Abstract
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.
Highlights
The formation and assembly of the flagellum, an essential motility apparatus in many bacteria, are well organized and hierarchically processed [1,2]
YdiV loses 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
A phosphate and a glycerol molecule appear in this groove in contact with Thr32, His33, Phe34, Thr45, Gln64, Gln85 and some water molecules (Figure 1D). They are partially parallel to the c-di-GMP molecule in TBD1265, implicating that other small molecules with similar structures may bind to YdiV within this groove and play a regulatory role to the function of YdiV
Summary
The formation and assembly of the flagellum, an essential motility apparatus in many bacteria, are well organized and hierarchically processed [1,2]. At the top of the hierarchy is the flhDC operon, whose expression is required for the transcription of class II flagellar operons. The products of flhDC operon, FlhD and FlhC, form a heterohexamer (D4C2) to bind to the upstream of class II promoters and promote class II genes transcription [3]. It has been reported that the FlhC subunit interacts with DNA, while the FlhD subunit strengthens the specificity to the FlhD4C2 box and the stability of the protein–DNA complex [5,6]. The two zinc-binding sites are located at both sides of the FlhDC heterohexamer complex, respectively. Wang et al [7] have suggested that the DNA may mainly wrap around the FlhD subunits when FlhD4C2 activates the downstream gene transcription.
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