The molecular mechanism of the type IVa pilus motors

Abstract

Type IVa pili are protein filaments essential for virulence in many bacterial pathogens; they extend and retract from the surface of bacterial cells to pull the bacteria forward. The motor ATPase PilB powers pilus assembly. Here we report the structures of the core ATPase domains of Geobacter metallireducens PilB bound to ADP and the non-hydrolysable ATP analogue, AMP-PNP, at 3.4 and 2.3 Å resolution, respectively. These structures reveal important differences in nucleotide binding between chains. Analysis of these differences reveals the sequential turnover of nucleotide, and the corresponding domain movements. Our data suggest a clockwise rotation of the central sub-pores of PilB, which through interactions with PilC, would support the assembly of a right-handed helical pilus. Our analysis also suggests a counterclockwise rotation of the C2 symmetric PilT that would enable right-handed pilus disassembly. The proposed model provides insight into how this family of ATPases can power pilus extension and retraction.

Figure 1. Identification and functional prediction of conserved residues in PilB.

(a) Overview of the phylogenetic analysis of PilT-like ATPase family members. Sequences from model systems are identified with a black circle and labelled. There are multiple circles for PilB, PilT and FlaI reflecting that there are multiple model systems from different species for these proteins. For a detailed view of the phylogenetic tree including the identity of branches not labelled here see Supplementary Fig. 1. Only branches with a >85% bootstrap value are shown (1,000 bootstraps). FtsK was used as an out-group. Protein sub-families, labelled as clades, are given a unique colour for clarity and to stratify the sequences for further analysis. (b) Sequence logo representation of conserved residues stratified based on the clade definitions above. Each dash indicates that there is no corresponding residue in PilB from G. metallireducens.

Figure 2. Structure of PilB.

(a) PilB:ADP hexamer, with each chain indicated in a different colour. ADP and formate are shown as black spheres, while magnesium and zinc are shown as grey spheres. A side view is shown with a grid drawn to emphasize the saddle-like shape. (b) Cartoon block illustrations of the PilB hexamer demonstrating the packing units observed, as well as defining the terminology used herein. In the cartoon on the far right, the interface between packing units is represented by a dashed line and the location of the ATP shown in stick representation. (c) PilB:AMP-PNP hexamer, with each chain coloured as in a. AMP-PNP and ADP are shown as black spheres, while magnesium and zinc are shown as grey spheres. A side view is shown with a grid drawn to emphasize the planar shape.

Figure 3. Surface representations of PilB from G. metallireducens.

(Left) The electrostatic surface of PilB calculated with missing side chains added in the most likely rotamer via Maestro (version 10.5, Schrödinger). (Right) The phylogenetic conservation of PilB residues mapped onto the surface of PilB using the ConSurf server.

Figure 4. ATP binding sites of PilB.

Direct polar contacts are shown as dashed lines. Magnesium is shown as a grey sphere. The green mesh represents the Feature Enhanced Map computed by PHENIX-FEM contoured at 2.0σ. (a) Cartoon clarifying the identity of domains in the following sub-Figures. The cartoon mirrors Fig. 2b. (b) Nucleotide binding site in the closed-ADP interface from PilB:ADP. (c) Nucleotide binding site in the open-APO interface from PilB:ADP. (d) Nucleotide binding site in the closed-AMP-PNP interface from PilB:AMP-PNP. (e) Nucleotide binding site in the closed-ADP interface from PilB:AMP-PNP. (f) Nucleotide binding site in the open-AMP-PNP interface from PilB:AMP-PNP.

Figure 5. Modeling the movements and functions of PilB and PilT ATPases.

(a) The PilB:ADP hexamer was aligning with the PilB:AMP-PNP hexamer to suggest the movements that may occur as two closed packing units open and two open packing units close during nucleotide exchange. Chains are coloured as in Fig. 2. (Bottom) A cross-section through the centre of PilB is shown with E400 displayed as spheres. (b) The PilT hexamer (PDB 2GSZ) was aligned with the same PilT hexamer rotated by one packing unit to suggest the movements that may occur as two closed packing units open and two open packing units close during nucleotide exchange. (Bottom) A cross-section through the centre of PilT is shown with E222 (the equivalent of E400 in PilB) and N260 from the midpoint of the extension on pore loop 3 (there is no equivalent in PilB) displayed as spheres. (c) The PilC^NTD dimer (PDB 2WHN) was manually placed in the two sub-pores of PilB, and the phylogenetic conservation of PilC residues were mapped onto the surface using the ConSurf server. (Bottom) A cross-section through the centre of PilB is shown with E400 displayed as spheres. The model is similar to the mode of PilC binding to PilB we proposed previously. (d) Working model for the molecular mechanism of the PilB motor. We propose that PilC is thrusted by PilB upwards towards the membrane, allowed to fall back, and rotated in 60° increments to facilitate helical PilA polymerization. (e) Working model for the molecular mechanism of the PilT motor. We propose that PilC is wrenched downward by PilT towards the cytoplasm, allowed to relax upwards, and rotated in 60° increments in the opposite direction of PilB to facilitate helical PilA depolymerization. The model presented in panels D and E refines our previous model that described how PilM-PilN interactions modulate PilB/PilT associations.