SET domain) methyltransferases and pave the way for studies seeking a deeper understanding of EftM's mechanism of action on EF-Tu.

Conformational heterogeneity in tails of DNA-binding proteins is augmented by proline containing repeats.

A cationic terminal extension or tail is a common feature of many DNA-binding proteins. We show that a particular type of tail rich in proline, alanine and lysine belongs to the class of 'flexible disorder' and consists of characteristic pentapeptide repeats. Our designed peptides, (AAKKA)1-4 and (PAKKA)1-4, represent the tails of several bacterial DNA-binding proteins. Enhanced conformational sampling of these representative peptides using accelerated molecular dynamic simulations supported by circular dichroism spectroscopy and nuclear magnetic resonance studies demonstrates the role of frequent and interspersed prolines in augmenting conformational heterogeneity of the peptide backbone. Analysis of circular variance of backbone dihedral angles indicates alternating regions of relative rigidity and flexibility along the peptide sequence due to prolines. Preferred placement of lysines in the regions of higher backbone flexibility might improve DNA-binding by conformational selection. Our results could be relevant for rational de novo design of disordered peptides.