Biology subjects

Brodersen, D. E.

Publications and source records attributed to Brodersen, D. E..

5 recordsLinked to original sources

bioRxiv2025

Catalytic Mechanism and Differential Alarmone Regulation of a Conserved Stringent Nucleosidase

Understanding how bacteria rapidly adapt their metabolism in response to external stimuli is key to addressing the present crisis of antibiotics-resistant infections. In the Gram-negative bacterium Escherichia coli, the universal stringent response is elicited in response to some antibiotics and involves production of the global alarmones, (p)ppGpp, which bind directly to many cellular targets. The nucleosidase PpnN that cleaves nucleotides into 5-phosphate ribose and nucleobase, was shown to be a target of (p)ppGpp and control the delicate balance of bacterial fitness and persistence to fluoroquinolone antibiotics, thus conferring optimal survival strategies to bacteria during antibiotic selective pressure. Although both pppGpp and ppGpp stimulate the enzymatic activity of PpnN, they exert distinct effects on the enzymes cooperativity. The molecular mechanism underlying this subtle difference as well as the precise catalytic mechanism of PpnN, remain obscure. In this study, we provide mechanistic insights into the interaction of PpnN with substrate analogue, reaction products and alarmone molecules, which allows us to understand the catalytic mechanism of this family of nucleosidases and the differential modes of regulation by ppGpp and pppGpp, respectively. Comparison with the homologous LOG proteins involved in cytokinin production in plants reveals an ancient, universal mechanism for cleaving purine monophosphates that bacteria have incorporated regulatory controls through alarmones upon stringent responses. IMPORTANCEThis study explores the distinct manners that the stringent alarmones pppGpp and ppGpp interact with the nucleosidase PpnN, which diverge from their typical, consistent effects on other target proteins. Furthermore, PpnN plays a key role in balancing bacterial fitness and tolerance to antibiotics, yet its catalytic mechanism has remained unclear. Through a combination of structural biology, molecular simulations, biochemistry, mutagenesis, and physiological analyses, this research uncovers the mechanistic differences in how both alarmones differentially impact enzyme cooperativity of PpnN and activation under stress conditions. Additionally, by analysing structures of PpnN complexed with substrate analogues, reaction products, and alarmones, as well as conducting bioinformatic comparisons, we propose a conserved catalytic mechanism shared with its homologue, LOG, a protein involved in cytokinin signalling - a critical growth hormone in plants. These insights underscore the nuanced regulatory roles of alarmones and PpnN/LOG group of nucleosidases, highlighting their evolutionary diversification to meet the varied environmental survival needs across organisms.

biochemistry↗

bioRxiv2024

Asymmetry of both repressor and operator is important for the transcriptional regulation of the P. putida Xre-RES system

Transcriptional regulation by binding of transcription factors to palindromic sequences in promoter regions is a fundamental process in bacteria. Some transcription factors have multiple dimeric DNA-binding domains, in principle enabling interaction with higher-order DNA structures; however, mechanistic and structural insights into this phenomenon remain limited. The Pseudomonas putida toxin-antitoxin (TA) system Xre-RES has an unusual 4:2 stoichiometry including two potential DNA-binding sites, compatible with a complex mechanism of transcriptional autoregulation. Here, we show that the Xre-RES complex interacts specifically with a palindromic DNA repeat in the promoter in a 1:1 molar ratio, leading to transcriptional repression. We determine the 2.7 [A] crystal structure of the protein-DNA complex, revealing an unexpected asymmetry in the interaction and suggesting the presence of a secondary binding site, which is supported by structural prediction of the binding to the intact promoter region. Additionally, we show that the antitoxin can be partially dislodged from the Xre-RES complex, resulting in Xre monomers and a 2:2 Xre-RES complex, neither of which repress transcription. These findings highlight a dynamic, concentration-dependent model of transcriptional autoregulation, in which the Xre-RES complex transitions between a non-binding (2:2) and a DNA-binding (4:2) form. AUTHOR SUMMARYBacteria regulate their gene expression to respond to environmental stress, to evade antibiotics, and to maintain population stability. In this study, we investigate how the xre-res toxin-antitoxin system from Pseudomonas putida controls its own expression. Using a combination of microbiology, structural biology, biophysical assays, and computational modelling, we discover how the Xre-RES protein complex represses its own transcription through direct binding to a specific DNA element in the promoter region. We show structurally that the Xre-RES complex adopts a unique 4:2 stoichiometry and binds DNA in an unusual asymmetrical manner. Moreover, the complex was found to shift between two different forms: one that binds DNA and represses transcription, and one that does not. We further demonstrate that this shift is dynamic and depends on the relative concentration of Xre antitoxin. Our findings provide new insight into how bacteria fine-tune gene expression and offers a model of transcriptional control based on protein stoichiometry and structural asymmetry.

microbiology↗

bioRxiv2022

Structural remodelling of the carbon-phosphorus lyase machinery by a dual ABC ATPase

Phosphorus is an essential macronutrient for all microorganisms and can be extracted from the environment by several metabolic pathways. In Escherichia coli, the 14-cistron phn operon encoding the carbon-phosphorus (C-P) lyase enzymatic machinery allows for extraction of phosphorus from a wide range of phosphonates characterised by the highly stable C-P bond.1, 2 As part of a complex, multi-step pathway, the PhnJ subunit was proposed to cleave the C-P bond via a radical reaction, however, the details of the mechanism were not immediately compatible with the structure of the 220 kDa PhnGHIJ C-P lyase core complex, leaving a significant gap in our understanding of phosphonate breakdown in bacteria.3, 4 Here we show using single-particle cryogenic-electron microscopy that PhnJ mediates binding of a unique double dimer of ATP-binding cassette (ABC) proteins, PhnK and PhnL to the core complex. ATP hydrolysis by PhnK induces drastic structural remodelling leading to opening of the core and reconfiguration of a metal-binding site located at the interface between the PhnI and PhnJ subunits. Our results offer new insights into the mechanism underlying C-P lyase and uncover a hitherto unknown configuration of ABCs that have wide-ranging implications for our understanding of the role of this module in biological systems.

biochemistry↗

bioRxiv2021

Phylogeny Reveals Novel HipA-Homologous Kinase Families and Toxin - Antitoxin Gene Organizations

Toxin - Antitoxin modules function in the genetic stability of mobile genetic elements, bacteriophage defense, and antibiotic tolerance. A gain-of-function mutation of the Escherichia coli K-12 hipBA module can induce antibiotic tolerance in a subpopulation of bacterial cells, a phenomenon known as persistence. HipA is a Ser/Thr kinase that phosphorylates and inactivates glutamyl tRNA synthetase, inhibiting cellular translation and inducing the stringent response. Additional characterized HipA homologues include HipT from pathogenic E. coli O127 and YjjJ of E. coli K-12, which are encoded by tri-cistronic hipBST and monocistronic operons, respectively. The apparent diversity of HipA homologues in bacterial genomes inspired us to investigate overall phylogeny. Here we present a comprehensive phylogenetic analysis of the Hip kinases in bacteria and archaea that expands on this diversity by revealing seven novel kinase families. Kinases of one family, encoded by monocistronic operons, consist of an N-terminal core kinase domain, a HipS-like domain and a HIRAN (HIP116 Rad5p N-terminal) domain. HIRAN domains bind single or double-stranded DNA ends. Moreover, five types of bicistronic kinase operons encode putative antitoxins with HipS-HIRAN, HipS, {gamma}{delta}-resolvase or Stl repressor-like domains. Finally, our analysis indicates that reversion of hipBA gene-order happened independently several times during evolution. ImportanceBacterial multidrug tolerance and persistence are problems of increasing scientific and medical significance. The first gene discovered to confer persistence was hipA, encoding the kinase toxin of the hipBA toxin-antitoxin (TA) module of E. coli. HipA-homologous kinases phosphorylate and thereby inactivate specific tRNA synthetases, thus inhibiting protein translation and cell proliferation. Here, we present a comprehensive phylogenetic analysis of bacterial Hip kinases and discover seven new families with novel operon structures and domains. Overall, Hip kinases are encoded by TA modules with at least 10 different genetic organizations, seven of which have not been described before. These results open up exciting avenues for the experimental analysis of the superfamily of Hip kinases.

microbiology↗

bioRxiv2019

Structure and function of the bacterial protein toxin phenomycin

Phenomycin is a bacterial mini-protein of 89 amino acids discovered more than 50 years ago with toxicity in the nanomolar regime towards mammalian cells. The protein inhibits the function of the eukaryotic ribosome in cell free systems and appears to target translation initiation. Several fundamental questions concerning the cellular activity of phenomycin have however remained unanswered. In this paper, we have used morphological profiling to show that direct inhibition of translation underlies the toxicity of phenomycin in cells. We have performed studies of the cellular uptake mechanism of phenomycin, showing that endosomal escape is the toxicity-limiting step, and we have solved a solution phase high-resolution structure of the protein using NMR spectroscopy. Through bioinformatic as well as functional comparisons between phenomycin and two homologs, we have identified a peptide segment, which constitutes one of two loops in the structure, that is critical for the toxicity of phenomycin.

cell biology↗