Bacterial chemoreceptor arrays are hexagonally packed trimers of receptor dimers networked by rings of kinase and coupling proteins

Abstract

Chemoreceptor arrays are supramolecular transmembrane machines of unknown structure that allow bacteria to sense their surroundings and respond by chemotaxis. We have combined X-ray crystallography of purified proteins with electron cryotomography of native arrays inside cells to reveal the arrangement of the component transmembrane receptors, histidine kinases (CheA) and CheW coupling proteins. Trimers of receptor dimers lie at the vertices of a hexagonal lattice in a "two-facing-two" configuration surrounding a ring of alternating CheA regulatory domains (P5) and CheW couplers. Whereas the CheA kinase domains (P4) project downward below the ring, the CheA dimerization domains (P3) link neighboring rings to form an extended, stable array. This highly interconnected protein architecture underlies the remarkable sensitivity and cooperative nature of transmembrane signaling in bacterial chemotaxis.

Fig. 1.

Architecture of native chemoreceptor arrays as seen by electron cryotomography. (Upper) Tomographic slice through the top of a S. enterica minicell. OM, outer membrane; IM, inner membrane. (Scale bar: 100 nm.) (Lower) Subtomogram averages of E. coli, H. hepaticus, S. enterica, and B. subtilis (from left to right) chemoreceptor arrays after application of sixfold symmetry. In all cases, the individual receptor dimers (asterisks) are clearly resolved, revealing a two-facing-two packing arrangement: A pair of dimers faces another pair of dimers at each interface around the ring, or to describe it in another way, trimers are oriented such that one receptor dimer points toward the center of each hexagon. The conserved architecture also shows that the cell lysis used to thin the E. coli and B. subtilis samples for high-resolution ECT did not perturb the arrays. (Scale bars: 12 nm.)

Fig. 2.

Model of a receptor trimer within the EM map. Two isosurfaces of the receptor region of the EM map are shown as blue and magenta grids (low and higher density, respectively) with an all-atom model of a receptor trimer fit to the map, seen from the side (Left, with back dimer removed for clarity) and in cross-section at three different positions (Right). The atomic model is based on a crystal structure of a truncated E. coli Tsr MCP which crystallized in a similar configuration (27). To fit that structure into the EM map, the four-helix coiled-coil was extended (based on the crystal structure of receptor Tm1143; ref. 29) to the junction of the HAMP domain (residues 264–514), separated slightly at the tips to better fit the electron density, and then refined against the EM data in reciprocal space (see Materials and Methods). The density clearly confirms the trimers-of-dimers architecture in vivo, but compared to the crystal structure, the receptors bend in the glycine hinge region and the four-helix coiled-coil extends to the level of the HAMP domain. The hexagonal order decreases toward the membrane. The additional density seen around the receptor tips (asterisks) is where the receptor bundle connects with the CheA/W baseplate.

Fig. 3.

Ternary complex crystal structure of T. maritima chemotaxis proteins. (A) Close-ups of the pseudosymmetric interactions made by the opposite ends of CheW (green ribbons) and P5 (blue ribbons), and the interaction between the receptor tip (magenta ribbons) and CheW. Inset shows a schematic of dimeric CheA:CheW, with the crystallized unit boxed. (B) Ring structure formed by the ternary complex crystals. Three CheW domains and three P5 domains generate a ring, and each CheW binds one receptor tip (pink) between subdomains 1 and 2 (Left). Similar interactions between P5 with the distal end of receptors (purple) link rings “head-to-head” in the unit cell (Right). The P4 domains (gray), of which only the core elements are visible, project above and below the double-ring structure at the junction to P5.

Fig. 4.

Structure of native chemoreceptor arrays. (A) Superposition of one ring of the crystal structure (P5 blue, CheW green) with its six receptor dimers (yellow helices) on the EM map (blue mesh) with its previously fit 18 receptor dimers (pink). The EM density in the CheA/W ring is only about half as intense as within the receptors, suggesting either lower occupancy or higher disorder. (B) Side view of the EM density (blue mesh) in the region of the CheA-P4 kinase domain (gray). (C) The CheA dimer links CheW/P5 rings. The two subunits of the CheA dimer (black and gray) provide one P5 to each of two neighboring rings. The P3 dimerization domain resides between the receptor bundles at the center of one hexagonal edge and the P4 domains reside beneath the interlocked rings. Views shown are in the plane of the rings (Left) and looking down from the membrane (Right). (D) The arrangement of components within the receptor array produces P6 point symmetry (P6 unit cell boxed in red, with the asymmetric unit in yellow; six-, three-, and twofold symmetry axes are designated in red). The lattice gives a CheA:CheW:MCP subunit stoichiometry of 1∶1∶6. If the “empty” hexagons instead contain six CheW proteins, the ratio would become 1∶2∶6. Movie S1 summarizes in a step-by-step animation how the new electron cryotomographical and X-ray crystallographic data were used to elucidate the array structure shown in D.