How to switch off a histidine kinase: crystal structure of Geobacillus stearothermophilus KinB with the inhibitor Sda

Abstract

Entry to sporulation in bacilli is governed by a histidine kinase phosphorelay, a variation of the predominant signal transduction mechanism in prokaryotes. Sda directly inhibits sporulation histidine kinases in response to DNA damage and replication defects. We determined a 2.0-A-resolution X-ray crystal structure of the intact cytoplasmic catalytic core [comprising the dimerization and histidine phosphotransfer domain (DHp domain), connected to the ATP binding catalytic domain] of the Geobacillus stearothermophilus sporulation kinase KinB complexed with Sda. Structural and biochemical analyses reveal that Sda binds to the base of the DHp domain and prevents molecular transactions with the DHp domain to which it is bound by acting as a simple molecular barricade. Sda acts to sterically block communication between the catalytic domain and the DHp domain, which is required for autophosphorylation, as well as to sterically block communication between the response regulator Spo0F and the DHp domain, which is required for phosphotransfer and phosphatase activities.

Fig. 1. The Gst Sda/KinB-CC complex

A) Schematic diagram illustrating structural features of the Bacillus sporulation HK KinB. The colored domains (DHp and CA domains), comprising the cytoplasmic CC, have been crystallized in this study. The HK functions as a homodimer (one monomer is colored dark red, the other orange). Autophosphorylation occurs in trans, with the orange CA domain phosphorylating the phosphoacceptor-His residue (labelled 'H') of the red DHp domain. B) Sda inhibits spontaneous autophosphorylation of KinB-CC. Incubation of KinB-CC with γ-[³²P]ATP results in autophosphorylation, as monitored by SDS-PAGE and phosphorimagery (lane 1). Addition of increasing concentrations of Sda [1, 5, or 25-fold molar excess over KinB-CC); lanes 2–4, respectively] inhibits the autophosphorylation reaction. C) Structure of the Sda/KinB-CC complex. Ribbon diagrams, with each protein chain color coded: KinB-CC monomer A, dark red; KinB-CC monomer B, orange; Sda, blue. The KinB DHp and CA domains are labeled. ADP molecules bound to the CA domains are shown in stick format, with carbon atoms colored yellow. The associated Mg²⁺ ions are shown as grey spheres. The side chain of the phosphoacceptor His213 is also shown. Disordered segments near the N-terminus of the KinB monomers are shown as spheres.

Fig. 2. Unusual structural features of KinB-CC

A) Comparison of the CC's of KinB (orange/red/yellow) and TM0853 (green/cyan/blue) . The two structures, shown as backbone worms, were superimposed on overlapping helices of the DHp domains, which superimpose closely (1.12 Å rmsd over 43 α-carbon positions corresponding to KinB chain B residues 211–231 and 239–260). The superposition reveals the different paths of the DHp-CA linkers (KinB-CC, red; TM0853, cyan) and the misalignment of the CA domains (KinB-CC, yellow; TM0853, blue). B) Solvent MPD molecules (shown in CPK format with carbon atoms in green) bound in a vestibule at the top of the DHp helices (orange), and surrounded by the DHp-CA linkers (red) and the CA domains (transparent yellow). The MPD molecules hydrogen bond to the carbonyl oxygens of Tyr260, disrupting the second DHp α-helix. C) Disordered N-terminal arm of KinB-CC. Rather than forming an α-helix at the N-terminus (as in TM0853, see Fig. 2A), the N-terminal 17 residues of KinB-CC form a sixth β-strand at the end of the CA domain β-sheet, which is connected to the beginning of the first DHp α-helix by a disordered loop (residues 195–207, cyan spheres).

Fig. 3. Structural and functional features of the Sda/KinB interface

A) (top) The KinB binding surface of Sda, viewed from the KinB perspective. The KinB chain A DHp helices are shown as a dark red backbone worm. Sda is shown as a blue backbone worm, along with a transparent molecular surface. Side chains that contact KinB (≤ 4 Å) are shown and color-coded (carbon, light grey; nitrogen, blue; oxygen, red; sufur, orange), except the carbon atoms of three residues (Leu21, Phe25, and Leu28) shown to be important for Bsu KinA binding are colored yellow. (bottom) Sequence conservation among 16 Sda orthologs. The sequence at the top shows the consensus sequence, while the histogram above it denotes the level of sequence conservation at each position (red bar, 100% conserved; dark blue bar, less than 20%). The sequences are shown in one-letter amino acid code and identified by species at the left (annotation of the species codes shown is given in the Supplement). The numbers at the beginning of each line indicate amino acid positions relative to the start of each protein sequence. The numbers at the top (underneath the consensus) indicate the amino acid position in Gst Sda. Positions in the alignment that share >50% identity with the consensus are indicated by red shading, while positions that share >50% homology are indicated by blue shading. The α-helices in the Gst Sda structure are indicated above the Sda sequences as blue rectangles, connecting loops are indicated by a continuous blue line. Gst Sda positions that contact KinB (≤4 Å) are denoted by grey dots, with the three residues shown to be important for Sda binding to Bsu KinA denoted by yellow dots. B) (top) The Sda binding surface of KinB, viewed from the Sda perspective. Sda is shown as a blue backbone worm. KinB is shown as a backbone worm (chain A, dark red; chain B, orange), along with a transparent molecular surface. Side chains that contact Sda (≤4 Å) are shown and color-coded (carbon, light grey; nitrogen, blue; oxygen, red), except the carbon atoms of three residues (Gly224, Phe225, and Leu228) shown to be important for Sda binding (Fig. 4A) are colored yellow. (bottom) Sequence conservation within the DHp domains of Bacillus sporulation HKs (top set of sequences) and other two-component, homodimeric sensor HKs (bottom set of sequences). The sequence at the top shows the consensus sequence, while the histogram above it denotes the level of sequence conservation at each position for just the sporulation HKs (red bar, 100% conserved; dark blue bar, less than 20%), while the histogram at the bottom denotes sequence conservation among all the HKs. The sequences are shown in one-letter amino acid code and identified by species at the left (Sco, Streptomyces coelicolor). The numbers at the beginning of each line indicate amino acid positions relative to the start of each protein sequence. The numbers at the top (underneath the consensus) indicate the amino acid position in Gst KinB. Positions in the alignment that share >50% identity with the consensus are indicated by red shading, while positions that share >50% homology are indicated by blue shading. The α-helices in the Gst KinB structure are indicated below the Gst KinB sequence as orange rectangles, connecting loops are indicated by a continuous orange line. Gst KinB positions that contact Sda (≤ 4 Å) are denoted by grey dots, with the three residues shown to be important for KinB binding to Sda (Fig. 4A) denoted by yellow dots. The H-box, a sequence motif conserved among all the HKs, is denoted by the horizontal purple line above and below the top and bottom histograms, respectively. The Sda binding motif conserved only among the sporulation HKs is denoted by the horizontal yellow line above the sporulation HK histogram.

Fig. 4. An Sda binding motif on the HK

A) Effect of Ala substitutions in KinB on Sda inhibition. Single Ala substitutions were introduced at residues of KinB making extensive contacts with Sda. Autophosphorylation of wild-type KinB-CC (blue) and mutants was monitored in reactions containing increasing concentrations of Sda (0, 1, 5, and 25-fold molar excess over KinB-CC). The results are expressed as normalized histograms (autophosphorylation activity in the absence of Sda for each HK was used as reference), with error bars denoting the standard error from a minimum of three experiments for each histogram. KinBs harboring Q227A (red) or E231A (yellow) substitutions were essentially as sensitive to Sda inhibition as wild-type KinB, while G224A (purple), F225A (orange), and L228A (brown) were highly resistant (T220A was intermediate). B) Three-residue Sda binding motif. Autophosphorylation by the CC of the homodimeric HK TM0853 is insensitive to Sda (1, 5, and 25-fold molar excess Sda, lanes 1, 2, and 3, respectively), while the triple-mutant TM0853* (A271G/Y272F/T275L, introducing side-chains corresponding to KinB G224, F225, and L228) is inhibited very effectively.

Fig. 5

Sda inhibits DHp function only of the DHp domain to which it is bound. Mixed dimers of His₆-KinB-CC and KinB-CC, containing either wild-type (Sda-sensitive) or Sda^R monomers, were generated and purified. The His₆-KinB-CC had a slightly lower mobility by SDS-PAGE, allowing the autophosphorylation of each monomer to be monitored as a function of Sda concentration (0, 1, 5, and 25-fold molar excess, left to right). In the left panel, the His₆-KinB-CC (orange monomer in the schematic) is Sda^R due to an F225A substitution (illustrated in the schematic by the magenta 'X'), while the KinB-CC (dark red) is competent to bind Sda. Phosphorylation of KinB-CC is inhibited by Sda, while phosphorylation of His₆-KinB-CC is resistant. In the right panel, the His₆-KinB-CC is competent to bind Sda, while the KinB-CC harbors the Sda^R mutation. In this case, phosphorylation of His₆-KinB-CC is inhibited by Sda, while phosphorylation of KinB-CC is resistant.

Fig. 6. Mechanism of Sda inhibition of autophosphorylation and phosphotransfer

A) Mechanism of Sda inhibition of autophosphorylation. The KinB DHp domain is shown as a ribbon diagram, with the side chain of the phosphoacceptor His213. Sda is shown in stick format. Superimposed on the Sda/KinB-DHp structure is a KinB CA domain (shown in stick format but with the ADP and Mg²⁺ shown as CPK spheres) poised for the autophosphorylation reaction (modeled according to Marina et al. 14). Sda is colored blue, except atoms within 4 Å of the modeled CA domain are colored green. the KinB CA domain is colored orange, except atoms within 4 Å of Sda are colored magenta. The green and magenta atoms on Sda and KinB-CA, respectively, illustrate the extend of steric clash between the bound Sda and the modeled CA domain, indicating that phosphorylation of His213 by the CA domain would be sterically blocked by the bound Sda. B) Mechanism of Sda inhibition of phosphotransfer to Spo0F. Sda (blue) and the KinB DHp domain (dark red and orange) are shown as a ribbon diagram, with the side chain of His213. Superimposed on the Sda/KinB-DHp structure is Spo0F (yellow ribbon, with the side chain of the phosphoacceptor Asp54) poised for phosphotransfer (modeled based on the Spo0F/Spo0B crystal structure 26). Extensive steric overlap between Sda and Spo0F indicates that the phosphotransfer reaction would be sterically blocked by the bound Sda.

Fig. 7

Sda inhibits the rate of phosphotransfer from KinB-CC^P to Spo0F, as well as KinB phosphatase activity towards Spo0F^P. (upper left panel) Autoradiographs showing the time course of phosphotransfer from KinB-CC^P to Spo0F in the absence (top) and presence (bottom) of a 25-fold molar excess of Sda. These experiments were repeated and quantitated in triplicate, and the results plotted in the lower panel (data points). The plot on the left shows the full time course (up to 0.5 hr), while the plot on the right shows the same data but with an expanded time scale. The lines show the results of the best fit rate parameters (shown in the table) assuming the reaction scheme described in the text.