Dynamic molecular machines: imaging the in situ architecture and transient binding of the flagella motor

Proteins are able to interact to form macromolecular nano-machines. Bacteria have evolved these
proteinaceous structures to perform many essential functions with precision regulated mechanisms. One such
nano-machine is the flagellar motor. Spanning two membranes, it assembles itself into a structure to harness
proton-motive force to drive rotation of a helical filament to act as a propeller. At the mechanistic heart of
the machine is a cytoplasmic associated C-ring. Yet, we do not understand how proteinaceous structures like
the C-ring enable mechanistic functions.
Different protein partners bind the C-ring and regulate mechanisms of the Campylobacter jejuni motor.
Flagella assembly is regulated by FlgS which detects the MS/C ring and autophosphorylates, phosphorylating
FlgR which up-regulates transcription of later stage components of the flagella motor. Polar
localisation/numeration is determined by FlhF which localises the nascent motor to the cell pole and FlhG
which limits construction to a single flagellum and directional switching from clockwise to counter-clockwise
rotation is mediated by CheY interacting with FliN in the C-ring. Binding location, function and architectural
effects are poorly understood for these mechanisms.
Electron cryo-tomography (ECT) allows nanometer resolution in situ investigation of the flagella. A sample is
flash frozen on an electron microscopy (EM) grid, maintaining a frozen-hydrated physiological state. The
sample is imaged incrementally in degrees in an electron microscope, rotating around a eucentric axis. This
provides 2D projections through the 3D object that can be reconstructed computationally to provide a 3D
volume. This produces nanometer level resolution of membrane embedded complexes. Resolution can be
increased by averaging the 3D structures to increase signal-to-noise ratios in a method known as
subtomogram averaging.
C. jejuni produces a single large stable motor at both cell poles and has a thin width enabling higher
resolution imaging through greater electron penetration. Genetically manipulatable, this medically relevant
bacteria has been optimised to be ideal for imaging and dissecting the flagella motor in situ. Here I propose
to use ECT to determine the in situ architecture of the C. jejuni C-ring, imaging the changing structures
during FlhFG, FlgS and CheY binding. This will provide insights into transient protein-protein interactions
affecting architectural changes and mechanistic function in protein nano-machines.
Structural/functional investigation of in situ C-ring architecture and transient binding will be done using ECT.
A high resolution (~3nm) WT C. jejuni motor structure will be determined by averaging 500 imaged motors.
This will be be the base for further imaging of tagging/deletion strains to localise and find stoichiometry of
FliGMNY in the C-ring. These tagged/deletion strains will require a resolution of 4.5 nm, achieved by
averaging ~150 flagella motors. Clockwise and counter-clockwise locked mutants will then be made and
imaged to determine architectural motor differences between the two rotational states. This will reveal the
mechanism of rotational directional switching in the flagella nano-machine.
The in situ binding sites of CheY, FlgS and FlhGF to the C-ring will be imaged by creating strains which bias
towards high occupancy C-ring binding. CheY will be upregulated and mutated with D8K, Y102W and S88I to
slow autodephosphorylation and CheZ dephosphorylation. FlgS will be over-expressed on a background of
FlgR deletion. FlhG and FlhF will be over-expressed and the FlhF overexpression will be made on a FlhG
deletion background. By imaging these strains and averaging ~150 motors to achieve 4.5nm resolution, these
transient interaction events can be imaged and shown in situ.