CryoEM and computer simulations reveal a novel kinase conformational switch in bacterial chemotaxis signaling
- University of Illinois at Urbana-Champaign, United States
- University of Pittsburgh School of Medicine, United States
Figures
Figure 1
Reconstitution of 2D arrays of the receptor signaling complex on lipid monolayers.
(A&B) Negatively stained electron micrographs of reconstituted lipid monolayers with TarCF only (A) or with TarCF/CheA/CheW (B). Inset, Fourier transform of a region from the monolayer array, …
Figure 2 with 2 supplements
CryoET of monolayer arrays of TarCF/CheA/CheW ternary signaling complex.
(A) A tomographic slice (1.2 nm thick) through the reconstituted monolayer arrays of TarCF/CheA/CheW, without CTF correction. Inset, The Fourier transform of a selected region, displaying Thon rings …
Figure 2—figure supplement 1
Classification of the sub-tomogram volumes.
The sub-tomograms containing the receptor hexagon (6 TODs, yellow circles) were subjected to alignment and classification. Sections of the sub-tomogram classes are shown at the receptor region (top …
Figure 2—figure supplement 2
Comparison of the chemotaxis arrays from native cells and from in vitro reconstituted monolayers.
(A) A tomographic slice (5.2 nm thick) of the native chemotaxis arrays from wild-type E. coli cells, overlaid with the positions of each sub-tomogram classified as either the CheA2-trimer unit …
Figure 3 with 3 supplements
CheA2-trimer and CheA2-hexamer density maps with molecular dynamics flexible fitting (MDFF) of computationally constructed T. maritima subunit models.
(A) Overall fitting of the CheA2-trimer density map contoured at 1.5σ. The three core signaling complexes are colored in pink, blue and green. (B) A sectional view of the boxed region in A, rotated …
Figure 3—figure supplement 1
Resolutions of the density maps.
(A) Gold-standard Fourier shell correlation (FSC) of the CheA2-trimer (left) and CheA2-hexamer (right) density maps. At FSC=0.143, the overall resolution of the CheA2-trimer map is 11.3 Å and that …
Figure 3—figure supplement 2
X-Z sectional views of the CheA2-trimer density map with MDFF model.
The positions of the sections are indicated in the last panel with an orthogonal view (X-Y plane).
Figure 3—figure supplement 3
A metric for the goodness of fit for the docking of the CheA-P4 domain.
(A) Distribution of 23 classes of fits for the P4 domain starting from random orientations. (B) The models from the top 9 highest cross-correlation classes are shown in panels1-9, with the …
Figure 4 with 5 supplements
CheA dimer conformational switch.
(A) Top and side views of the core-signaling unit, consisting of two receptor TODs (red), a single CheA dimer (blue), and four CheW monomers (green). (B) Two distinct classes, undipped (top) and …
Figure 4—figure supplement 1
Nomenclatures.
(A) TOD: receptor trimer of dimers. (B) Core-signaling unit/complex: 2 TOD, 1 CheA dimer, 4 CheW. (C) Unit cell: 3 coupled core-signaling units. (D) CheA2-trimer: 3 core-signaling units, pseudo …
Figure 4—figure supplement 2
Overview of molecular modeling and simulation strategy taken in this study.
(A) High resolution X-ray structures from T. maritima were taken as inputs for the generation of models corresponding to the array’s core components, namely the receptor trimer-of-dimers, coupled …
Figure 4—figure supplement 3
Computational modeling of the extended chemosensory array structure.
(A) Molecular dynamics (MD) simulations show that T. maritima receptors form a stable trimer-of-dimers (TOD). Side view (left) and top view (right) of the highly conserved protein interaction tip, …
Figure 4—figure supplement 4
Overview of key all-atom molecular dynamics simulations conducted in this study.
(A) Table summarizing simulations for each molecular system with atom number reported roughly in millions (M) of atoms (including protein, solvent, and ions) and simulation duration in nanoseconds …
Figure 4—figure supplement 5
Time series of CheA dimer conformations extracted from CheA2-trimer simulations.
Traces track the projection of the conformations of 30 CheA dimers onto the first principal component of the 'dipping' motion. Colored traces track CheA dimers that undergo an extended (>10 ns) …
Figure 5 with 1 supplement
Biochemical validation of alternative CheA conformations in E. coli.
(A) Swimming ability of E. coli cells with mutations in the CheA-P3 and Tsr interface (I304/N405 and D316/R394) and in the 'dipped' CheA-P4 and Tsr interface (E361/R394). Swimming activities are …
Figure 5—figure supplement 1
CryoEM images of plunge-frozen E.
coli cells expressing WT Tsr and WT CheA (A&B), R265A CheA (C), R265C CheA (D), R265S CheA (E), and R265E CheA (F). The arrays are marked with white curved arrows. Scale bars, 100 nm.
Author response image 1
Effect of missing cone on the tomography density map.
Shown in gray solid surface, the ideal density map without missing cone, and maps with different amount of missing cone applied (colored meshes). Left, green mesh 12° cone (this study, Figure …
Author response image 2
CheA dimers from multiple initial conformations converge to same fitted model.
Red, blue, and green traces track RMSD of CheA dimers as measured from the initial MDFF-refined model. Though, the dimer conformations diversity during the 120 ns production run, a 20 ns MDFF …
Author response image 3
Comparison of density maps containing full-length wt CheA and wt CheA-P345.
Densities are contoured at 2σ. Solid blue, with wt CheA-P345; red mesh, with full-length wt CheA. The density with CheA-P345 appears elongated along the Z direction, due to the missing wedge effect …
Author response image 4
Resolution anisotropy.
(A) Gold-standard Fourier shell correlation (FSC) of the CheA2-trimer density map. The overall FSC of the map is plotted as a solid black line. The FSC curves for the conical Fourier shells along …
Author response image 5
A metric for the goodness of fit for the docking of the CheA-P4 domain.
(A) Distribution of 23 classes of fits for the P4 domain starting from random orientations. (B) The models from the top 9 highest cross-correlation classes are shown in panels 1-9, with the …
Videos
Video 1
Tomographic slices of monolayer arrays.
Related to Figure 2.
Video 2
MDFF model fitting of the CheA2-trimer density map.
Related to Figure 3.
Video 3
Molecular dynamics simulation of array unit cell.
Shown here is a 75 ns clip of a wild type unit cell trajectory, illustrating the dynamics of the 1.2 million atom model, including 6 receptor TODs (red), 3 CheA dimers (blue), and 12 CheW monomers …
Video 4
Molecular dynamics simulations reveal conformational switch in CheA P4 domain.
Shown here is one of four 'dipping' events observed in the wild type unit cell simulations, leading to modified contacts between the CheA dimer and receptor TODs. Strong contacts between P3 and …
Additional files
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Supplementary file 1
Summary of protein-protein interactions at key interfaces of equilibrated T. maritima unit cell model.
Residues participating in a given interface but not associated with particular partners are listed separately for each domain. Residues that interact significantly (>50% of frames) are listed as a pair in a separate row. Interactions unique to this study are listed in green. Where ambiguous, residue pairs involving a receptor bound to CheA-P5, CheW from a CheA-P4/CheW ring or CheW from a CheW-only ring are denoted with a (1), (2) or (3) respectively. ** Signifies interfaces taken directly from experimental structures. Recent references pertaining to each protein-protein interface are given.
- https://doi.org/10.7554/eLife.08419.023
