Bacterial flagellar motility, an important virulence factor, is energized by a rotary motor localized within the flagellar basal body. The rotor module consists of a large framework (C-ring), composed of the FliG, FliM and FliN proteins. FliN and FliM contacts the FliG torque ring to control the direction of flagellar rotation. We report that structure-based models constrained only by residue coevolution can recover the binding interface of atomic X-ray dimer complexes with remarkable accuracy (ca. 1 Å RMSD). We propose a model for FliM-FliN heterodimerization, which agrees accurately with homologous interfaces as well as in-situ cross-linking experiments, and hence supports a proposed architecture for the lower portion of the C-ring. Furthermore, this approach allowed the identification of two discrete and interchangeable homodimerization interfaces between FliM middle domains that agree with experimental measurements and might be associated with C-ring directional switching dynamics triggered upon binding of CheY signal protein. Our findings provide structural details of complex formation at the C-ring that have been difficult to obtain with previous methodologies and clarify the architectural principle that underpins the ultra-sensitive allostery exhibited by this ring assembly that controls the clockwise (CW) or counterclockwise (CCW) rotation of flagella.
Chemical Cross-Links in FliM homodimerization
Chemical Cross-links reported in literature and used to support the predicted models for FliM middle domain homodimerization.
cross_linking_FliMm_all
MSA of FliM middle domain family
multiple sequence alignment used to compute coevolution signals in the middle domain of FliM protein from T. maritma
FLIM_Thermotoga_maritma_filter30gap.afa
MSA for FliM/N c-terminal family
Multiple Sequence Alignment used to compute coevolution signals in FliN C-terminal and FliM C-terminal domains. These couplings were mapped to predict FliN C-terminal homodimerization in validation process and to predict a model for FliN/FliM C-terminal heterodimerization.
FliMNcterminal_PF01052_uniprot_format.afa
Prediction of FliN C-terminal homodimer
Prediction of FliN C-terminal homodimer through simulations with structure-based models driven by coevolution signals. The final model presented a RMSD of 0.81Å when compared to experimental X-ray structure (1O6A). This prediction was used as a validation step to support subsequent models.
FliN_homodimer_predicted.pdb
prediction of FliNM heterodimerization
Prediction of FliN/FliM C-terminal heterodimer through simulations with structure-based models driven by coevolution signals.
FliNM_heterodimer_predicted.pdb
predicted FliM homodimer model I
Predicted homodimerization interface of FliM middle domain in a parallel configuration that is compatible to the interaction mode of one rotational state suggested by cryo-EM data fitting and chemical cross-linking experiments.
FliM_homodimer_cluster1.pdb
predicted FliM homodimer model II
Predicted homodimerization interface of FliM middle domain in a twisted configuration that is supported by chemical cross-linking experiments and can be related to an alternative conformational state of flagellum rotation.
FliM_homodimer_cluster2.pdb
Recurring Chemical Cross-Links in FliN/FliM C-terminal homodimerization
Chemical Cross-Links in FliN/FliM C-terminal dimerization that are present in both rotational states of flagellar motor. This dataset from literature was used to support predicted model for FliN/FliM C-terminal heterodimer.
both_modes
Chemical Cross-Links observed in the CW state of FliN/FliM C-terminal dimer
Chemical Cross-Links in FliN/FliM C-terminal dimerization that are observed only in CW rotational states of flagellar motor. This dataset from literature was used to support predicted model for FliN/FliM C-terminal heterodimer.
CW_mode
Chemical Cross-Links observed in the CCW state of FliN/FliM C-terminal dimer
Chemical Cross-Links in FliN/FliM C-terminal dimerization that are observed only in CCW rotational states of flagellar motor. This dataset from literature was used to support predicted model for FliN/FliM C-terminal heterodimer.
CCW_mode
DCA for FliM models
Coevolution couplings derived from Direct-Coupling Analysis used to predict FliM middle domain homodimers (parallel and twisted models).
DCA_FliM_middle
DCA for FliM/FliN model
Coevolution couplings derived from Direct-Coupling Analysis used to predict the dimerization interface of FliM/FliN C-terminal domain.
DCA_FliN_FliM_Cterminal
DCA for FliN homodimer model
Coevolution couplings derived from Direct-Coupling Analysis used to predict the dimerization interface of FliN C-terminal domain (validation of methodology).
DCA_FliN_Cterminal