A scissor blade-like closing mechanism implicated in transmembrane signaling in a Bacteroides hybrid two-component system

Abstract

Signaling across the membrane in response to extracellular stimuli is essential for survival of all cells. In bacteria, responses to environmental changes are predominantly mediated by two-component systems, which are typically composed of a membrane-spanning sensor histidine kinase and a cytoplasmic response regulator. In the human gut symbiont Bacteroides thetaiotaomicron, hybrid two-component systems are a key part of the bacterium's ability to sense and degrade complex carbohydrates in the gut. Here, we identify the activating ligand of the hybrid two-component system, BT4663, which controls heparin and heparan sulfate acquisition and degradation in this prominent gut microbe, and report the crystal structure of the extracellular sensor domain in both apo and ligand-bound forms. Current models for signal transduction across the membrane involve either a piston-like or rotational displacement of the transmembrane helices to modulate activity of the linked cytoplasmic kinases. The structures of the BT4663 sensor domain reveal a significant conformational change in the homodimer on ligand binding, which results in a scissor-like closing of the C-termini of each protomer. We propose this movement activates the attached intracellular kinase domains and represents an allosteric mechanism for bacterial transmembrane signaling distinct from previously described models, thus expanding our understanding of signal transduction across the membrane, a fundamental requirement in many important biological processes.

Fig. 1.

Periplasmic domain of BT4663 HTCS binds unsaturated disaccharides that are derived from heparin and HS. (A) (Upper) Bt heparin/HS locus structure with genes of known function labeled. GH, glycoside hydrolase; PL, polysaccharide lyase [Carbohydrate-Active EnZymes database (34)]. (Lower) Domain organization of BT4663. Domain predictions, including the type I signal peptide (SP1), Reg_prop repeats, Y_Y_Y, TM, phosphoacceptor (Pfam HisKA), ATPase (Pfam HATPase_c), receiver (Pfam REC), and DNA binding (Pfam HTH_AraC) domains, are from SMART (35). (B) ITC data showing binding of the unsaturated disaccharide ΔUA-GlcNAc6S to the periplasmic sensor domain of BT4663 and the structure of the sugar ligand. The upper part of the ITC panel shows the raw heats of binding and the lower part the integrated heats fit to a single-site binding model.

Fig. 2.

Crystal structure of the apo form of BT4663 periplasmic sensor domain dimer. (A) One protomer is color-ramped from N-terminus–C-terminus (blue-red) with a pale blue surface; the surface of the second protomer is shown in green. The gray bar represents the cytoplasmic membrane. (B) Enlarged view of the Y_Y_Y domains, showing the strand labels and Y_Y_Y dimer interface. (C) Side view of the BT4663 sensor domain dimer and cartoon of the relative domain arrangements (Inset). The solid lines symbolize the planes of the propellers (Materials and Methods). The axes of rotation are the intersection between two planes and perpendicular to the plane of the paper.

Fig. 3.

Location of the ligand binding sites within BT4663 sensor dimer. (A) Electron density difference map (F_obs-F_calc) into which the ligand (ΔUA-GlcNAc6S; yellow sticks) was built (final model shown). (B) Side and top views of the ligand-bound BT4663 sensor domain dimer. The ligand is represented as spheres. (C) Binding site with disaccharide ligand (ΔUA-GlcNAc6S; carbons shown as yellow sticks, sulfur shown in light green) indicating potential polar interactions with the two BT4663 protomers (blue and green).

Fig. 4.

Conformational changes in the BT4663 dimer on ligand binding. (A) Apo dimer (Left) and ligand-bound dimer (Right, ligand shown as spheres). In each case, one protomer is color-ramped N-terminus–C-terminus (blue-red), with the other protomer shown in green. (B) View of the Y_Y_Y domains looking up through the membrane to illustrate the movement of the C-termini on ligand binding. The Y_Y_Y domain of chain A of the apo (gray) and liganded (red) dimers is superimposed to show the relative movement of chain B. The Cα of residue Ile-779, the last residue for which secondary structure can be unambiguously defined, is represented as a blue sphere. (C) Overall shape envelope representing the solution structure of the apo (gray envelope) and liganded (yellow envelope) BT4663, respectively, giving the lowest χ-values (Table S3) and the fit to the experimental data represented in Fig. S5. The front view (Upper) and side view (Lower) of the dimer are shown.

Fig. 5.

Scissor model for TM signaling in HTCSs. Cartoon of proposed model for HTCS activation. Ligand binding drives a scissor-like closing movement of the C-termini of the Y_Y_Y domains in the dimer, leading to activation of the associated locus by either by bringing the attached cytoplasmic HK domains close enough together to allow autophosphorylation (A) or resulting in rearrangement of an existing kinase dimer in the cytoplasm to drive autophosphorylation (B). Chains A and B are colored blue and green, respectively. The red arrows indicate the direction of movement of the C-termini of the Y_Y_Y domains on ligand binding. Note that the attached RR domains of the HTCS are omitted for clarity.