Molecular Mechanism for Rotational Switching of the Bacterial Flagellar Motor

Abstract

23The bacterial flagellar motor is a remarkable nanomachine that can rapidly rotate in both 24 counter-clockwise (CCW) and clockwise (CW) senses. The transitions between CCW and CW 25 rotation are critical for chemotaxis, and they are controlled by a signaling protein (CheY-P) 26 that interacts with a switch complex at the cytoplasmic side of the flagellar motor. However, 27 the exact molecular mechanism by which CheY-P controls the motor rotational switch remains 28 enigmatic. Here, we use the Lyme disease spirochete, Borrelia burgdorferi, as the model 29 system to dissect the mechanism underlying flagellar rotational switching. We first determined 30 high resolution in situ motor structures in the cheX and cheY3 mutants in which motors are 31 genetically locked in CCW or CW rotation. The structures showed that the CheY3 protein of 32 B. burgdorferi interacts directly with the FliM protein of the switch complex in a 33 phosphorylation-dependent manner. The binding of CheY3-P to FliM induces a major 34 remodeling of the switch protein FliG2 that alters its interaction with the torque generator. 35Because the remodeling of FliG2 is directly correlated with the rotational direction, our data 36 lead to a model for flagellar function in which the torque generator rotates in response to an 37 inward flow of H + driven by the proton motive force. Rapid conformational changes of FliG2 38 allow the switch complex to interact with opposite sides of the rotating torque generator, 39 thereby facilitating rotational switching between CW and CCW. 40 assembling the collar, stator complexes, and C-ring together, we revealed a complex 130 architecture of the CW-rotating flagellar motor with unprecedented details (Fig. 1h, i). 131 132 CheY3-P binds to the FliM protein of the C-ring 133The well-defined C-ring in the ∆cheX mutant was found to be associated with two previously 134 unidentified densities (arrowheads indicated in Fig. 1d, e). We hypothesized that these 135 densities represent bound CheY3-P, as high levels of CheY3-P are expected in the ∆cheX 136 cells 14 . To characterize CheY3-P and its interaction with the ∆cheX motor, we replaced the 137 cheX-cheY3 genes with cheY3-gfp, generating a cheX::cheY3-GFP mutant (Extended Data Fig. 138

The flagellar motor of Vibrio alginolyticus undergoes major structural remodeling during rotational switching

“…The cells should have endogenous CheY, as there has been no alteration to the CheY gene. Second, density similar in location and shape has been shown two recent studies to be CheY-P. Chang et al have used GFP-tagged CheY-P to show that, in Borrelia burgdorferi , CheY-P occupies the same position ( Chang et al, 2020 ). Rossmann et al also identified similar density for the CheY homolog, CleD in Caulobacter crescentus ( Rossmann et al, 2020 ).…”

“…However, we observed ~21 Å increase in the diameter and ~40 Å lateral change at FliG C of the CW-motor. In a parallel study, Chang et al used a B. burgdorferi with a CheX deletion or CheY3 deletion to investigate the switching mechanism of spirochetes ( Chang et al, 2020 ). Similar conformational changes of the C-ring were observed in both species; however, the stator complexes were resolved in B. burgdorferi, as they appear to be less dynamic than those in V. alginolyticus.…”

“…Chang et al have used GFP-tagged CheY-P to show that, in Borrelia burgdorferi, CheY-P occupies the similar position 68 . Rossmann et al also identified similar density for the CheY homolog, CleD in Caulobacter crescentus 69 .…”

Site-directed crosslinking identifies the stator-rotor interaction surfaces in a hybrid bacterial flagellar motor

“…The crosslinking data indicate that this surface, which contains the RxxGΦΦxLE motif, interacts closely with FliG and is important for stator assembly and motor rotation. Recently, a rotational gear model for stator function has been proposed by several groups (43,44,54). A similar rotational model has been reported for ExbB/ExbD in the Ton molecular motor.…”

The promise and the challenges of cryo‐electron tomography

“…Cryo‐ET has provided insights into the structure and function of viruses [9–12,92–97], nuclear pore complexes [64,98–100], ribosomes [101–103], coated vesicles [104–109], nucleosome arrangement in chromatin [110], cytoskeletal elements [111–113], bacterial secretion systems [114–117], chemotaxis [118,119], flagellar motors [120], and S‐layers [121]. Obtaining information about the spatial arrangement of proteins by cryo‐ET has also found use in assessing protein denaturation at the air‐buffer interface for single‐particle analysis [122].…”