Thread by @NMITaylorLab

1/ Here is the #Tweetorial presenting the story behind our paper on the structure and function of #StatorUnits of the bacterial #FlagellarMotor published in @CellCellPress!
cell.com/cell/fulltext/…

2/ Many bacteria swim by rotating their flagella, a motion crucial for survival and pathogenicity. Flagellar rotation is bidirectional: in Salmonella and E. coli, counterclockwise (CCW) rotation causes the cell to swim forward, while clockwise (CW) rotation causes it to tumble.

3/ The flagellum is made of an external filament, a flexible hook and a motor. This motor consists of a rotor surrounded by a ring of stator units (MotAB). #MotAB is an ion channel that powers the rotation of the motor using energy from the transmembrane ion gradient (H+ or Na+).

4/ We were amazed by this biological motor and wanted to understand how its bidirectional rotation is powered, so we set out to solve the structure of MotAB, reveal its conformational changes upon ion transport and elucidate how these changes power rotation of the flagellum.

5/ To do so, we used cryo-electron microscopy (#Cryo-EM), a revolutionary electron microscopic technique in which we plunge-freeze the sample to cryogenic temperatures in order to visualize it with the least possible distortion.

6/ We screened several stator units of different organisms, the winner was Campylobacter jejuni with a resolution of 3.1 Å. The ratio of MotA:MotB was suggested to be 4:2, so we were astonished to see that it is actually 5:2! We saw the same ratio in MotAB of two other species.

7/ If you look closer, you will see that in this structure CjMotB is interacting through its plug helices with MotA at the latter’s periplasmic surface. This structure corresponds to the plugged, inactive state of MotAB. The plug region is important to prevent proton leakage.

8/ We also wanted to reveal the active state, and to do so, we made a deletion of the 20 amino acids corresponding to the plug motif. Again, using #Cryo-EM we solved the structure of the unplugged stator unit at 3.0 Å resolution.

9/ When comparing plugged and unplugged structures, we saw a difference in the universally conserved CjMotB D22 residue, responsible for ion transport. This residue in CjMotB chain 1 (in black) would be protonatable, but the same residue in CjMotB chain 2 (in grey) would not.

10/ This suggests that there is an access pathway for protons from the periplasm. Comparing other residues, we see that such a pathway appears to exist from the side of MotA between chains 1 and 2, which is shielded by the hydrophobic residue CjMotA F186 in the plugged structure.

11/ Then we wanted to study the conformational changes that the stator unit undergoes upon proton transport. Check what happened when we combined the unplugging mutation with a CjMotB(D22N) mutation that mimics protonation of D22. Spoiler alert: N22 is in a different position!

12/ Taking all together, we proposed a rotational model for torque generation, where MotA rotates around MotB. Rotation of MotA around MotB (in a clockwise direction, from the periplasmic side) occurs in steps of 36°, where MotB D22 of both chains transport ions alternately.

13/ This is how we think it goes: when MotB D22 is engaged, it can drive a power stroke (when the charge of the other MotB D22 becomes neutralized). When it is not engaged, it picks up a proton from the channel and inches to the position where it can drive the power stroke.

14/ Finally, we wanted to explain how CW rotation of MotA around MotB could translate to both CW and CCW rotation of the motor. For this, we need to introduce another component of the rotor, FliG, whose C-terminal domain (FliGCC) interacts with the cytoplasmic domain of MotA.

15/ The story goes like this: chemotactic signaling (trough CheY-P) causes FliGCC to make a 180° turn relative to MotAB (called switching), which allows the rotor to turn in the other direction. The same CW rotation of MotA now powers rotation of the rotor in the other direction.

16/ In summary, and as @NavishWadhwa once tweeted: ‘A tiny motor rotates bacterial flagella, but what rotates the flagellar motor? An even tinier rotary motor!’ We hope that our results provide a robust structure-based framework for many experiments to come on the #FlagellarMotor

17/ Thanks so much for reading! If you want more, check out recent papers from @LeaLabTweets (nature.com/articles/s4156…) and @JunLiuLab (nature.com/articles/s4159…), converging as well on a “tiny rotary motor” model for powering bidirectional rotation of the flagellar motor. /END