An atypical receiver domain controls the dynamic polar localization of the Myxococcus xanthus social motility protein FrzS

Abstract

The Myxococcus xanthus FrzS protein transits from pole-to-pole within the cell, accumulating at the pole that defines the direction of movement in social (S) motility. Here we show using atomic-resolution crystallography and NMR that the FrzS receiver domain (RD) displays the conserved switch Tyr102 in an unusual conformation, lacks the conserved Asp phosphorylation site, and fails to bind Mg(2+) or the phosphoryl analogue, Mg(2+) x BeF(3). Mutation of Asp55, closest to the canonical site of RD phosphorylation, showed no motility phenotype in vivo, demonstrating that phosphorylation at this site is not necessary for domain function. In contrast, the Tyr102Ala and His92Phe substitutions on the canonical output face of the FrzS RD abolished S-motility in vivo. Single-cell fluorescence microscopy measurements revealed a striking mislocalization of these mutant FrzS proteins to the trailing cell pole in vivo. The crystal structures of the mutants suggested that the observed conformation of Tyr102 in the wild-type FrzS RD is not sufficient for function. These results support the model that FrzS contains a novel 'pseudo-receiver domain' whose function requires recognition of the RD output face but not Asp phosphorylation.

Fig. 1

The FrzS RD sequence differs from canonical RDs and pseudo-RDs. Sequence alignment of representative canonical RDs, FrzS RD and KaiA pseudo-RD. The schematic tree shown at the left is representative of the sequence groupings (red = canonical-RD, blue = FrzS-group-RD, green = pseudo-RD), but branch lengths differ from the quantitative tree (Supplemental Fig. S1). Sequence alignment was generated using the MUSTANG alignment algorithm (Konagurthu et al., 2006). Regions of poor structural alignment delineating KaiA from canonical receivers and FrzS are indicated in light orange. Important RD sites are shown in blue, with deviations from canonical residues in KaiA and FrzS shown in dark orange. Conserved hydrophobic residues are highlighted in light green, conserved polar and charged residues in pink, and conserved prolines and glycines in yellow. The secondary structure of FrzS is indicated underneath the alignment.

Fig. 2. Crystal structure of the FrzS RD

A. 2F_o-F_c electron density (1 σ– light blue) and F_o-F_c difference electron density (2 σ– green) at 1.0 Å resolution showing the putative switch Tyr102 and the neighbouring His92. Difference electron density indicates that there is a hydrogen bond between His92-Nδ1 and Tyr102-O. B. Overlay of the main chain of the FrzS RD in the hexagonal form crystals (blue) with three independent chains of the tetragonal form (red, magenta, purple) shows a high degree of similarity between independent structures of the FrzS RD. There are two conformers of the β3–α3 loop in the hexagonal form, one of which corresponds to the conformation seen in all three tetragonal monomers. The switch Tyr102 is in the same rotamer in all four chains and highlighted by a yellow circle. The position of Phe89, which renders the canonical ‘inward facing’ Tyr102 rotamer inaccessible, is highlighted by a green circle. C. Plot of main chain RMSD (Å) between the four independent FrzS RD chains highlights the variability in the β3-α3 loop and the similarities elsewhere in the structure. D. Multiple conformations of the β3-α3 loop seen in the 2F_o-F_c electron density (1.5 σ– light blue) at 1.0 Å resolution in the hexagonal form of the FrzS RD reveal coordinated motions of this loop region.

Fig. 3. Comparison of the FrzS RD and CheY structures

A. FrzS and CheY have similar global structures, but the FrzS ‘switch’ Tyr102 is in a different position in the two RDs. Ribbon representation showing FrzS (blue) and meta-active CheY (1jbe, red). The two conformations representing inactive and active states of CheY Tyr106 differ from the distinct rotamer of FrzS Tyr102 (sticks). B. Superimposed FrzS (blue) and phosphorylated CheY (1fqw, red) contain the switch Tyr pointed into distinct pockets in the respective proteins. The FrzS structure reveals that this RD does not support Asp phosphorylation or allosteric coupling to the Asp triad. FrzS Ala53 replaces the CheY Asp57 phosphorylation site, and the FrzS Asp55 Cα is shifted > 5 Å relative to CheY Asp57. FrzS Ser10 replaces CheY Asp13, which is required for Mg²⁺ binding in CheY. FrzS also lacks the Thr87 side chain required to functionally link the Asp57 phosphorylation to Tyr106 in CheY. Residues are shown in sticks labelled according to their identities in FrzS/CheY. BeF₃ is shown as yellow/green sticks and Mg²⁺ is shown as a purple sphere.

Fig. 4

Mg²⁺ does not bind the FrzS RD. ¹⁵N-HSQC spectra of the FrzS RD in the presence of 0 mM (black), 30 mM (red) and 60 mM (green) MgCl₂. Chemical shifts of the backbone amide peaks showed no changes associated with Mg²⁺ addition. This insensitivity suggested that FrzS failed to bind Mg²⁺, which is a signature of competence for Asp phosphorylation of canonical RDs.

Fig. 5. FrzS mutations Tyr102Ala and His92Phe, but not Asp55Ala, abolished S-motility in vivo

A. Myxococcus xanthus S-motility phenotypes of FrzS RD point mutants. FrzS-GFP (TM3), ΔFrzS (DZ4526), FrzS^ΔRec–GFP (DZ4535), FrzS^Asp55Ala (DZ4539), FrzS^Tyr102Ala (DZ4538) and FrzS^His92Phe (DZ4550) were spotted at 4 × 10⁷ cells ml⁻¹ on nutrient rich CYE media containing 0.5% agar and incubated for 24 h at 32°C. Scale bar equals 5 mm. The Asp55Ala FrzS mutant supported wild-type levels of S-motility, while the phenotypes of the Tyr102Ala and His92Phe FrzS mutants were indistinguishable from the deletions of the RD or all of FrzS. B. Anti-FrzS Western blot of whole cell lysates of wild-type M. xanthus and the strains shown in A. DZ4539 is not shown. The Asp55Ala, His92Phe and Tyr102Ala FrzS variants were expressed at equivalent levels in vivo.

Fig. 6

Subcellular localization of FrzS-GFP variants during Myxococcus xanthus movement correlates with S-motility phenotypes and crystal structures of FrzS RD variants. A. Time-lapse image sequences showing the cell location (top) and GFP fluorescence signal (bottom). Arrows indicate the direction of cell movement. ‘R’ indicates the frame in which a cell reversal took place. Scale bar = 5 μm. B. Time-lapse image sequence showing the subcellular localization of FrzS^Tyr102Ala-GFP during Myxococcus xanthus movement. The mutant FrzS was localized at the lagging pole, rather than the leading pole. Scale bar = 5 μm. C. Time-lapse image sequence showing the subcellular localization of FrzS^His92Phe-GFP during Myxococcus xanthus movement. The localization pattern of His92Phe FrzS matched that of the Tyr102Ala mutant, indicating that both of these RD residues are required for normal FrzS localization. Scale bar = 5 μm. D. The structures of RD mutants Tyr102Ala (orange) and His92Phe (magenta) are globally similar along the α4-β5-α5 output face to the structure of WT FrzS (blue). This similarity suggests the mutant and wild-type structures are in the same signalling state, and that the buried nature of the Tyr102 rotamer is critical in switching the signalling or binding state of FrzS.