Crystal structures of C4-dicarboxylate ligand complexes with sensor domains of histidine kinases DcuS and DctB

Abstract

Two-component signaling systems allow bacteria to adapt to changing environments. Typically, a chemical or other stimulus is detected by the periplasmic sensor domain of a transmembrane histidine kinase sensor, which in turn relays a signal through a phosphotransfer cascade to the cognate cytoplasmic response regulator. Such systems lead ultimately to changes in gene expression or cell motility. Mechanisms of ligand binding and signal transduction through the cell membrane in histidine kinases are not fully understood. In an effort to further understand such processes, we have solved the crystal structures of the periplasmic sensor domains of Escherichia coli DcuS and of Vibrio cholerae DctB in complex with the respective cognate ligands, malate and succinate. Both proteins are involved in the regulation of the transport and metabolism of C(4)-dicarboxylates, but they are not highly related by sequence similarity. Our work reveals that despite disparate sizes, both structures contain a similar characteristic alpha/beta PDC (PhoQ-DcuS-CitA) sensor-domain fold and display similar modes of ligand binding, suggesting similar mechanisms of function.

FIGURE 1.

Structures of the malate-DcuS-(42-181) and succinate-DctB-(28-286) ligand complexes. A, overall structure of DcuS-(42-181) is shown as a ribbon diagram with secondary structure elements labeled in black. Bound malate is shown in ball-and-stick representation with carbon atoms in yellow and oxygen atoms in red. B, stereo plot of a C^α trace of DcuS-(42-181). Every 10th C^α atom is depicted as a black sphere and labeled accordingly. Malate was omitted for greater clarity. C, overall structure of DctB-(28-286) is shown as a ribbon diagram with secondary structure elements labeled in black. Bound succinate is shown in ball-and-stick representation with carbon atoms in yellow and oxygen atoms in red. Calcium ions are shown as magenta spheres. D, stereo plot of a C^α trace of DctB-(28-286). Every 10th C^α atom is depicted as a black sphere and labeled accordingly. Succinate was left out for greater clarity. The diagrams were created using MolScript (41) and BobScript (42).

FIGURE 2.

Structure-based sequenced alignments. A, structure-based sequence alignment between the two subdomains of the V. cholerae DctB periplasmic sensor domain. Secondary structure elements for the distal subdomain 1 (D1) and the proximal subdomain 2 (D2) are labeled above and below the sequences, respectively, and are colored olive (helices) and light blue (strands). Conserved residues are shown in red. The shaded regions represent aligned regions. B, structure-based sequence alignment of DcuS-(42-181) and DctB-(28-286) distal subdomain 1 (D1) to each other and to the sensor domains of CitA and PhoQ. Conserved residues are shown in red. The secondary structure elements of DcuS-(42-181) are labeled above the alignment, whereas that for the other structures are shown as shaded regions that overlay the sequences themselves. Secondary structure elements are colored olive (helices) and light blue (strands). Organism names are abbreviated in italics (Ec for Escherichia coli, Kp for Klebsiella pneumoniae, and Vc for Vibrio cholerae).

FIGURE 3.

Superimpositions of PDC sensor domains. PDC sensor domains are shown superimposed upon DcuS in stereo. Only the first N-terminal helix and all β-strands for each structure are shown in ribbon representation. All other segments are shown in worm representation. In each panel, DcuS is shown in yellow, and the superimposed PDC sensor is shown in blue. A, CitA is superimposed with DcuS, with an r.m.s.d. of 1.38Å over 117 C^α residues. B, PhoQ is superimposed with DcuS, with an r.m.s.d. of 1.50Å over 63 C^α residues. C, distal domain D1 of DctB is superimposed with DcuS, with an r.m.s.d. of 1.54Å over 115 C^α residues. D, proximal domain D2 of DctB is superimposed with DcuS, with an r.m.s.d. of 1.68Å over 67 C^α residues. The diagrams were created using MolScript (41).

FIGURE 4.

Stereodrawings of ligand-binding sites of DcuS and DctB. A, malate-binding site of DcuS. The protein backbone is shown in a ribbon representation, except that loop residues 139-144 are shown in full stick representation. An F_o - F_c omit map contoured at the 4σ contour level, colored purple, is shown around malate. Malate and its contacting protein side chain and main chain atoms are shown in stick representation and labeled accordingly. Hydrogen bonds are depicted as gray dots. Carbon backbone atoms of the ligand are colored yellow for greater clarity. All other atoms are colored by atom type: carbon, black; oxygen, red; nitrogen, blue. Water is depicted in turquoise. B, succinate-binding site of DctB. The protein backbone is shown in a ribbon representation, except that loop residues 147-154 are shown in full stick representation. An F_o - F_c omit map at the 4σ contour level, colored purple, is shown around succinate. Succinate and its contacting protein side chain and main chain atoms are shown labeled in stick representation, with hydrogen bonds depicted as gray dots. The same coloring scheme for atom types in A is used. The diagrams were created using MolScript (41).

FIGURE 5.

Ribbon diagrams of the DcuS-(42-181) crystallographic dimer. A, side view of the dimer showing relative orientations of the N and C termini. B, top view of the dimer showing packing between helices H1b and H3b of opposing protomers. Individual protomers are colored green and blue with malate shown in ball-and-stick representation. The figure was created using MolScript (41).

FIGURE 6.

Equilibrium centrifugation analysis of DcuS-(42-181) oligomerization. The black open circles represent absorbance (280 nm) data taken at 17,000 rpm, starting with a loading concentration of 45 mg/ml (2.8 mm). The smooth red curve through the circles represents the model for a monomer/dimer/tetramer reversible equilibrium. The distribution of residuals for a least squares fit to the model is shown above the plot. The yellow, green, and pink curves represent the absorbance calculated for the individual monomer, dimer, and tetramer species, respectively. The data can be fit with a K_d of dimerization of 9.7 ± 3.7 mm.