bioRxiv Science⌕ Search

bioRxiv · 10.64898/2025.11.30.691423

IcmX Plug Ejection in L. pneumophila's Type IV Secretion System: Is It Possible?

Abstract

1Legionella pneumophila is a gram-negative bacterial pathogen that is the causative agent of several infectious diseases, notably the severe form of pneumonia known as Legionnaires Disease. L. pneumophila targets amoebas and, in humans, alveolar macrophages. L. pneumophila infects its host by envelopment into the host cell through phagocytosis followed by the activation of the Type IV Secretion System (T4SS). The T4SS secretes effector proteins into the host cell which deactivate cell defenses and reprogram cell function to support L. pneumophila reproduction. After reproduction, L. pneumophila lyses the host cell and the cycle repeats, causing swelling and destroying a primary cell in the hosts immune system--infections caused by L. pneumophila are notoriously hard to treat. The stages of the infection process are known but the physical mechanisms are poorly understood. The structure of the T4SS includes a 13 member polymer, DotG which protrudes from the outer leaflet of the bacterial outer membrane. Given this exposure and the necessity of T4SS for infection, DotG is a promising drug target. However, designing an effective drug requires understanding the physical mechanisms. Via MD simulations we have tested hypotheses regarding its function. Specifically, we show the feasibility that an applied force on IcmX, a plug-like penta-mer that initially blocks transport through the T4SS channel, can cause a structural change in DotG and allow for protein release. Further, this applied force could reasonably come from a build up of pressure in the channel from other effector proteins. We conclude that the process of secretion is dependent on the induced structural change in DotG. Inhibiting this change provides a possible drug development direction.

Explore related subjects

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Hamilton, C. J., Bills, S., Stark, B., Hart, G. L. W.. 2025-12-02. IcmX Plug Ejection in L. pneumophila's Type IV Secretion System: Is It Possible?. https://doi.org/10.64898/2025.11.30.691423

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

Mechanism of molecular recognition revealed through dynamic drug binding pathways to SARS-CoV-2 main protease

Characterization of drug-binding pathways remains experimentally limited by transient intermediates and computationally challenging due to long timescales intractable for conventional molecular dynamics. To address these challenges, we combined solution NMR titrations with weighted ensemble (WE) enhanced sampling simulations to resolve atomistic pathways of nirmatrelvir binding to the SARS-CoV-2 main protease. NMR titration revealed residue-dependent heterogeneity spanning fast, intermediate, and slow exchange regimes. WE simulations complement the NMR by providing insights into unassigned residues and adding time-resolved and three-dimensional structural context. We map key interactions along two distinct binding pathways, provide dynamic explanations for residues involved in resistance, and capture unique backbone conformations compared to those sampled in unbound or bound states. Our comprehensive binding model is consistent with a combined conformational selection and induced fit mechanism in which early transient contacts are made with residues E47 and L50 and allosteric motions are centered around residue V204 of the distal domain. This synergistic application of WE and titration NMR enables a more comprehensive characterization of drug binding than either method alone, providing an integrated framework that may have broader applicability to defining structure-kinetic relationships and guiding design of next-generation inhibitors.

biophysics↗

A Minimally Perturbative DARPin Probe for Quantitative Fluorescence Imaging of the Human TCR-CD3 Complex

Fluorescence microscopy is a powerful tool for dissecting the molecular mechanisms of T-cell antigen recognition in living cells, but its quantitative insight critically depends on non-perturbative, high-quality probes. Here, we repurpose a small (~15 kDa) CD3epsilon-binding DARPin (designed ankyrin repeat proteins) to a fluorescent label for T-cell receptor (TCR)/CD3 complexes on primary human CD8+ T-cells, with the aim of generating a powerful tool for quantitative analysis, single-molecule tracking, and advanced imaging of TCR dynamics. We show that the DARPin binds CD3{varepsilon} with high affinity and selectivity and using single molecule tracking and brightness analysis, we characterize the TCR-CD3 diffusion behavior and show that the DARPin binds to both CD3epsilon; subunits. Importantly, labeling preserves antigen sensitivity: on supported lipid bilayers presenting cognate pMHC, T-cells remain responsive, assemble synapses, form TCR microclusters, and initiate signaling similar to unlabeled controls. We further demonstrate compatibility with lattice light-sheet microscopy for volumetric imaging of T-cell - APC interactions in living cells. Together, these results establish DARPins as versatile, minimally perturbative probes for high resolution, quantitative studies of T cell synapse organization and signaling.

biophysics↗

Monitoring intramolecular dynamics across two regions of the mouse prion protein during misfolding and oligomerization using fluorescence correlation spectroscopy

It is important to determine whether native state dynamics drive the misfolding and oligomerization of the prion protein, which are important events in prion disease, and how they are modulated by conformational conversion. Native (N) mouse prion protein (moPrP) is known to form small (OS) and large (OL) oligomers rich in {beta}-sheet, and in this study, photoinduced electron transfer-fluorescence correlation spectroscopy (PET-FCS) has been used to characterize intramolecular dynamics within individual monomeric units in both isolated OS and OL, as well as the diffusion properties of the oligomers. It is estimated that OS and OL comprise of about 15 and 55 monomeric units, respectively. Microsecond dynamics at each of the two regions that are the 1-3 and 2-3 interfaces of native protein are distinct in N, OS and OL, although they occur on very similar timescales. Analysis of the evolution of the distribution of diffusion times, determined using the maximum entropy method, indicates heterogeneity in the oligomerization reaction. Analysis of the change in the fluctuations which occur in two different timescales in the native state ensemble shows that they are damped more at the erstwhile 1-3 interface than the erstwhile 2-3 interface. The difference in the extent of damping at the erstwhile 1-3 and 2-3 interfaces can be explained on the basis of the structural changes known to occur across each region. The changes in dynamics occur concurrently in both regions, indicating that the structural changes accompanying conformational conversion also occur simultaneously during the oligomerization of moPrP.

biophysics↗