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

Lead Research Organisation: Imperial College London
Department Name: Life Sciences

Abstract

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.

Publications

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Henderson LD (2018) High-Throughput Electron Cryo-tomography of Protein Complexes and Their Assembly. in Methods in molecular biology (Clifton, N.J.)

Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011178/1 01/10/2015 30/09/2023
1656988 Studentship BB/M011178/1 03/10/2015 30/09/2019 Louie Derek Henderson
 
Description Within the bacterium Campylobacter jejuni, it has been discovered that its motility apparatus (the bacterial flagella motor) has a novel architecture in the region projecting into the interior of the cell known as the C-ring.

I have successfully developed an algorithm which fuses proteins in silico for use with developing tags for in situ study.
Exploitation Route The flagella work may be useful in understanding diversity and evolution of larger structures in bacterial rotary machines. The software development may have applications in in situ structural work in answering many questions and might also have biotechnological applications in the construction of bifunctional novel proteins.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Title Protagger: rational protein tag algorithm 
Description Protagger is a software I've developed to add aditional protein densities to macromolecular protein complexes. It achives this through a spatial RMSD minimisation of potential tags to a given tag site. 
Type Of Material Improvements to research infrastructure 
Year Produced 2017 
Provided To Others? No  
Impact Nothing as of yet 
 
Description Imperial festival Beeby lab stand 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact As a lab, we run a stall in the Imperial fesitval in which we exhibit lego models of our structures to convey our scince to the general public.
Year(s) Of Engagement Activity 2016,2017,2018