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ACS Omega Mar 2024Recent advances in iterative neural network analyses (e.g., AlphaFold2 and RoseTTA fold) have been revolutionary for protein 3D structure prediction, especially for...
Molecular Dynamics of Outer Membrane-Embedded Polysaccharide Secretion Porins Reveals Closed Resting-State Surface Gates Targetable by Virtual Fragment Screening for Drug Hotspot Identification.
Recent advances in iterative neural network analyses (e.g., AlphaFold2 and RoseTTA fold) have been revolutionary for protein 3D structure prediction, especially for difficult-to-manipulate α-helical/β-barrel integral membrane proteins. These model structures are calculated based on the coevolution of amino acids within the protein of interest and similarities to existing protein structures; the local effects of the membrane on folding and stability of the calculated model structures are not considered. We recently reported the discovery, 3D modeling, and characterization of 18-β-stranded outer-membrane (OM) WzpX, WzpS, and WzpB β-barrel secretion porins for the exopolysaccharide (EPS), major spore coat polysaccharide (MASC), and biosurfactant polysaccharide (BPS) pathways (respectively) in the Gram-negative social predatory bacterium DZ2. However, information was not obtained regarding the dynamic behavior of surface-gating WzpX/S/B loop domains or on potential treatments to inactivate these porins. Herein, we developed a molecular dynamics (MD) protocol to study the core stability and loop dynamism of neural network-based integral membrane protein structure models embedded in an asymmetric OM bilayer, using the WzpX, WzpS, and WzpB proteins as test candidates. This was accomplished through integration of the CHARMM-graphical user interface (GUI) and Molecular Operating Environment (MOE) workflows to allow for a rapid simulation system setup and facilitate data analysis. In addition to serving as a method of model structure validation, our molecular dynamics simulations revealed a minimal movement of extracellular WzpX/S/B loops in the absence of an external stimulus as well as druggable cavities between the loops. Virtual screening of a commercial fragment library against these cavities revealed putative fragment-binding hotspots on the cell-surface face of each β-barrel, along with key interacting residues, and identified promising hits for the design of potential binders capable of plugging the β-barrels and inhibiting polysaccharide secretion.
PubMed: 38524450
DOI: 10.1021/acsomega.3c09970 -
The Journal of Biological Chemistry Apr 2024Cell polarity oscillations in Myxococcus xanthus motility are driven by a prokaryotic small Ras-like GTPase, mutual gliding protein A (MglA), which switches from one...
Cell polarity oscillations in Myxococcus xanthus motility are driven by a prokaryotic small Ras-like GTPase, mutual gliding protein A (MglA), which switches from one cell pole to the other in response to extracellular signals. MglA dynamics is regulated by MglB, which functions both as a GTPase activating protein (GAP) and a guanine nucleotide exchange factor (GEF) for MglA. With an aim to dissect the asymmetric role of the two MglB protomers in the dual GAP and GEF activities, we generated a functional MglAB complex by coexpressing MglB with a linked construct of MglA and MglB. This strategy enabled us to generate mutations of individual MglB protomers (MglB or MglB linked to MglA) and delineate their role in GEF and GAP activities. We establish that the C-terminal helix of MglB, but not MglB, stimulates nucleotide exchange through a site away from the nucleotide-binding pocket, confirming an allosteric mechanism. Interaction between the N-terminal β-strand of MglB and β of MglA is essential for the optimal GEF activity of MglB. Specific residues of MglB which interact with Switch-I of MglA, partially contribute to its GAP activity. Thus, the role of the MglB protomer in the GAP activity of MglB is limited to restricting the conformation of MglA active site loops. The direct demonstration of the allosteric mechanism of GEF action provides us new insights into the regulation of small Ras-like GTPases, a feature potentially present in many uncharacterized GEFs.
Topics: Bacterial Proteins; Enzyme Activation; GTPase-Activating Proteins; Guanine Nucleotide Exchange Factors; Myxococcus xanthus; Protein Multimerization; Models, Molecular; Protein Structure, Quaternary
PubMed: 38508314
DOI: 10.1016/j.jbc.2024.107197 -
BioRxiv : the Preprint Server For... Feb 2024Protein capsids are a widespread form of compartmentalisation in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry...
Protein capsids are a widespread form of compartmentalisation in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry maximises the internal volume that can be enclosed within. Despite the strong preference for icosahedral symmetry, we show that simple point mutations in a virus-like capsid can drive the assembly of novel symmetry-reduced structures. Starting with the encapsulin from , a 180-mer bacterial capsid that adopts the well-studied viral HK97 fold, we use mass photometry and native charge detection mass spectrometry to identify a triple histidine point mutant that forms smaller dimorphic assemblies. Using cryo-EM, we determine the structures of a precedented 60-mer icosahedral assembly and an unprecedented 36-mer tetrahedron that features significant geometric rearrangements around a novel interaction surface between capsid protomers. We subsequently find that the tetrahedral assembly can be generated by triple point mutation to various amino acids, and that even a single histidine point mutation is sufficient to form tetrahedra. These findings represent the first example of tetrahedral geometry across all characterised encapsulins, HK97-like capsids, or indeed any virus-derived capsids reported in the Protein Data Bank, revealing the surprising plasticity of capsid self-assembly that can be accessed through minimal changes in protein sequence.
PubMed: 38370832
DOI: 10.1101/2024.02.05.579038 -
Frontiers in Microbiology 2024Bacterial predators are widely distributed across a variety of natural environments. Understanding predatory interactions is of great importance since they play a... (Review)
Review
Bacterial predators are widely distributed across a variety of natural environments. Understanding predatory interactions is of great importance since they play a defining role in shaping microbial communities in habitats such as soils. is a soil-dwelling bacterial predator that can prey on Gram-positive and Gram-negative bacteria and even on eukaryotic microorganisms. This model organism has been studied for many decades for its unusual lifecycle, characterized by the formation of multicellular fruiting bodies filled with myxospores. However, less is known about its predatory behavior despite being an integral part of its lifecycle. Predation in is a multifactorial process that involves several mechanisms working synergistically, including motility systems to efficiently track and hunt prey, and a combination of short-range and contact-dependent mechanisms to achieve prey death and feed on them. In the short-range attack, is best known for the collective production of secondary metabolites and hydrolytic enzymes to kill prey and degrade cellular components. On the other hand, contact-dependent killing is a cell-to-cell process that relies on Tad-like and type III secretion systems. Furthermore, recent research has revealed that metals also play an important role during predation, either by inducing oxidative stress in the prey, or by competing for essential metals. In this paper, we review the current knowledge about predation, focusing on the different mechanisms used to hunt, kill, and feed on its prey, considering the most recent discoveries and the transcriptomic data available.
PubMed: 38328431
DOI: 10.3389/fmicb.2024.1339696 -
PLoS Biology Jan 2024Ecological variation influences the character of many biotic interactions, but examples of predator-prey reversal mediated by abiotic context are few. We show that the...
Ecological variation influences the character of many biotic interactions, but examples of predator-prey reversal mediated by abiotic context are few. We show that the temperature at which prey grow before interacting with a bacterial predator can determine the very direction of predation, reversing predator and prey identities. While Pseudomonas fluorescens reared at 32°C was extensively killed by the generalist predator Myxococcus xanthus, P. fluorescens reared at 22°C became the predator, slaughtering M. xanthus to extinction and growing on its remains. Beyond M. xanthus, diffusible molecules in P. fluorescens supernatant also killed 2 other phylogenetically distant species among several examined. Our results suggest that the sign of lethal microbial antagonisms may often change across abiotic gradients in natural microbial communities, with important ecological and evolutionary implications. They also suggest that a larger proportion of microbial warfare results in predation-the killing and consumption of organisms-than is generally recognized.
Topics: Animals; Predatory Behavior; Antibiosis; Biological Evolution; Microbiota; Myxococcus xanthus
PubMed: 38261596
DOI: 10.1371/journal.pbio.3002454 -
Frontiers in Microbiology 2023Adjusting motility patterns according to environmental cues is important for bacterial survival. , a bacterium moving on surfaces by gliding and twitching mechanisms,...
Adjusting motility patterns according to environmental cues is important for bacterial survival. , a bacterium moving on surfaces by gliding and twitching mechanisms, modulates the reversal frequency of its front-back polarity in response to mechanical cues like substrate stiffness and cell-cell contact. In this study, we propose that 's gliding machinery senses environmental mechanical cues during force generation and modulates cell reversal accordingly. To examine our hypothesis, we expand an existing mathematical model for periodic polarity reversal in , incorporating the experimental data on the intracellular dynamics of the gliding machinery and the interaction between the gliding machinery and a key polarity regulator. The model successfully reproduces the dependence of cell reversal frequency on substrate stiffness observed in gliding. We further propose reversal control networks between the gliding and twitching motility machineries to explain the opposite reversal responses observed in wild type cells that possess both motility mechanisms. These results provide testable predictions for future experimental investigations. In conclusion, our model suggests that the gliding machinery in can function as a mechanosensor, which transduces mechanical cues into a cell reversal signal.
PubMed: 38260904
DOI: 10.3389/fmicb.2023.1294631 -
International Journal of Biological... Feb 2024Prolyl endopeptidases (PEP) from Sphingomonas capsulata (sc) and Myxococcus xanthus (mx) selectively degrade gluten peptides in vitro, offering a potential therapeutic...
Prolyl endopeptidases (PEP) from Sphingomonas capsulata (sc) and Myxococcus xanthus (mx) selectively degrade gluten peptides in vitro, offering a potential therapeutic strategy for celiac disease. However, the mechanisms governing the interaction of these enzymes with their substrates remain unclear. In this study, conventional molecular dynamics simulations with a microsecond timescale and targeted molecular dynamics simulations were performed to investigate the native states of mxPEP and scPEP enzymes, as well as their allosteric binding with a representative substrate, namely, Z-Ala-Pro-p-nitroanilide (pNA). The simulations reveal that the native scPEP is in an open state, while the native mxPEP is in a closed state. When pNA approaches a closed mxPEP, it binds to an allosteric pocket located at the first and second β-sheet of the β-propeller domain, inducing the opening of this enzyme. Neither enzyme is active in the open or partly-open states. Enzymatic activity is enabled only when the catalytic pocket in the closed state fully accommodates the substrates. The internal capacity of the catalytic pocket of PEP in the closed state determines the maximum size of the gluten peptides that the enzymes can catalyze. The present work provides essential molecular dynamics information for the redesign or engineering of PEP enzymes.
Topics: Humans; Prolyl Oligopeptidases; Celiac Disease; Serine Endopeptidases; Molecular Dynamics Simulation; Glutens; Peptides
PubMed: 38216012
DOI: 10.1016/j.ijbiomac.2024.129313 -
BioRxiv : the Preprint Server For... Dec 2023Ecological context often modifies biotic interactions, yet effects of ecological history are poorly understood. In experiments with the bacterium , resource-level...
Ecological context often modifies biotic interactions, yet effects of ecological history are poorly understood. In experiments with the bacterium , resource-level histories of genotypes interacting during cooperative multicellular development were found to strongly regulate social fitness. Yet how developmental spore production responded to variation in resource-level histories between interactants differed greatly between cooperators and cheaters; relative-fitness advantages gained by cheating after high-resource growth were generally reduced or absent if one or both parties experienced low-resource growth. Low-resource growth also eliminated facultative exploitation in some pairwise mixes of cooperation-proficient natural isolates that occurs when both strains have grown under resource abundance. Our results contrast with previous studies in which cooperator fitness correlated positively with resource level and suggest that resource-level variation may be important in regulating whether exploitation of cooperators occurs in a natural context.
PubMed: 38168390
DOI: 10.1101/2023.12.14.571652 -
Frontiers in Microbiology 2023and represent a well-studied microbial predator-prey pair frequently examined in laboratory settings. While significant progress has been made in comprehending the...
and represent a well-studied microbial predator-prey pair frequently examined in laboratory settings. While significant progress has been made in comprehending the mechanisms governing predation, various aspects of the response and defensive mechanisms of as prey remain elusive. In this study, the MG1655 large-scale chromosome deletion library was screened, and a mutant designated as ME5012 was identified to possess significantly reduced susceptibility to predation by . Within the deleted region of ME5012 encompassing seven genes, the significance of and genes in driving the observed phenotype became apparent. Specifically, the deletion of resulted in a notable reduction in flagellum production in , contributing to a certain level of resistance against predation by . Meanwhile, the removal of in led to diminished inducibility of myxovirescin A production by , accompanied by a slight decrease in susceptibility to myxovirescin A. These findings shed light on the molecular mechanisms underlying the complex interaction between and in a predatory context.
PubMed: 38116529
DOI: 10.3389/fmicb.2023.1304874 -
Molecular Omics Feb 2024The outer membrane vesicles (OMVs) secreted by some Gram-negative bacteria contain RNA cargo, which can be introduced into target cells, affecting their cellular...
The outer membrane vesicles (OMVs) secreted by some Gram-negative bacteria contain RNA cargo, which can be introduced into target cells, affecting their cellular processes. To test whether the antimicrobial OMVs secreted by predatory myxobacteria might contain cargo RNA with a role in prey killing, we purified OMVs and cells from four different strains of spp. for RNA-seq transcriptome sequencing. Myxobacterial OMVs contained distinct sets of RNA molecules. The abundance of major cellular transcripts correlated strongly with their abundance in OMVs, suggesting non-specific packaging into OMVs. However, many major cellular transcripts were absent entirely from OMVs and some transcripts were found exclusively in OMVs, suggesting OMV RNA cargo loading is not simply a consequence of sampling the cellular transcriptome. Despite considerable variation in OMV RNA cargo between biological replicates, a small number of transcripts were found consistently in replicate OMV preparations. These 'core' OMV transcripts were often found in the OMVs from multiple strains, and sometimes enriched relative to their abundance in cellular transcriptomes. In addition to providing the first transcriptomes for myxobacterial OMVs, and the first cellular transcriptomes for three strains of spp., we highlight five transcripts for further study. These transcripts are 'core' for at least two of the three strains of studied, and encode two alkyl hydroperoxidase proteins (AhpC and AhpD), two ribosome-associated inhibitors (RaiA-like) and a DO-family protease. It will be interesting to test whether the transcripts serve a biological function within OMVs, potentially being transported into prey cells for translation into toxic proteins.
Topics: Myxococcus; RNA
PubMed: 38098456
DOI: 10.1039/d3mo00222e