Molecular structure of EmbR, a response element of Ser/Thr kinase signaling in Mycobacterium tuberculosis

Abstract

Ser/Thr phosphorylation has emerged as a critical regulatory mechanism in a number of bacteria, including Mycobacterium tuberculosis. This problematic pathogen encodes 11 eukaryotic-like Ser/Thr kinases, yet few substrates or signaling targets have been characterized. Here, we report the structure of EmbR (2.0 A), a putative transcriptional regulator of key arabinosyltransferases (EmbC, -A, and -B), and an endogenous substrate of the Ser/Thr-kinase PknH. EmbR presents a unique domain architecture: the N-terminal winged-helix DNA-binding domain forms an extensive interface with the all-helical central bacterial transcriptional activation domain and is positioned adjacent to the regulatory C-terminal forkhead-associated (FHA) domain, which mediates binding to a Thr-phosphorylated site in PknH. The structure in complex with a phospho-peptide (1.9 A) reveals a conserved mode of phospho-threonine recognition by the FHA domain and evidence for specific recognition of the cognate kinase. The present structures suggest hypotheses as to how EmbR might propagate the phospho-relay signal from its cognate kinase, while serving as a template for the structurally uncharacterized Streptomyces antibiotic regulatory protein family of transcription factors.

Fig. 1.

Linear domain composition of SARP-family proteins. The SARP-family proteins are S. peucetius DnrI and S. coelicolor AfsR. DBD, DNA-binding domain.

Fig. 2.

Overall structure of EmbR and comparison of domains. (A) Ribbon diagram of phospho-peptide-bound EmbR. The DNA-binding domain is colored in blue (helices) and green (strands) and the BTA and FHA domains in yellow and red, respectively. Secondary structure elements are labeled according to the homologous domains in B–D. Trp residues are indicated as sticks in magenta, and Trp-63 is labeled. (B–D) Superposition of EmbR domains (colored as in A) with structural neighbors (shown in violet and determined by Dali) (24): DNA-binding (B), BTA (C), and FHA (D). The superimposed domains are the PhoB DNA-binding domain bound to pho box (1GXP) (21), the TPR domain of protein phosphatase pp5 (1A17) (23), and the FHA domain of Rad53p (1G6G) (28), respectively.

Fig. 3.

Surface potential and domain interfaces. (A) “Front” and “back” view of the electrostatic surface of EmbR in the peptide-bound form. (B) Stereo view of interdomain contacts formed by the T2 to -3 loop (purple) and helix T3 (yellow). Selected side chains mentioned in the text are shown and labeled by sequence number. Ribbon colors are as in Fig. 2A.

Fig. 4.

Phosphorylation and phosphopeptide binding of EmbR. (A) A 2D-gel electrophoresis of EmbR postphosphorylation by PknH. ³³P-labeled EmbR is visualized by autoradiography. (B) Intensity of Trp fluorescence emission at 330 nm as a function of concentration of the Rad53p-ligand (SLEVpTEADT). (Inset) Shift of the emission peak with increasing peptide concentrations.

Fig. 5.

Phosphopeptide complex. (A) View of the peptide bound to chain A (shown with carbon atoms in yellow). The σ_A-weighted 2Fo–Fc (1.9 Å, 0.8 σ, black) and Fo–Fc maps (2.5 σ, green) were calculated with model phases before building the peptide. Peptide residues (orange) are labeled relative to the pT position. (B and C) View of the peptide termini, illustrating the putative specificity-determining regions. Shown are the ligands for chains A (B) and B (C). The σ_A-weighted 2Fo–Fc map (0.8 σ) is contoured before (black) and after (cyan), including the peptide atoms in the phase calculation. Selected side chains of apo EmbR are shown in magenta.