Biology subjects

Zeytuni, N.

Publications and source records attributed to Zeytuni, N..

9 recordsLinked to original sources

bioRxiv2026

Structural tuning of native type V fimbriae shapes mechanical specialization in Porphyromonas gingivalis

Fimbriae are proteinaceous filaments central to bacterial adhesion, colonization, and biofilm formation. Porphyromonas gingivalis, a keystone periodontal pathogen, utilizes two type V fimbriae systems, the major Fim and the minor Mfa fimbriae, that support distinct adhesive and biofilm-associated functions. Here, we determine the high-resolution cryo-electron microscopy structures of natively assembled FimA and Mfa1 stalks purified directly from P. gingivalis. Despite a shared donor-strand exchange mechanism, the two stalks exhibit distinct helical geometries and donor-strand environments. FimA has a larger helical pitch, whereas the Mfa1 donor strand is more extensively buried and further shielded by an ordered N-terminal latch. Comparison with monomeric stalk pilin structures reveals distinct assembly-associated remodeling around the donor-strand interface. Deletion of the Mfa1 latch reduces heat-resistant oligomer accumulation and produces species consistent with incomplete processing, yet recovered filaments retain the donor-strand-exchanged stalk architecture and exhibit initial force peaks comparable to native Mfa1 during simulated axial extension. Atomic force microscopy reveals greater apparent local stiffness of FimA under indentation, whereas steered molecular dynamics simulations show multiple lower-force axial transitions in FimA but a dominant higher-force transition in Mfa1. In both systems, the exchanged donor strands remain engaged during simulated extension while distinct surrounding contact networks rearrange. Together, these findings demonstrate how a conserved polymerization linkage can support structurally and mechanically distinct adhesive filaments, providing a molecular basis for mechanical diversification of type V fimbriae.

molecular biology↗

bioRxiv2026

A Sensory Pathway Controls Motile Microcolony Formation and Spatial Organization in Gliding Bacteria

How cells transition between distinct modes of collective organization is a fundamental question in biology. We uncover the regulatory landscape governing the choice between bacterial swarming and zorbing. In Flavobacterium johnsoniae, zorbs are spherical, biofilm-like microcolonies that migrate collectively, whereas swarms are multilayered and ordered populations that expand planarly. We identify RgzAB, an unorthodox sensor kinase-response regulator pair governing this transition; suppressor and knockout mutants establish the importance of RgzA phosphorelay and RgzB as a downstream component. Transcriptomic and perturbation analyses link this switch to iron availability, connecting environmental sensing to collective behavior. Remarkably, RgzA activity also governs spatial organization within mixed populations: activated cells encapsulate wildtype neighbors into segregated co-zorbs, within which wildtype cells assemble into networks undergoing an abundance-dependent, percolation-like transition. Together, these findings reveal that one sensory circuit governs bacterial collectives at nested scales, from motility choice to the internal architecture of the collective.

microbiology↗

bioRxiv2026

Resolving hidden stoichiometries in Bacterial proteasome activator (Bpa)-substrate complexes by cryo-EM and charge detection mass spectrometry

Bpa (Bacterial proteasome activator) is a regulatory particle within the Mycobacterium tuberculosis (Mtb) proteasome system that that facilitates ATP-independent substrate engagement and delivery to the 20S core particle (CP) for degradation. The best characterized Bpa substrate is HspR, a transcriptional repressor of Mtb stress-response genes whose Bpa-dependent degradation is required for pathogen virulence. However, the stoichiometry of the Bpa:HspR complex, the molecular mechanism of substrate engagement, and the heterogeneity of the resulting assemblies remain unclear. Here, we combine charge detection mass spectrometry (CDMS) and single-particle electron cryomicroscopy (cryo-EM) as complementary approaches to characterize both apo and HspR-bound Bpa. CDMS revealed a previously unreported undecameric apo species and defines a Bpa12:HspR2 complex stoichiometry with minimal heterogeneity. Cryo-EM, performed without the employment of cross-linking reagents, independently confirmed the presence of undecameric Bpa in solution and localized substrate-associated density to the C-terminal H4 helix of Bpa. Together, these complementary single-particle approaches inform future efforts to target the Mtb proteasome system and provide new molecular insight into proteasomal substrate recognition in prokaryotes. Table of Content Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/722288v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@be4bb6org.highwire.dtl.DTLVardef@15ccf73org.highwire.dtl.DTLVardef@379a2eorg.highwire.dtl.DTLVardef@6b7b90_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Structural basis of substrate recognition for proteasome degradation by prokaryotic ubiquitin-like protein ligase PafA

Selective protein degradation in some bacteria is performed by the Pup-proteasome system, in which the ligase PafA tags hundreds of substrates for proteasomal degradation. How a single enzyme achieves such broad substrate specificity in the absence of conserved sequence motifs has remained unclear. Here we determine structures of PafA in complex with a pupylated substrate and show that substrate recognition is mediated by a minimal and highly distributed interface. PafA samples an ensemble of closely related conformations that collectively position the target lysine residue for modification. This recognition mechanism arises from a combination of structured contacts and dynamic elements on both the enzyme and substrate, enabling geometric compatibility rather than sequence-specific interactions. These findings reveal an ensemble-driven mechanism of molecular recognition that explains how broad substrate specificity is achieved and provides a framework for understanding selective protein degradation in prokaryotes.

biochemistry↗

bioRxiv2026

Structural and mechanistic basis of ubiquitous bacterial kinase signaling identifies PorX as a noncanonical substrate in Porphyromonas gingivalis

Protein phosphorylation enables bacteria to coordinate regulatory networks underlying virulence and environmental adaptation. The ubiquitous bacterial kinase family comprises atypical kinases with dual serine threonine and tyrosine specificity, yet their structural organization, catalytic mechanisms, and physiological roles remain incompletely defined. In the anaerobic oral pathogen Porphyromonas gingivalis, the sole UbK homolog, UbK1, was previously shown to phosphorylate the orphan response regulator RprY, linking UbK1 to virulence-associated pathways. Here, we present the crystal structure of UbK1, revealing the conserved Walker A, HxDxYR, SPT/S, and EW motifs arranged around the ATP-binding site. Structure-guided mutagenesis establishes essential roles for these motifs in ATP hydrolysis and kinase activity. Phosphosite mapping identifies multiple autophosphorylation sites, with the flexible SPT/S loop showing the strongest occupancy and supporting a model in which loop centered autophosphorylation is a major feature of UbK1 cycling while additional sites arise through intermolecular phosphotransfer in trans. Consistent with this, biochemical assays demonstrate that UbK1 undergoes autophosphorylation both in cis and in trans, arguing against a strictly intramolecular autokinase mechanism. Using conserved gene neighborhood analysis, we identified the orphan response regulator PorX as a previously unrecognized UbK1 substrate, consistent with the relaxed substrate specificity reported for UbK homologs. UbK1 catalyzes PorX transphosphorylation in vitro at a single tyrosine residue within the receiver domain, independent of PorX oligomeric state. In vivo, ubk1 deletion causes a modest reduction in gingipain secretion, whereas mutation of the UbK1 dependent PorX phosphosite does not measurably affect T9SS mediated cargo export, indicating that UbK1-PorX signaling is separable from PorX canonical gingipain secretion output. Together, these findings establish a structural and mechanistic framework for UbK1 function, expand the UbK substrate repertoire, and support a model in which UbK1 contributes to regulatory pathways in P. gingivalis that extend beyond canonical secretion associated outputs.

molecular biology↗

bioRxiv2024

Mechanism of allosteric activation in human mitochondrial ClpP protease

Human ClpP protease contributes to mitochondrial protein quality control by degrading misfolded proteins. ClpP is overexpressed in cancers such as acute myeloid leukemia (AML), where its inhibition leads to the accumulation of damaged respiratory chain subunits and cell death. Conversely, hyperactivating ClpP with small-molecule activators, such as the recently-discovered ONC201, disrupts mitochondrial protein degradation and impairs respiration in cancer cells. Despite its critical role in human health, the mechanism underlying the structural and functional properties of human ClpP remain elusive. Notably, human ClpP is paradoxically activated by active-site inhibitors. All available structures of human ClpP published to date are in the inactive compact or compressed states, surprisingly even when ClpP is bound to an activator molecule such as ONC201. Here, we present the first structures of human mitochondrial ClpP in the active extended state, including a pair of structures where ClpP is bound to an active-site inhibitor. We demonstrate that amino acid substitutions in the handle region (A192E and E196R) recreate a conserved salt bridge found in bacterial ClpP, stabilizing the extended active state and significantly enhancing ClpP activity. We elucidate the ClpP activation mechanism, highlighting a hormetic effect where sub-stoichiometric inhibitor binding triggers an allosteric transition that drives ClpP into its active extended state. Our findings link the conformational dynamics of ClpP to its catalytic function and provide high-resolution structures for the rational design of potent and specific ClpP inhibitors, with implications for targeting AML and other disorders with ClpP involvement. Significance statementHuman ClpP protease is essential for maintaining mitochondrial protein quality by degrading damaged proteins. In cancers like acute myeloid leukemia (AML), ClpP is overexpressed, and inhibiting it causes cancer cell death by disrupting mitochondrial function. Conversely, activating ClpP with small molecules, such as ONC201, also leads to cancer cell death by impairing mitochondrial respiration. However, the structural details of ClpP activation have been elusive. Our research presents the first structures of human ClpP in its active state, revealing a novel activation mechanism where inhibitors unexpectedly trigger activity through allosteric changes. These insights provide a foundation for designing targeted therapies for AML and other diseases where ClpP plays a crucial role.

biochemistry↗

bioRxiv2024

Structural basis for allosteric regulation of the proteasome core particle

Intracellular protein degradation is vital across all domains of life1. In eukaryotes, the ubiquitin proteasome system performs most non-lysosomal protein degradation and influences numerous cellular processes. Some bacteria, including the human pathogen Mycobacterium tuberculosis (Mtb), encode a proteasome system that selectively degrades damaged or misfolded proteins crucial for the pathogens survival within host macrophages2-7. Consequently, the 20S core particle (CP), the central component of the proteasome system, has emerged as a viable target for tuberculosis treatment strategies2,8-10. Both eukaryotic and Mtb proteasome systems are allosterically regulated11-13, yet the specific conformations involved have not been captured in high-resolution structures to date. Here we present the first structure of Mtb 20S CP, and indeed any 20S CP, in an inactive state called 20SOFF, distinguished from the canonical active state, 20SON, by the conformation of switch helices I and II. The rearrangement of these helices collapses the S1 pocket, effectively inhibiting substrate binding. The switch helices are conserved and regulate the activity of HslV protease, the proteasomes ancestral enzyme in bacteria, and a diverse family of serine/threonine protein phosphatases. Our results highlight the potential of harnessing allostery to develop therapeutics against the 20S CP in Mtb and eukaryotic systems.

biochemistry↗

bioRxiv2024

Unveiling the Molecular Mechanisms of the Type-IX Secretion System's Response Regulator: Structural and Functional Insights

The Type-IX secretion system (T9SS) is a nanomachinery utilized by bacterial pathogens to facilitate infection. The system is regulated by a signaling cascade serving as its activation switch. A pivotal member in this cascade, the response regulator protein PorX, represents a promising drug target to prevent the secretion of virulence factors. Here, we provide a comprehensive characterization of PorX both in vitro and in vivo. First, our structural studies revealed PorX harbours a unique enzymatic effector domain, which, surprisingly, shares structural similarities with the alkaline phosphatase superfamily, involved in nucleotide and lipid signaling pathways. Importantly, such pathways have not been associated with the T9SS until now. Enzymatic characterization of PorXs effector domain revealed a zinc-dependent phosphodiesterase activity, with active site dimensions suitable to accommodate a large substrate. Unlike typical response regulators that dimerize via their receiver domain upon phosphorylation, we found that zinc can also induce conformational changes and promote PorXs dimerization via an unexpected interface. These findings suggest that PorX can serve as a cellular zinc sensor, broadening our understanding of its regulatory mechanisms. Despite the strict conservation of PorX in T9SS-utilizing bacteria, we demonstrate that PorX is essential for virulence factors secretion in Porphyromonas gingivalis and affects metabolic enzymes secretion in the non-pathogenic Flavobacterium johnsoniae, but not for the secretion of gliding adhesins. Overall, this study advances our structural and functional understanding of PorX, highlighting its potential as a druggable target for intervention strategies aimed at disrupting the T9SS and mitigating virulence in pathogenic species.

biochemistry↗

bioRxiv2022

Cryo-EM structure of the Agrobacterium tumefaciens T-pilus reveals the importance of positive charges in the lumen

Agrobacterium tumefaciens is a natural genetic engineer that transfers DNA into plants and this is the most frequently applied process for the generation of genetically modified plants. DNA transfer is mediated by a type IV secretion system localized in the cell envelope and extracellular T-pili. We here report the cryo-electron microscopic structures of the T-pilus at 3.2[A] resolution and that of the related plasmid pKM101-determined N-pilus at 3[A] resolution. Both pili contain a main pilus protein (VirB2 in A. tumefaciens and TraM in pKM101) and phospholipids arranged in a 5-start helical assembly. They contain positively charged amino acids in the pilus lumen and the lipids are positively charged in the T-pilus (phosphatidylcholine) conferring overall positive charge to the lumen. Mutagenesis of the lumen-exposed Arg91 residue in VirB2 resulted in protein destabilization and loss of pilus formation. Our results reveal that different phospholipids can be incorporated into type IV secretion system pili and that the charge of the lumen is of functional importance.

biochemistry↗