Biology subjects

Megrian, D.

Publications and source records attributed to Megrian, D..

10 recordsLinked to original sources

bioRxiv2026

Structural basis of MfpD, a versatile pathogeny protein from the mfp conservon of Mycobacterium tuberculosis

The mfp conservon of Mycobacterium tuberculosis has been associated with fluoroquinolone resistance and encodes five conserved proteins, including the small GTPase MfpB and its regulatory partner MfpD. In this study, we combined phylogenetic, structural, and biophysical approaches to define the molecular basis of MfpD function. MfpD adopts a Roadblock/LC7-like /{beta} fold and forms a stable dimer in solution, with hydrophobic 2-helix interactions stabilizing the interface. Additional biophysical analyses and AlphaFold3 modeling suggest that MfpD may promote GTP hydrolysis by MfpB through a noncanonical Switch I-dependent mechanism. These findings establish the first structural framework for MfpD-MfpB interactions, building on previously identified in vitro catalytic properties and proposing new insights into MfpDs non-catalytic pathogenesis activity of MfpD in macrophages. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/709265v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@77b2d2org.highwire.dtl.DTLVardef@7d1908org.highwire.dtl.DTLVardef@f66eeforg.highwire.dtl.DTLVardef@ed159c_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG

biochemistry↗

bioRxiv2025

Charge segregation in the C-terminal linker of FtsZ enables Z-ring scaling with prokaryotic cell width

Bacterial cytokinesis is orchestrated by the Z-ring, a cytoskeletal structure formed by treadmilling filaments of the tubulin-like GTPase FtsZ. During assembly, filament curvature must match the cell diameter, yet how curvature is encoded in FtsZ remains unclear. Using in vitro reconstitution with purified proteins and supported lipid bilayers, we identify FtsZs intrinsically disordered C-terminal linker (CTL) as a curvature tuner that determines the Z-ring diameter. Increasing CTL charge segregation ({kappa}) progressively reduces Z-ring diameter without altering treadmilling dynamics, monomer turnover, or GTPase activity, indicating that {kappa} modulates filament curvature rather than polymerization kinetics. Linker-dilution experiments, in which wild-type FtsZ is mixed with a CTL-deletion mutant ({Delta}CTL), further show that inter-CTL electrostatic interactions straighten filaments, while weakening these contacts increases curvature and compacts rings. To test whether this mechanism is conserved, we analyzed 4,603 prokaryotic FtsZ sequences and found that CTL charge segregation quantified by {kappa} - but not CTL length, fraction of charged residues, or net charge per residue - correlates with cell width: at low {kappa}, cells show a broad range of widths, whereas high {kappa} values exclude wide cells. Co-expression of {Delta}CTL variants in vivo perturbs Z-ring positioning and impairs growth, consistent with a curvature mismatch and defects during assembly. Together, these results suggest a simple evolutionary mechanism in which CTL charge patterning encodes intrinsic filament curvature, enabling Z-ring geometry to scale with cell diameter and linking protein sequences to bacterial morphology.

biochemistry↗

bioRxiv2025

Functional dissection of Wag31 domains for septal recruitment and polar distribution during the cell cycle

Bacterial cell morphogenesis is controlled by the synthesis and organization of peptidoglycan and driven by multi-protein complexes such as the divisome and elongasome. Here we investigate the role of the Corynebacterium glutamicum DivIVA homologue, Wag31, the elongasome scaffold essential for polar growth in Corynebacteriales. Conditional depletion of Wag31 results in viable but coccoid-shaped cells, showing that Wag31 is essential for rod shape maintenance. Our structural phylogenetic analyses of DivIVA homologues revealed that in Actinobacteria, unlike Firmicutes, an intrinsically disordered region spatially separates the N-terminal lipid-binding domain (LBD) from the C-terminal coiled-coil domain (CCD). We show that the LBD is necessary and sufficient for septum localization, independent of its membrane-binding properties, while the CCD domain mediates self-interaction and polar accumulation. Our findings suggest that Wag31 is recruited specifically to the septum through protein-protein interactions, priming the future pole and allowing for a timely divisome-elongasome transition at cytokinesis. Once the pole is formed the self-aggregative properties of the C-terminal CCD dominate and form a stable structure that likely organizes the pole for cell wall biosynthesis.

microbiology↗

bioRxiv2025

Evolutionary repurposing of a DNA segregation machinery into a cytoskeletal system controlling cyanobacterial cell shape

Bacteria, like eukaryotes, use conserved cytoskeletal systems for intracellular organization. The plasmid-encoded ParMRC system forms actin-like filaments that segregate low-copy plasmids. In multicellular cyanobacteria like Anabaena sp., we found that a chromosomally-encoded ParMR has evolved into a novel cytoskeleton-termed CorMR-with a function in cell shape control rather than DNA segregation. Using live-cell imaging, in vitro reconstitution and Cryo-EM, we demonstrate that CorM forms dynamically unstable, antiparallel double-stranded filaments, which are recruited to the membrane by CorR via an amphipathic helix conserved in multicellular cyanobacteria. CorMR filaments are regulated by MinC, which excludes them from the poles and division plane. Comparative genomics reveal that the repurposing of ParMR and Min systems co-evolved with cyanobacterial multicellularity, highlighting the evolutionary plasticity of cytoskeletal systems in bacteria.

microbiology↗

bioRxiv2025

Mechanistic insights into the allosteric regulation of cell wall hydrolase RipA in Mycobacterium tuberculosis.

D,L-endopeptidase RipA is the major PG hydrolase required for daughter cell separation in Mycobacterium tuberculosis (Mtb), as RipA defects severely hinder cell division and increase antibiotic vulnerability. Despite extensive studies, the mechanisms governing Mtb RipA regulation remain controversial and poorly understood. Here, we report an integrative structural and functional analysis of the SteAB system, a regulatory complex that has been shown to modulate cell separation in the model organism Corynebacterium glutamicum (Cglu) and is conserved across Mycobacteriales. Although Mtb SteB was previously proposed to act as a mycobacterial outer membrane copper transporter, the crystal structures of the homodimeric protein, alone and in complex with the RipA coiled-coil (CC) domain, rule out this hypothesis. Instead, the high-affinity SteB-RipA interaction, together with computational and biophysical data, strongly supports the role of SteB as a direct RipA activator that releases enzyme autoinhibition upon complex formation. In addition, crystallographic characterization of the cytoplasmic core of SteA revealed a homodimeric organization harboring a conserved functional pocket similar to the phosphonucleotide-binding site of thiamine pyrophosphokinase. These data, coupled with the in vivo phenotypic analysis of a steAB knockout mutant of Cglu, support a model in which the transmembrane SteAB heterotetramer, driven by cytoplasmic ligand binding, orchestrates the productive periplasmic positioning of RipA, leading to PG hydrolysis activation. These findings shed new light on the regulation of mycobacterial cell wall remodeling, with implications for understanding Mtb pathogenesis and identifying novel antimicrobial targets.

microbiology↗

bioRxiv2024

Cryo-EM structure and polar assembly of the PS2 S-layer of Corynebacterium glutamicum

The polar-growing Corynebacteriales have a complex cell envelope architecture characterized by the presence of a specialized outer membrane composed of mycolic acids. In some Corynebacteriales, this mycomembrane is further supported by a proteinaceous surface layer or S-layer, whose function, structure and mode of assembly remain largely enigmatic. Here, we isolated ex vivo PS2 S-layers from the industrially important Corynebacterium glutamicum and determined its atomic structure by 3D cryoEM reconstruction. PS2 monomers consist of a six-helix bundle core, a three-helix bundle arm, and a C-terminal transmembrane (TM) helix. The PS2 core oligomerizes into hexameric units anchored in the mycomembrane by a channel-like coiled-coil of the TM helices. The PS2 arms mediate trimeric lattice contacts, crystallizing the hexameric units into an intricate semipermeable lattice. Using pulse-chase live cell imaging, we show that the PS2 lattice is incorporated at the poles, coincident with the actinobacterial elongasome. Finally, phylogenetic analysis shows a paraphyletic distribution and dispersed chromosomal location of PS2 in Corynebacteriales as a result of multiple recombination events and losses. These findings expand our understanding of S-layer biology and enable applications of membrane-supported self-assembling bioengineered materials.

microbiology↗

bioRxiv2024

Evolutionary plasticity and functional repurposing of the essential metabolic enzyme MoeA

MoeA, or gephyrin in higher eukaryotes, is crucial for molybdenum cofactor biosynthesis required in redox reactions. Gephyrin is a moonlighting protein also involved in postsynaptic receptor clustering, a feature thought to be a recent evolutionary trait. We showed previously that a repurposed copy of MoeA (Glp) is involved in bacterial cell division. To investigate how MoeA acquired multifunctionality, we used phylogenetic inference and protein structure analyses to understand the diversity and evolutionary history of MoeA. Glp-expressing Bacteria have at least two copies of the gene, and our analysis suggests that Glp has lost its enzymatic role. In Archaea we identified an ancestral duplication where one of the paralogs might bind tungsten instead of molybdenum. In eukaryotes, the acquisition of the moonlighting activity of gephyrin comprised three major events: first, MoeA was obtained from Bacteria by early eukaryotes, second, MogA was fused to the N-terminus of MoeA in the ancestor of opisthokonts, and finally, it acquired the function of anchoring GlyR receptors in neurons. Our results support the functional versatility and adaptive nature of the MoeA scaffold, which has been repurposed independently both in eukaryotes and bacteria to carry out analogous functions in network organization at the cell membrane.

evolutionary biology↗

bioRxiv2023

High resolution cryo-EM and crystallographic snapshots of the large actinobacterial 2-oxoglutarate dehydrogenase: an all-in-one fusion with unique properties

Actinobacteria possess unique ways to regulate the oxoglutarate node located in the central position of the tricarboxylic acid cycle, a crossroad between energy conservation and nitrogen metabolism. Here, we studied the decarboxylative oxidation route that leads, through the 2-oxoglutarate dehydrogenase (ODH) complex, to the generation of succinyl-CoA and reduced equivalents to feed the respiratory chain. Compared to most organisms in which the oxidative decarboxylation and reductive acylation steps are carried out by different enzymes within the ODH complex, actinobacteria rely on an all-in-one protein (OdhA) in which both activities are carried out by the same polypeptide. We describe high-resolution cryo-EM and X-ray crystallography snapshots of representative enzymes from Mycobacterium smegmatis and Corynebacterium glutamicum, showing that OdhA is an 800-kDa homohexamer that folds into a three-blade propeller shape. The obligate trimeric and dimeric states of the acyltransferase and dehydrogenase domains, respectively, are critical for maintaining the overall assembly, where both domains interact via subtle readjustments of their interfaces. Complexes obtained with substrate analogues, reaction products and allosteric regulators illustrate how these domains operate. Furthermore, we provide additional insights into the phosphorylation-dependent regulation of this enzymatic machinery by the FHA (Fork-Head Associated) signalling protein OdhI, delivering new molecular details on how this actinobacterial-specific switching mechanism operates. Overall, the quaternary organization of OdhA represents a new piece of the fascinating puzzle of the synergistic, mixed pyruvate dehydrogenase/2-oxoglutarate dehydrogenase actinobacterial supercomplex.

biochemistry↗

bioRxiv2023

Eukaryotic-like gephyrin and cognate membrane receptor coordinate corynebacterial cell division and polar elongation.

The order Corynebacteriales includes major industrial and pathogenic actinobacteria such as Corynebacterium glutamicum or Mycobacterium tuberculosis. Their elaborate multi-layered cell wall, composed primarily of the mycolyl-arabinogalactan-peptidoglycan complex, and their polar growth mode impose a stringent coordination between the septal divisome, organized around the tubulin-like protein FtsZ, and the polar elongasome, assembled around the tropomyosin-like protein Wag31. Here, we report the identification of two new divisome members, a gephyrin-like repurposed molybdotransferase (GLP) and its membrane receptor (GLPR). We show that the interplay between the GLPR/GLP module, FtsZ and Wag31 is crucial for orchestrating cell cycle progression. Our results provide a detailed molecular understanding of the crosstalk between two essential machineries, the divisome and elongasome, and reveal that Corynebacteriales have evolved a protein scaffold to control cell division and morphogenesis similar to the gephyrin/GlyR system that in higher eukaryotes mediates synaptic signaling through network organization of membrane receptors and the microtubule cytoskeleton.

microbiology↗

bioRxiv2020

SepF is the FtsZ-anchor in Archaea: implications for cell division in the Last Universal Common Ancestor

The Archaea present profound differences compared to Bacteria in fundamental molecular and cellular processes. While most Archaea divide by binary fission using an FtsZ-based system similar to Bacteria, they lack the majority of the components forming the complex bacterial divisome. Moreover, how FtsZ precisely functions and interacts with other proteins to assemble the archaeal division machinery remains largely unknown. Notably, among the multiple bacterial factors that tether FtsZ to the membrane during cell constriction, Archaea only possess SepF-like homologues, but their function has not been demonstrated. Here, we combine structural, cellular, and evolutionary approaches to demonstrate that SepF is the FtsZ anchor in the human-associated archaeon Methanobrevibacter smithii. 3D super-resolution microscopy of immunolabeled cells shows that M. smithii SepF co-localizes with FtsZ at the division plane. We also show that M. smithii SepF binds both to membranes and FtsZ, inducing filament bundling. High-resolution crystal structures of archaeal SepF alone and in complex with FtsZCTD reveal that SepF forms a dimer with a specific homodimerization interface. This drives a strikingly different binding mode from what is observed in Bacteria. Finally, analysis of the distribution and phylogeny of SepF and FtsZ indicates that these proteins date back to the Last Universal Common Ancestor (LUCA) and that Archaea may have retained features of an ancestral minimal cell division system, while Bacteria likely diverged to accommodate the emergence of the complex machinery required to coordinate cytokinesis with the rigid peptidoglycan cell wall and the appearance of additional FtsZ tethers. Our results contribute key insights into the largely understudied mechanisms of archaeal cell division, and pave the way for a better understanding of the processes underlying the divide between the two prokaryotic domains.

microbiology↗