Journal: bioRxiv

Article Title: Structural modeling reveals the mechanism of motor ATPase coordination during type IV pilus retraction

doi: 10.1101/2025.10.30.685630

Figure Lengend Snippet: AF3 models (left) of the indicated V. cholerae proteins with their respective PAE maps and confidence scores (right). ( A ) PilT(x6)-PilC(x3) and ( B ) PilT(x6)-PilU(x6) were used to generate the PilC-PilT-PilU hybrid complex shown in . ( C ) The PilU(x6)-PilC(x3) AF3 model had very low confidence metrics, suggesting that these proteins may not directly interact.

Article Snippet: Molecular dynamics simulation of the AF3 PilT-PilU model. A 100-ns span of the simulation is show for PilT K36 -PilU D366,E368 .

Techniques:

Journal: bioRxiv

Article Title: Structural modeling reveals the mechanism of motor ATPase coordination during type IV pilus retraction

doi: 10.1101/2025.10.30.685630

Figure Lengend Snippet: ( A ) AF3 model of PilT (green)-PilU (cyan) interactions. Inset highlights the interaction interfaces targeted for mutagenesis. The PilT residues that putatively participate in intermolecular salt bridges are shown in magenta. ( B ) NT assays of the indicated V. cholerae strains. Dark gray bars denote strains that report on PilT-dependent retraction ( i.e. , in a pilT + background where PilT ATPase activity is intact), while light grey bars denote strains that report on PilT-PilU-dependent retraction ( i.e. , in a pilT WA background where retraction is dependent on PilU). ( C ) Representative images of surface piliation (top panel) and cellular aggregation (bottom panel) for the indicated strains. Scale bars on micrographs, 3µm. Data are representative of two independent experiments. ( D ) Motor localization assay of cells expressing mCherry-pilU WA and the indicated pilT alleles. For each strain, the percentage of cells ( n = 120 cells analyzed) with one or more fluorescent mCherry-PilU WA foci is reported. ( E ) Motor localization assay of ΔpilU cells expressing an mCherry-pilT WA construct with the additional mutations indicated. For each strain, the percentage of cells ( n = 120 cells analyzed) with one or more fluorescent mCherry-PilT WA foci is reported. Data in B , D , and E are from 4 independent biological replicates and shown as the mean ± SD.

Article Snippet: Molecular dynamics simulation of the AF3 PilT-PilU model. A 100-ns span of the simulation is show for PilT K36 -PilU D366,E368 .

Techniques: Mutagenesis, Activity Assay, Expressing, Construct

Journal: bioRxiv

Article Title: Structural modeling reveals the mechanism of motor ATPase coordination during type IV pilus retraction

doi: 10.1101/2025.10.30.685630

Figure Lengend Snippet: AF3 model of the PilT (green)-PilU (cyan) complex. Insets show the residues involved in the three putative intermolecular salt bridges with PilT residues colored in magenta and PilU residues colored in blue.

Article Snippet: Molecular dynamics simulation of the AF3 PilT-PilU model. A 100-ns span of the simulation is show for PilT K36 -PilU D366,E368 .

Techniques:

Journal: bioRxiv

Article Title: Structural modeling reveals the mechanism of motor ATPase coordination during type IV pilus retraction

doi: 10.1101/2025.10.30.685630

Figure Lengend Snippet: ( A ) Multiple sequence alignment of PilT and PilU homologs from the following bacterial species: Vc = Vibrio cholerae , Ab = Acinetobacter baylyli , Pa = Pseudomonas aeruginosa , Lp = Legionella pneumophila , and Xf = Xylella fastidiosa . Red boxes denote conservation of the PilT Vc residues studied (D2, R35, K36, and E53), the PilT AIRNLIRE motif, and the extended C-terminus that is unique to PilU homologs. The red asterisk highlights conservation of the PilU Vc K348 residue among PilU homologs. ( B ) AF3 models of PilT-PilU complexes from the indicated species highlights the predicted conservation of the experimentally validated PilT Vc D2,E53 -PilU Vc K348 interfacial salt bridge.

Article Snippet: Molecular dynamics simulation of the AF3 PilT-PilU model. A 100-ns span of the simulation is show for PilT K36 -PilU D366,E368 .

Techniques: Sequencing, Residue

Journal: bioRxiv

Article Title: Structural modeling reveals the mechanism of motor ATPase coordination during type IV pilus retraction

doi: 10.1101/2025.10.30.685630

Figure Lengend Snippet: ( A ) AF3 model of PilT (green)-PilU (cyan) interactions. The inset highlights the residues that participate in the three putative salt bridges targeted for mutagenesis. PilT residues are shown in magenta with nitrogen atoms colored blue, and PilU residues are shown in dark blue with oxygen atoms colored red. ( B ) NT assay of the indicated V. cholerae strains. Data are from five independent biological replicates and shown as the mean ± SD. ( C ) Representative images of surface piliation (top panel) and cellular aggregation (bottom panel) for the indicated strains. Scale bar for micrographs, 3µm. Data are representative of two independent experiments. ( D ) Distance frequency histogram for the indicated residue pairs during the MD simulation of the AF3 PilT-PilU model. Distances between the side chains of the indicated residues were measured at 0.1-ns intervals over the 500-ns simulation. Percentage on graph denotes the amount of time that the indicated side chains are <3.5Å (dotted line). Data are compiled from two independent MD simulations.

Article Snippet: Molecular dynamics simulation of the AF3 PilT-PilU model. A 100-ns span of the simulation is show for PilT K36 -PilU D366,E368 .

Techniques: Mutagenesis, Residue

Journal: bioRxiv

Article Title: Structural modeling reveals the mechanism of motor ATPase coordination during type IV pilus retraction

doi: 10.1101/2025.10.30.685630

Figure Lengend Snippet: ( A ) Distance frequency histogram for the indicated residue pairs during the MD simulation of the AF3 PilT-PilU model. Distances between the side chains of the indicated residues were measured at 0.1-ns intervals over the 500 ns simulation. Percentage on graph denotes the amount of time that the indicated side chains are <3.5Å (dotted line). Data are compiled from two independent MD simulations. ( B ) Time-course scatter plots of distance measurements for the indicated residue pairs. Trajectories 1 and 2 represent two independent replicates of the MD simulations.

Article Snippet: Molecular dynamics simulation of the AF3 PilT-PilU model. A 100-ns span of the simulation is show for PilT K36 -PilU D366,E368 .

Techniques: Residue

Journal: bioRxiv

Article Title: Structural modeling reveals the mechanism of motor ATPase coordination during type IV pilus retraction

doi: 10.1101/2025.10.30.685630

Figure Lengend Snippet: ( A ) AF3 model of PilT Ab (orange)- PilU Ab (pink) interactions. Inset shows the conserved PilT Ab D2,D53 -PilU Ab K351 salt bridge. ( B ) NT assays of the indicated A. baylyi strains. Dark gray bars denote strains that report on PilT-dependent retraction ( i.e. , in a pilT + background where PilT ATPase activity is intact), while light grey bars denote strains that report on PilT-PilU-dependent retraction ( i.e. , in a pilT WA background where retraction is dependent on PilU). Data are from three independent biological replicates and shown as the mean ± SD.

Article Snippet: Molecular dynamics simulation of the AF3 PilT-PilU model. A 100-ns span of the simulation is show for PilT K36 -PilU D366,E368 .

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Structural modeling reveals the mechanism of motor ATPase coordination during type IV pilus retraction

doi: 10.1101/2025.10.30.685630

Figure Lengend Snippet: AF3 model of the PilC(3x)-PilT(6x) complex. Residues of the AIRNLIRE motif (PilT A288-E295 ) are colored in magenta. The inset shows a slice of the PilT-PilC model for a better view of the AIRNLIRE motif at the PilC-PilT interface.

Article Snippet: Molecular dynamics simulation of the AF3 PilT-PilU model. A 100-ns span of the simulation is show for PilT K36 -PilU D366,E368 .

Techniques: