Research ArticleStructural Biology

Structural insights into the functional versatility of an FHA domain protein in mycobacterial signaling

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Science Signaling  07 May 2019:
Vol. 12, Issue 580, eaav9504
DOI: 10.1126/scisignal.aav9504
  • Fig. 1 GarA regulates glutamate metabolism in Actinobacteria.

    (A) GarA consists of a C-terminal FHA domain and an N-terminal peptide extension that contains two adjacent phosphorylatable threonine residues: Thr21 and Thr22 in M. tuberculosis GarA (18, 20) [equivalent to Thr14 and Thr15 in the C. glutamicum homolog OdhI (17)]. PknG phosphorylates the first Thr in each protein, whereas PknB targets the second Thr residue in each protein. (B) Mycobacterial signaling pathway involving GarA, which functions as a phospho-dependent (ON/OFF) molecular switch (26). In the ON state, nonphosphorylated GarA represses or stimulates the activity of three distinct metabolic enzymes and redirects the metabolic flux (thick gray lines) toward the synthesis of glutamate. GarA binding to its downstream targets does not depend on a pThr residue in the target, which is rare among FHA domains. Phosphorylation of GarA by PknG or PknB triggers an intramolecular interaction, resulting in a closed (autoinhibited) conformation that blocks the pThr-binding site of the FHA domain and switches off the regulatory properties of GarA. Dephosphorylation of GarA by PstP, the conserved transmembrane protein serine-threonine phosphatase in Actinobacteria, is assumed based on PstP-mediated dephosphorylation of the GarA homolog OdhI in C. glutamicum (63). OG, 2-oxoglutarate; Glu, glutamate; Gln, glutamine; SucCoA, succinyl–coenzyme A.

  • Fig. 2 Structure of the ternary GarA-PknB-nucleotide complex.

    (A) Overall view of the GarA-PknBCD,L33E complex. The complex was crystallized in the presence of the non-hydrolyzable ATP analog AMP-PCP. GarA is shown in yellow, the PknB kinase activation loop is shown in violet, and the two PknB helices involved in the interface are shown in green. The non-hydrolyzable ATP analog and the residues involved in Thr-phosphate binding are depicted as sticks. (B) The N-terminal GarA peptide (residues 26 to 34) mediates the interaction between a patch of basic residues on the FHA domain surface (Arg62, Arg143) and the αG helix of PknB. (C) Close-up view of the pThr171-binding site, showing all phosphate oxygens involved in hydrogen-bonding interactions. (D) Structural differences in the PknB helix αC between the PknB-GarA complex (green) and the back-to-back PknB homodimer (blue, PDB code 1O6Y). (E) Kinase activity of PknBCD and PknBCD,L33A on full-length GarA (orange) and on the 17-residue N-terminal peptide substrate (green). (F) Kinase activity of PknBCD and PknBCD,L33A on the peptide substrate in the absence (light green) and presence (dark green) of the GarA FHA domain. In (E) and (F), values are the mean of three independent measurements (n = 3), and error bars represent the SEM. Significance of differences between the mean values (E and F) was analyzed statistically (*P < 0.05, Student’s t test).

  • Fig. 3 Structure of the GarA FHA domain (GarAΔ44)–E1o (KGDΔ360) complex.

    (A) Crystal structure of the GarA FHA domain (GarAΔ44) bound to the E1o domain of KGD (KGDΔ360). Two GarA FHA domain molecules (yellow) bind the outer helices, 475–500 and 785–813 (purple), of the E1o homodimer (magenta). The cofactor thiamine diphosphate (ThDP) at the E1o active site is shown in sphere representation (orange). (B) Overall view of the protein-protein interface, showing the molecular surface of KGD color-coded according to electrostatic charge, and GarA FHA domain in ribbon representation. The electrostatic surface is represented color-coded from acidic (red) to basic (blue). All protein residues involved in intermolecular hydrogen bonds are labeled (black label for GarA residues and white labels for surface KGD residues). (C) Close-up view of the pThr-binding pocket showing atomic interactions of the phosphomimetic residue Asp795 in KGD. Color-coding as in (A). (D) Hydrogen bonds between GarA FHA domain Arg62 and Arg143 (yellow) and main-chain carbonyl groups from the KGD loop 586–594 (in violet, side chains are omitted for clarity). (E) Catalytic activity of wild-type KGDΔ360 (red squares) and three interface mutants: D795A (black open circles), S484R/A488Q (black filled circles), and R802A (green circles) in the presence of the GarA FHA domain. Values are the mean of three independent measurements (n = 3); SEM values (not shown) are in the range from 0.1 to 0.5.

  • Fig. 4 Dual binding specificity of the GarA FHA domain.

    The same region of the GarA FHA domain (colored molecular surface) mediates both phosphorylation-dependent and phosphorylation-independent interactions with binding partners. The structures shown are those of (A) the closed conformation of autoinhibited GarA, (B) the GarA-PknBCD,L33A complex, and (C) the GarAΔ44-KGDΔ360 complex. The FHA domain is shown in a similar orientation in each complex.

  • Table 1 Binding affinities of KGDΔ360 and PknBCD for GarA point mutants.

    The binding affinities of M. tuberculosis PknB catalytic domain (MtPknBCD) and M. smegmatis KGD (Ms KGDΔ360) for different MsGarA point mutants were determined by SPR. Values represent KD ± SE from a representative experiment (fig. S8) of three or more independent experiments. n.d., not determined (no reliable fit).

    GarAKGD KD (μM)PknBCD KD (μM)
    Wild type1.92 ± 0.1512.2 ± 1.0
    R62An.d.14.7 ± 1.5
    S95A1.89 ± 0.11n.d.
    K141En.d.10.8 ± 0.8
    R143An.d.12.4 ± 1.1

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/580/eaav9504/DC1

    Fig. S1. Alignment of GarA homologs in selected Actinobacteria.

    Fig. S2. Continuous sedimentation coefficient distribution analysis of the PknBCD-GarA complex.

    Fig. S3. ITC characterization of the interaction between autophosphorylated PknBCD and GarA.

    Fig. S4. The N-terminal GarA extension occupies a similar position in different GarA structures.

    Fig. S5. Conserved mode of phosphopeptide recognition in different M. tuberculosis FHA domains.

    Fig. S6. Detailed structure of the PknB activation loop bound to GarA.

    Fig. S7. Formation of the enamine-ThDP covalent adduct in the presence of GarA.

    Fig. S8. SPR studies of protein-protein interactions.

    Table S1. Data collection and refinement statistics.

    References (64, 65)

  • This PDF file includes:

    • Fig. S1. Alignment of GarA homologs in selected Actinobacteria.
    • Fig. S2. Continuous sedimentation coefficient distribution analysis of the PknBCD-GarA complex.
    • Fig. S3. ITC characterization of the interaction between autophosphorylated PknBCD and GarA.
    • Fig. S4. The N-terminal GarA extension occupies a similar position in different GarA structures.
    • Fig. S5. Conserved mode of phosphopeptide recognition in different M. tuberculosis FHA domains.
    • Fig. S6. Detailed structure of the PknB activation loop bound to GarA.
    • Fig. S7. Formation of the enamine-ThDP covalent adduct in the presence of GarA.
    • Fig. S8. SPR studies of protein-protein interactions.
    • Table S1. Data collection and refinement statistics.
    • References (64, 65)

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