Regulation of the chemotaxis histidine kinase CheA: A structural perspective

Abstract

Bacteria sense and respond to their environment through a highly conserved assembly of transmembrane chemoreceptors (MCPs), the histidine kinase CheA, and the coupling protein CheW, hereafter termed "the chemosensory array". In recent years, great strides have been made in understanding the architecture of the chemosensory array and how this assembly engenders sensitive and cooperative responses. Nonetheless, a central outstanding question surrounds how receptors modulate the activity of the CheA kinase, the enzymatic output of the sensory system. With a focus on recent advances, we summarize the current understanding of array structure and function to comment on the molecular mechanism by which CheA, receptors and CheW generate the high sensitivity, gain and dynamic range emblematic of bacterial chemotaxis. The complexity of the chemosensory arrays has motivated investigation with many different approaches. In particular, structural methods, genetics, cellular activity assays, nanodisc technology and cryo-electron tomography have provided advances that bridge length scales and connect molecular mechanism to cellular function. Given the high degree of component integration in the chemosensory arrays, we ultimately aim to understand how such networked molecular interactions generate a whole that is truly greater than the sum of its parts. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins.

Keywords: Enzymology; Histidine kinase; Membrane proteins; Phosphorelay; Protein structure; Signal transduction.

Fig. 1.

The bacterial chemotaxis system of E. coli. Attractants and repellents stimulate transmembrane chemoreceptors that function in a trimer-of-dimers. Binding of ligands to the extracellular domain elicits a conformational change in the receptor that is propagated through the HAMP to the intracellular protein interaction region (PIR) where the receptor directly interacts with the adaptor protein CheW (yellow) and the dimeric histidine kinase CheA (green, purple, orange). CheA has five domains (P1–P5) and once activated by receptors, the P4 kinase domain (orange) will trans phosphorylate the P1 substrate domain. CheA binds the response regulator CheY through the P2 domain (purple) and activates CheY by phospho-transfer. Phospho-CheY interacts directly with the flagellar motor at the C-ring to induce clockwise rotation of the flagella and cell tumbling. The concentration of phospho-CheY is modulated by the phosphatase CheZ. CheA also phosphorylates the methyl-esterase CheB, which removes methyl groups on glutamate residues located at the receptor adaptation region. The SAM-dependent methyltransferase CheR adds methyl groups to glutamate residues to reactivate receptors inhibited by attractant.

Fig 2.

The chemosensory arrays. (A) Schematic of the bacterial chemotaxis transmembrane array viewed from the membrane. CheA P5 (blue) and CheW (green) interact to form hexagonal rings that are linked together by dimerization of the CheA P3 domain (dark blue). One CheA dimer is shown in the dashed circle. The rings are anchored to the membrane and further stabilized by direct interaction with chemoreceptors (grey), which form a trimer-of-dimer oligomeric state. Within the trimer-of-dimer module, the subunits of the receptor dimer either interact with an adjacent receptor subunit within the trimer or with P5/CheW. In areas of the lattice where there is no CheA P5 domain, CheW assembles into all-CheW rings. (B) ECT of Ec in vivo arrays show trimer-of-receptor dimers; crystal structures of chemotaxis protein fragments are fit into the electron density [27].

Fig 3.

Crystal structures of Tm CheA domains. (A) Crystal structures of CheA fragments P1 (PDB ID: 1TQG), P2 (PDB ID: 1U0S), and P3P4P5 (PDB ID: 1B3Q) arranged to represent a full-length kinase. Dashed lines denote flexible linkers between the P1/P2 and P2/P3 domains with undetermined structure. (B) The P1 structure (PDB ID:1TQG) consists of helices A-D. Residues D93, M94 and R97 (red) participate in non-product interactions with P4. Residues near the H45 substrate residue (green) mediate productive interactions with P4. E67 (cyan) activates H45 for phosphorylation.

Fig. 4.

The core complex and key interactions of the chemosensory array. (A) Model of the P4 kinase domain (grey) bound to the P1 substrate domain (purple) [37]. The ATP lid (orange) shown in an open configuration may mediate interactions among nucleotide and substrate. (B) Crystal structures of P5 bound to CheW (PDB ID:3UR1) reveals conserved hydrophobic interfaces (1 and 2) that form hexagonal rings that are apparent in vivo [35]. (C) View from the membrane of a core complex composed of one CheA dimer, 2 CheW molecules and two receptor trimmers of dimers. Chemoreceptors interact with both CheA P5 and CheW at the junction between two β-barrels. (D) Side view of (C). The P4 domain resides below the P5:CheW ring and is relatively mobile [27]. P4 may move in the different activation states of CheA and perhaps interact with the P5-CheW layer to modulate access of substrates to the nucleotide-binding site. P1 and P2 are not shown.

Fig. 5.

Electron density maps of the Ec chemotaxis core complex in a kinase-off state generated by ECT [102]. Increased density that resembles a ‘keel’ resides below the receptor tips in a full-length kinase (grey) but is no longer present when the arrays are generated from a CheA variant that does not contain the P1 and P2 domains (teal).

Fig. 6.

A hypothetical cartoon model for regulation of the CheA kinase. Two trimers-of-receptor dimers (magenta) are shown bound to an extended core complex of one CheA, 2 CheWs proteins, plus additional CheW and P5 domains that extend the array through interface 2. CheA P3 resides between the receptors. For detailed molecular interactions, see Fig. 4. (A) In the kinase-off state, interactions within the P5-CheW layer are relatively weakened and encourage greater interactions of P4 within the core complexes, causing either occlusion of the ATP pocket (orange) or interference with ATP binding determinants, such as the ATP-lid. P1 and P2 also associate with P3 and P4 in the sequestered conformation with P1 forming self-interactions or docking to an inhibitory site on P4 (arrows and dashed linker indicate alternative P1 docking sites). (B) In the kinase-on state, conformational signals from the receptor are transmitted through the receptor:CheW interface and perhaps the P3:receptor interface to alter the P5-CheW layer and increase interactions within both interface 1 and interface 2. Effects on the P3–P4 and P4–P5 linkers release the P4 domain, increase mobility of P1 and P2, and expose the ATP binding pocket and/or release of the lid (orange) to facilitate ATP binding (blue diamond) and P1 trans autophosphorylation (red circle).