Molecular Mechanism By Which YdiV Negatively Regulate Flagellum Biogenesis And Motility

Bacterial flagellum is important locomotive organelle. It propels cells through liquid envirionment to look for more favorable growing site, and play important roles in the process of infection produced by many pathogenic bacteria. More than50genes reuquired for flagellar formation have been identified in Salmonella enterica serovar Typhimurium and Escherichia coli. These flagellar genes belong to more than15operons that have been divided into three classes according to their transcriptional hierarchy. At the top of the hierarchy is the flhDC operon, whose expression is required for the transcription of all other flagellar operons. YdiV, a member of the EAL protein family, does not show anticipatory catalytic activity to c-di-GMPbut acts as a negative regulator of cell motility of Escherichia coli. It represses bacterium motility by interacting with F1hD4C2complex and shutting down the downstream genes transcription. So far, how YdiV turn off the downstream genes transcription is unclear.We first report the crystal structure of YdiV using selenium single-wavelength anomalous diffraction (SAD) at1.9A resolution. The YdiV monomer consists of ten a helices, eight P strands and two short310helices, which exhibits a modified TIM-like barrel fold. Although YdiV shares low sequence identities with other EAL domain proteins, the topology of structures is the same. The α8helix of YdiV, which is essential for dimerization in other EAL structures, reveals particular transformation. Notably, although YdiV lost most of the residues coordinating with c-di-GMP, a similar groove that is responsible for c-di-GMP binding in other EAL structures is still retained. Interestingly, a phosphate and a glycerol molecule appear in this groove. They are partially parallel to the c-di-GMP molecule in TBD1265indicating that other small molecules with similar structures may bind to YdiV with this groove and play a regulatory role to the function of YdiV. The interface between both monomers is not as large as that of the standard EAL protein dimers. We therefore speculate YdiV does not form stable dimer in solution. Our speculation was then confirmed by the result of size-exclusion chromatography of purified YdiV. The inability to pursue dimerizalion indicates YdiV perform its function in a unique way quite different from other EAL proteins.Pull down assay showed that YdiV binds to the F1hD4C2complex through interaction with the F1hD subunit and the binding of YdiV does not lead to a separation of the F1hD and F1hC subunits of F1hD4C2.We further characterized ihe minimal YdiV-interacting domain from F1hD using purified recombinant proteins from E. coli. Four fragments of F1hD (1-71aa,1-82aa,1-98aa and1-106aa) are tested with YdiV. All fragments can form stable complex with YidV which was proved by the nickel column pulled down assay. We found YdiV forms many kinds of complexes with F1hDC and it inhibits F1hD4C2binding to DNA in a concentration dependent manner.The structure of YdiV-F1hD complex was determined at2.9A resolution with the molecular replacement approach using the F1hD structure and our YdiV structure. The final model contains four YdiV molecules and four F1hD molecules in the asymmetric unit, with every YdiV interacting with one corresponding F1hD in a uniform binding manner. YdiV-F1hD complex may form a stable tetramer in solution through a tightly coupled F1hD dimer core. The dimerization pattern of FlhD in the YdiV-FlhD complex is very similar to that of F1hD homodimers. The size-exclusion chromatography results also show that YdiV-F1hD complex is tetramer in solution. The016, a7and α8of YdiV and al and α4of F1hD directly participate in interaction and compose a highly firm interface. Remarkably, α8of YdiV provides most of the interacting residues. The high conservation of residues composing the interface of YdiV and FlhD across Entewbacleriaceae group suggests that YdiV from other Enierobacteriaceae group members may also interact with F1hD in the same way and down regulate flagella biogenesis and motility. We designed three mutants of F1hC to verify the DNA binding mechanism. EMSA result strongly suggests that target DNA directly interacts with the positive charge enriched region of F1hC while F1hD is necessary for DNA binding by keeping the ring-like structure of F1hD4C2. Our data also suggest that the anti-F1hD4C2function of YdiV is directly caused by F1hD binding:the mutants which did not bind to F1hD also lose all their inhibition function to the DNA binding affinity of F1hD4C2and did not inhibit cell motility in vivo; the ones with decreasing in the affinity of FlhD binding presented decline in their inhibition function in the same degree.EAL proteins have been known to be dimers in solution and catalyze the hydrolysis of the secondary messenger molecule c-di-GMP. In this regard, YdiV is an alternative. Sequence analysis shows eight out of the ten conserved catalytic residues are mutated. Structure analysis indicates, due to the key residue mutations, the potential substrate binding site is no longer compatible for c-di-GMP binding. More importantly, the a8of YdiV differs from typical EAL proteins extensively and eventually abolish the dimerization activity of YdiV. Ironically, it is the α8of YdiV that makes the greatest contribution to the interaction with F1hD. Previous bioinfomiatic studies showed450out of1805EAL-only proteins lack key catalytic residues and do not hydrolyze c-di-GMP Similar to YdiV, those unconventional EAL proteins may also function in a way apart from c-di-GMP turnover.It has been reported that YdiV can mediate the interaction between the two quorum sensing systems in E. coli in cooperation with its transcription activators SdiA and cAMP. This raises a question that if there is crosstalk between quorum sensing system and F1hDC transcriptional activity through YdiV. Although YdiV cannot bind to c-di-GMP, a large hydrophobic groove is still observed at the potential active site. Electron density map clear shows the existence of a phosphate and a glycerol molecule in this groove. So small molecules, such as cGMP or cAMP, may bind to YdiV. It reasonable to expect that binding of ligand may induce significant conformational change around the active site of YdiV. α6. α7and α8of YdiV locate around the binding groove and could be affected during this process. Consequently, YdiV may loss binding affinity to F1hD4C2complex.F1hD4C2complex contains four YdiV binding sites. Two of them are exposed and always ready for YdiV binding. The other two sites are buried in the ring-like structure. YdiV begins to squeeze into the ring-like structure to occupy these two site only when its concentration reaches a certain threshold and saturates the two peripheral binding sites. We have proved that DNA binds to F1hD4C2complex through wrapping around F1hC subunits rather than F1hD subunits. Occupation of the two peripheral binding sites by YdiV does not affect the DNA binding ability of F1hD4C complex. Our data strongly suggest YdiV regulate F1hD4C2transcriptional activity in a concentration dependent manner.The unique mechanism by which YdiV regulate F1hD4C2complex also raises a question that why bacteria adopt the concentration dependent mechanism and if bacteria can benefit from this kind of mechanism. Flagella biogenesis is an energy-consuming and time-consuming process. The switch between the motile and sessile lifestyles is a significant decision for bacteria. In order to prevent unnecessary energy waste, bacteria should not turn on or shutdown the corresponding gene transcription too frequently when the external environment is always changing rapidly. Only the signal is strong enough and maintain long enough, the intracellular concentration of YdiV becomes high, then flagella biogenesis shutdown completely. In another words, bacteria only change their life style when they have to.A very recent paper by takaya el al reported that YdiV not only strip F1hD4C2from DNA but also facilitate C1pXP protease mediated F1hD4C2degradation. In this regard, the breakage of the ring-like structure of F1hD4C2complex when the intracellular concentration of YdiV reaches a threshold will makes YdiV4-F1hD4C; expose the recognition site of C1pXP protease and become easy to be degraded. Based on our data and previous research, we propose a model in which YdiV negatively regulates transcriptional activity of F1hD4C2. At beginning. F1hD4C? binds to the promoter region of flagellar operons, the corresponding genes remain in the open state. Then the expression of YdiV is triggered by external signal and starts to bind to the peripheral binding sites of F1hD4C2. At this stage, gene transcription is not affected. However, if the external signal is strong enough and maintains long enough, the intracellular YdiV concentration eventually reaches a threshold and YdiV begins to squeeze into the ring-like structure of F1hD4C2complex. Finally, DNA is displaced from the F1hD4C2and F1hD4C2is degraded by C1pXP protease. As a result, the subsequent expression of flagellar and motility is repressed.