Structural insight of a concentration-dependent mechanism by which YdiV inhibits Escherichia coli flagellum biogenesis and motility

YdiV is a negative regulator of cell motility. It interacts with FlhD4C2 complex, a product of flagellar master operon, which works as the transcription activator of all other flagellar operons. Here, we report the crystal structures of YdiV and YdiV2–FlhD2 complex at 1.9 Å and 2.9 Å resolutions, respectively. Interestingly, YdiV formed multiple types of complexes with FlhD4C2. YdiV1–FlhD4C2 and YdiV2–FlhD4C2 still bound to DNA, while YdiV3–FlhD4C2 and YdiV4–FlhD4C2 did not. DNA bound FlhD4C2 through wrapping around the FlhC subunit rather than the FlhD subunit. Structural analysis showed that only two peripheral FlhD subunits were accessible for YdiV binding, forming the YdiV2–FlhD4C2 complex without affecting the integrity of ring-like structure. YdiV2–FlhD2 structure and the negative staining electron microscopy reconstruction of YdiV4–FlhD4C2 suggested that the third and fourth YdiV molecule bound to the FlhD4C2 complex through squeezing into the ring-like structure of FlhD4C2 between the two internal D subunits. Consequently, the ring-like structure opened up, and the complex lost DNA-binding ability. Thus, YdiV inhibits FlhD4C2 only at relatively high concentrations.

inhibit or activate YdiV through some unknown mechanism and then turn on or turn off flagellar biosynthesis. In this regard, motility of bacteria can be regulated by quorum sensing signals or nutrition signals receipting by YdiV and acting through the interaction between YdiV and FlhD 4 C 2 . Generously allowed (%) 0.5 0.8 Disallowed (%) 0 0 *Highest resolution shell is shown in parenthesis. One crystal was used for each dataset.

Protein expression and purification
The ydiV and flhD genes were PCR amplified from Escherichia coli str. K-12 substr. Whole flhDC operon was cloned into pET21b in which FlhC contained a C-terminal his tag.
FlhD 4 C 2 complex were obtained by co-expression and further purified by Ni 2+ -NTA affinity column, ion exchange column Source Q and Superdex 200.

Crystallization and Structure Determination
YdiV were concentrated to 9 mg/ml. Crystals were grown Five Se sites were found using the program SOLVE (34). Initial single anomalous dispersion phases were then improved and the chain was automatically traced using the program RESOLVE (35). The atomic model was built using COOT (36) and refined using PHENIX (37). Data collection and structure refinement statistics are summarized in Table 1. The structure of YdiV-FlhD was determined at 2.9Å resolution, with the molecular replacement approach using PHASER (38) with the FlhD structure (PDB code: 1G8E) and our YdiV structure as searching models. The final model contains four YdiV and four FlhD molecules in the asymmetric unit. Structural figures were generated using PyMol (http://www.pymol.org).

Size-exclusion chromatography
Purified proteins (about 0.4 mg) were injected to size exclusion chromatography using a superdex 200 column equilibrated by a buffer contains 10 mM Tris-HCl (pH 8.0) and 100 mM NaCl. Three proteins (BSA, VibB and ViuP) were used as standards. For complex study, two proteins (YdiV and FlhD 4 C 2 ) were mixed in different mixing ratio (5:1~1:1) for 10 min at room temperature and injected to size exclusion chromatography using a superdex 200 column. All data were processed by Origin.

Protein Pull-down Assay
Bait protein (with his tag) were prepared as described above. His-tag of prey protein were

EMSA Experiment
A 49 bp DNA fragment containing the FlhD 4 C 2 box of the flhB promoter was synthesized as target DNA. 10 pmol DNA was pre-incubated with different radios proteins in a reaction buffer contains 20 mM Tris-HCl (pH8.0), 100 mM NaCl,1 mM MgCl 2 , 1 mM ZnCl 2 and 4%(v/v) glycerol for 10 minutes. Then samples were analysed using a native 5% polyacrylamide gel at 4 o C in 0.5TBE buffer (46 mM Tris base, 46 mM boric acid, 1 mM EDTA) for 1 h and dyed by EB for 10 min. Some gels were also dyed by coomassie brilliant blue.

Swarming Motility Assay
Motility was evaluated using 0.3% or 0.5% soft agar plates as described before. Briefly, single colonies were poked into the plates using toothpicks and incubated for 6 h at 37 o C. For motility assays requiring the induction of pGEX construct, colonies were picked from plates that contained 1 mM IPTG. At least six independent colonies were checked for each strain.

Negative stain EM sample preparation and single particle image analysis
The gel-filtration purified FlhDC-YdiV complex were diluted to 50-80 nM in the elution buffer and immediately applied to glow-discharged holey carbon grids with a thin layer of carbon over the holes. After 1 min, the samples were stained consecutively in 3 droplets of 2% (w/v) uranyl acetate solution and the remaining stain was removed by gentle blotting with filter paper. The samples were examined using an FEI F20 electron microscope equipped with a field emission gun operated at 200 kV acceleration voltage using a nominal magnification of 50,000. Images were recorded on a 4k x 4k Ultrascan4000 CCD camera (Gatan) using low-dose mode with an exposure dose of 20-30 e/Å 2 . The defocus used to collect the raw image was -1.2 μm to -1.5 μm. The electron micrographs had a pixel size of 2.2 Å and were directly used for image processing. We used EMAN2 package to perform semi-automatic particle picking and to box the particles from the raw micrographs into boxes of 80 x 80 square pixels for the samples (39). The particles were normalized and high-and low-pass filtered prior all image processing procedures. About 30,000 raw particles of FlhDC-YdiV complexes were collected to two-dimensional reference-free alignment and classification using multivariate statistical analysis and multireference alignment in IMAGIC-4D (40) to a total of 200 classes.