Bacillus subtilis RapA phosphatase domain interaction with its substrate, phosphorylated Spo0F, and its inhibitor, the PhrA peptide

Abstract

Rap proteins in Bacillus subtilis regulate the phosphorylation level or the DNA-binding activity of response regulators such as Spo0F, involved in sporulation initiation, or ComA, regulating competence development. Rap proteins can be inhibited by specific peptides generated by the export-import processing pathway of the Phr proteins. Rap proteins have a modular organization comprising an amino-terminal alpha-helical domain connected to a domain formed by six tetratricopeptide repeats (TPR). In this study, the molecular basis for the specificity of the RapA phosphatase for its substrate, phosphorylated Spo0F (Spo0F∼P), and its inhibitor pentapeptide, PhrA, was analyzed in part by generating chimeric proteins with RapC, which targets the DNA-binding domain of ComA, rather than Spo0F∼P, and is inhibited by the PhrC pentapeptide. In vivo analysis of sporulation efficiency or competence-induced gene expression, as well as in vitro biochemical assays, allowed the identification of the amino-terminal 60 amino acids as sufficient to determine Rap specificity for its substrate and the central TPR3 to TPR5 (TPR3-5) repeats as providing binding specificity toward the Phr peptide inhibitor. The results allowed the prediction and testing of key residues in RapA that are essential for PhrA binding and specificity, thus demonstrating how the widespread structural fold of the TPR is highly versatile, using a common interaction mechanism for a variety of functions in eukaryotic and prokaryotic organisms.

Fig 1

Kinetic analysis of RapA dephosphorylation of Spo0F∼P and inhibition by PhrA. (A and B) The rates of dephosphorylation were obtained at 10 concentrations of Spo0F∼P (0.33, 0.5, 1, 1.33, 2, 2.5, 3.33, 5, 7.5, and 10 μM). Seven time points were taken for each substrate concentration, and the remaining Spo0F∼P was measured by exposing the gels to a PhosphorImager screen and analyzing the data with the ImageQuant software program. The percentage of remaining Spo0F∼P was plotted versus time, and the slope of each reaction (calculated as shown in Materials and Methods) multiplied by the substrate concentration gave the rate. The rate at each substrate concentration was plotted as a Michaelis-Menten (A) or Lineweaver-Burk (B) graph. (C and D) Time points of Spo0F∼P desphosphorylation by RapA were collected in the presence of four concentrations of the PhrA inhibitor and six concentrations of the substrate. The rates for each reaction were calculated as described above and in Materials and Methods. The best-fit analysis was carried out with the SigmaPlot software program, and the Michaelis-Menten (C) and the Lineweaver-Burk (D) graphs of the inhibition equations that best fit the data are shown. The remaining graphs of the curve fit analysis are shown in Fig. S1 in the supplemental material.

Fig 2

Amino acid sequence alignment of RapA and RapC. The alignment was obtained with the ClustalW program. Asterisks indicate identical residues; colons and periods indicate conserved and semiconserved substitutions, respectively. The six TPR domains as defined by amino acid sequence conservation are in the gray boxes (31). The extent of the two α-helices that include each TPR domain, as determined by the crystal structure of RapH, is indicated by the green line (α1) or the yellow line (α2) (27). The position of the RapAC fusions is indicated by the red connectors, while the position of the RapCA fusion is shown by the blue connector. The residues corresponding to the regions in RapH that form four α-helices in the N terminus and the two α-helices in the connecting region between TPR5 and TPR6, identified by the crystal structure of the RapH-Spo0F complex, are shown by the black lines (27). The six residues affected in PhrA binding are shown in red (D192, Y224, N225, N228, H260, and P259).

Fig 3

Effect of the multicopy plasmids expressing the RapAC1 or RapCA1 hybrid protein on rapC transcription. Strain JH19278 (spo0A abrB rapC amyE::rapC-lacZ) was transformed with the multicopy plasmids pBS19 (●), pBS19-RapAC1 (▲), and pBS19-RapCA1 (■). Cells were grown in Schaeffer's sporulation medium, and samples were taken at hourly intervals before and after the transition (T₀) from the vegetative to the sporulation phase.

Fig 4

Interaction of the RapAC1 and RapCA1 hybrid proteins with the Spo0F and ComA response regulators. The 10% Tris-Tricine-EDTA native gel assay was carried out as described in Materials and Methods. Each protein was used at a 10 μM final concentration.

Fig 5

Time courses of Spo0F∼P dephosphorylation by RapA wt or RapAC1 and inhibition by Phr peptides. Purified Spo0F∼P (0.5 μM) was incubated alone or in the presence of RapA wt (A and B) or RapAC1 (C and D) (0.5 μM). The PhrA (A and C) or PhrC (B and D) peptides were added at a 1 μM final concentration. Samples were analyzed by 15% SDS-PAGE and quantitated by the ImageQuant software program after exposure to a PhosphorImager screen. Symbols: Spo0F∼P alone, ●; Spo0F∼P and Rap protein, ▲; Spo0F∼P, Rap protein, and Phr peptide, ■.

Fig 6

Activities of the RapAC2 and RapAC3 hybrid proteins. Time courses of dephosphorylation of Spo0F∼P by RapAC2 and RapAC3 were carried out in vitro as described in Materials and Methods. Proteins were used at a 1.5 μM concentration. The RapAC2 hybrid protein was also tested in the presence of PhrC (3 μM). The samples were run on a 15% SDS-PAGE gel, exposed to a PhosphorImager screen, and quantified by the ImageQuant software program. Symbols: ●, Spo0F∼P alone; ■, Spo0F∼P and RapA wt; ▼, Spo0F∼P and RapAC2; ◆, Spo0F∼P and RapAC3; ▲, Spo0F∼P, RapAC2, and PhrC.

Fig 7

Time course of Spo0F∼P dephosphorylation by RapAC4 (A) or RapAC5 (B) and inhibition by PhrA or PhrC. Spo0F∼P (2.5 μM) was incubated in the absence (●) or presence (▲) of the RapAC proteins (2.5 μM) and the PhrA (■) or PhrC (◆) peptide at a 5 μM final concentration. Samples were analyzed by 15% SDS-PAGE and quantitated by the Image Quant software program after exposure to a PhosphorImager screen.

Fig 8

Time courses of Spo0F∼P dephosphorylation by RapA wt or alanine mutant proteins and inhibition by Phr peptides. Purified Spo0F∼P (0.5 μM) was incubated alone or in the presence of RapA wt or RapA Y224A (A), RapA N225A (B), RapA N228A (C), or RapA H260A (D). The PhrA peptide was added at a 1 μM final concentration. Samples were analyzed by 15% SDS-PAGE and quantitated by the ImageQuant software program after exposure to a PhosphorImager screen. Symbols: no RapA, ●; RapAwt, ▲; RapA wt plus PhrA, ▵-; RapA mutant, ■-; RapA mutant plus PhrA, □-.

Fig 9

Alanine scanning mutagenesis reveals the PhrA peptide binding pocket on RapA. (A) Shown is the structure of B. subtilis RapA (khaki), homology modeled on B. subtilis RapH in complex with Spo0F (gray). RapA alanine mutants unable to bind and respond to PhrA are in red, demonstrating that the PhrA peptide binding site is distal from the Spo0F binding site and on the concave face of the TPR domain. (B, C, and D Stereo view overlay (B) or side-by-side view (C and D) of TPR2-5 of RapA (khaki) with the equivalent of PlcR (blue); compare the position of alanine mutants unable to bind PhrA peptides (red) to the position of the PapR peptide ligand (magenta). Sites on PlcR that correspond to the RapA alanine mutants are in green. Hydrogen bonds between the peptide and the displayed PlcR residues are in yellow. Amino- and carboxy-terminal ends of the PapR peptide are as labeled.

Fig 10

The N-terminal helices of Rap phosphatases are structurally reminiscent of the four-helix bundle of the Spo0B protein. Previous alanine scanning mutagenesis of the entire surface exposed residues of Spo0F revealed an extensive overlap of mutants unable to interact with either Spo0B or the Rap phosphatases (42). Overlaying the Spo0F molecule of the Spo0F-Spo0B complex (blue) (43) and that of the Spo0F-RapH complex (yellow) (27) shows that the N-terminal helices α1, α2, and α3 of the Rap phosphatases (orange) structurally mimic the homodimeric four-helix bundle of Spo0B (turquoise), which forms extensive interactions with helix α1 of Spo0F. Helix α5, the first helix of the first TPR domain of the Rap phosphatase, completes the four-helix bundle-like structure; however, it is slanted by 40° in respect to helix α2 in Spo0B. Phosphorylation sites on Spo0B (H30) and on Spo0F (D54) and the catalytic residue on RapH (Q47) are displayed.