Alternative modes of contact between PilZ and the FimX EAL domain observed in different crystal structures.

A-C)Left: The principal crystal contacts observed between PilZ and FimX subunits in the A) PilZ-FimXEAL-c-di-GMP complex from X. citri (PDB: 4FOU; FimXEAL colored in orange and PilZ colored in blue), B) PilZ-FimXEAL-c-di-GMP complex from X. campestris pv. campestris (PDB: 4F48; FimXEAL colored in green and PilZ colored in yellow) and C) PilZΔ107-117-FimXGGDEF-EAL-c-di-GMP complex from X. citri (this study; FimXEAL colored in magenta and PilZ colored in cyan). The dashed black circles and dashed red boxes delimit the two principal crystal contacts (interface 1 and 2, respectively). Right: Detailed view of interface 2 in the three structures. D) Superposition of one FimXEAL domain with the two contacting PilZ subunits via interfaces 1 and 2 observed in the three structures shown in A-C with the same coloring scheme. Note the different orientations of the first alpha helix in the X. citri and X. campestris pv. campestris FimX EAL domains. This explains the reduced contact surface at interface 1 in the latter structure (see part E). E) Buried surface areas at the two interfaces in the three structures as calculated by the PISA server [37]. F) Size exclusion chromatography of PilZ-FimXGGDEF-EAL binary complexes containing wild-type PilZ (PilZWt) and its mutants (PilZF49E/L51E, PilZI10E and PilZD46A/E47A). In each chromatogram, the elution profile of the FimXGGDEF-EAL−PilZ mixture (1:1.5 molar ratio) was performed in the absence (black line) and in the presence (red line) of a 2-fold excess of c-di-GMP to FimXGGDEF-EAL. In these experiments, FimXGGDEF-EAL has an N-terminal 6xHis-tag. Each experiment was performed at least three times and representative results are shown. These chromatograms are reproduced in S5A Fig with the respective SDS-PAGE analysis of the relevant fractions.