Browsing by Author "Gentile, Rocco"

Filter results by typing the first few letters
Now showing 1 - 2 of 2
  • No Thumbnail Available
    Item
    Data for "Molecular Insights into CLD Domain Dynamics and Toxin Recruitment of the HlyA E. coli T1SS"
    (N/A, 2025) Gentile, Rocco; Schott-Verdugo, Stephan; Khosa, Sakshi; Bonus, Michele; Reiners, Jens; Smits, Sander H.J.; Schmitt, Lutz; Gohlke, Holger
    Escherichia coli is a Gram-negative opportunistic pathogen causing nosocomial infections through the production of various virulence factors. Type 1 secretion systems (T1SS) contribute to virulence by mediating one-step secretion of unfolded substrates directly into the extracellular space, bypassing the periplasm. A well-studied example is the hemolysin A (HlyA) system, which secretes the HlyA toxin in an unfolded state across the inner and outer membranes. T1SS typically comprise a homodimeric ABC transporter (HlyB), a membrane fusion protein (HlyD), and the outer membrane protein TolC. Some ABC transporters in T1SS also contain N-terminal C39 peptidase or peptidase-like (CLD) domains implicated in substrate interaction. Recent cryo-EM studies have resolved the inner-membrane complex as a trimer of HlyB homodimers with associated HlyD protomers. However, a full structural model including TolC remains unavailable. We present the first complete structural model of the HlyA T1SS, constructed using template- and MSA-based information and validated by SAXS. Molecular dynamics simulations provide insights into the function of the CLD domains, which are partially absent from existing cryo-EM structures. These domains may modulate transport by stabilizing specific conformations of the complex. Simulations with a C-terminal fragment of HlyA indicate that toxin binding occurs in the occluded conformation of HlyB, potentially initiating substrate transport through a single HlyB protomer before transitioning to an inward-facing state. HlyA binding also induces allosteric effects on HlyD, altering key residues involved in TolC recruitment. These results indicate how substrate recognition and transport are coupled and may support the development of antimicrobial strategies targeting the T1SS.
  • No Thumbnail Available
    Item
    MD simulation data for: "Molecular Mechanisms Underlying Medium-Chain Free Fatty Acid-regulated Activity of the Phospholipase PlaF from Pseudomonas aeruginosa"
    (N/A, 2023-11) Gentile, Rocco; Schott-Verdugo, Stephan; Gohlke, Holger
    PlaF is a membrane-bound phospholipase A1 from P. aeruginosa that is involved in remodeling membrane glycerophospholipids (GPLs) and modulation of virulence-associated signaling and metabolic pathways. Previously, we identified the role of medium-chain free fatty acids (FFA) in inhibiting PlaF activity and promoting homodimerization, yet the underlying molecular mechanism remained elusive. Here, we used unbiased and biased molecular dynamics simulations and free energy computations to assess how PlaF interacts with FFAs localized in the water milieu surrounding the bilayer or within the bilayer, and how these interactions regulate PlaF activity. Medium-chain FFAs localized in the upper bilayer leaflet can stabilize inactive dimeric PlaF, likely through interactions with charged surface residues as experimentally validated. Potential of mean force (PMF) computations indicate that membrane-bound FFAs may facilitate the activation of monomeric PlaF by lowering the activation barrier of changing into a tilted, active configuration. We estimated that the coupled equilibria of PlaF monomerization-dimerization and tilting at the physiological concentration of PlaF lead to the majority of PlaF forming inactive dimers when in a cell membrane loaded with decanoic acid (C10). This is in agreement with a suggested in vivo product feedback loop and GC-MS profiling results indicating that PlaF catalyzes the release of C10 from P. aeruginosa membranes. Additionally, we found that C10 in the water milieu can access the catalytic site of active monomeric PlaF, contributing to the competitive component of C10-mediated PlaF inhibition. Our study provides mechanistic insights into how medium-chain FFA may regulate the activity of PlaF, a potential bacterial drug target.