PilB sequence conservation and interactions.

A) Sequence logo generated from an alignment of 50 non-redundant PilB N-terminal sequences that contain ND0 and ND1 sub-domains. The X. citri PilB N-terminal (residues 1–190) sequence is shown below the alignment (highlighted in green). The secondary structure elements observed in the crystal structure of X. citri PilB (residues 12–163) are indicated above the alignment. B-D) Surface residues of PilB12-163 colored according to degree of conservation. Panel B shows the PilB12-163 surface oriented towards PilZ in the PilB12-163-PilZ complex. * Indicates residues involved in direct contacts with PilZ. Panels C and D show PilB12-163 after 120o and 240o rotations with respect to A. The surface in B presents residues proposed to be involved in the interaction with PilM. E) Possible mode of interaction between PilB, PilZ and PilM. Shown is the superposition of the X. citri PilB12-163-PilZ complex with homologous EpsEND1-EpsL complex (PDB ID: 2BH1) using the ND1 sub-domains as reference (see S12 and S13 Figs). PilZ and the EpsL cytoplasmic domain (PilM homolog) are shown in ribbon representation colored in yellow and blue, respectively. F) Proposed model for the PilB-PilZ-FimX ternary complex based on the superposition of the PilB12-163-PilZ complex and the PilZ-FimXEAL complex (interface 2). PilZ is the reference in the superposition. G) Above: SEC analysis of the mixture of full-length PilB-PilZ complex and full-length FimX shows that these proteins form a ternary complex (black continuous line). Addition of ATPγS and c-di-GMP results in a shift in the elution profile of the ternary complex (red continuous line). Also shown is the elution profile of the PilBK343A/E407A-PilZ and FimX mixture in absence (dashed black line) and presence (dashed red line) of ATPγS and cdi-GMP. In all cases, a 1:1 molar ratio of PilB-PilZ complex to FimX was used. The elution profiles of full-length FimX (blue continuous line) and the PilB-PilZ complex (green continuous line) are also shown. To facilitate comparison, peak heights were normalized. Below: SDS-PAGE of the fractions of the major peak observed for the PilB-PilZ-FimX ternary complex in presence of ATPγS and cdi-GMP. H) ATPase activity of the PilB-PilZ complex in the absence and presence of full-length FimX. ATPase activity was determined by measuring the production of inorganic phosphate (Pi) at 30°C using 1 μM of PilB-PilZ complex, 1 mM de ATP in the absence and presence of 1 μM FimX. Where indicated, c-di-GMP was added to a final concentration of 20 μM. When wild-type PilB was substituted with the PilBK343A/E407A mutant, no significant ATPase activity was observed. Each experiment was performed at least three times. Error bars: standard deviation.