Auto-activation mechanism of the Mycobacterium tuberculosis PknB receptor Ser/Thr kinase

Abstract

Many Ser/Thr protein kinases are activated by autophosphorylation, but the mechanism of this process has not been defined. We determined the crystal structure of a mutant of the Ser/Thr kinase domain (KD) of the mycobacterial sensor kinase PknB in complex with an ATP competitive inhibitor and discovered features consistent with an activation complex. The complex formed an asymmetric dimer, with the G helix and the ordered activation loop of one KD in contact with the G helix of the other. The activation loop of this putative 'substrate' KD was disordered, with the ends positioned at the entrance to the partner KD active site. Single amino-acid substitutions in the G-helix interface reduced activation-loop phosphorylation, and multiple replacements abolished KD phosphorylation and kinase activation. Phosphorylation of an inactive mutant KD was reduced by G-helix substitutions in both active and inactive KDs, as predicted by the idea that the asymmetric dimer mimics a trans-autophosphorylation complex. These results support a model in which a structurally and functionally asymmetric, 'front-to-front' association mediates autophosphorylation of PknB and homologous kinases.

Figure 1

Complex of PknD surrogate KD with KT5720. (A) Autoradiograms showing MyBP phosphorylation catalysed by the KDs (100 nM) of PknD, PknB and the Met145Leu/Met155Val double mutant of PknB. In this double mutant ‘PknD surrogate', all the ATP contact residues except Tyr94 (bottom) matched those in PknD. Each set of three lanes shows the reactions without inhibitor and in the presence of 10 μM staurosporine and 10 μM KT5720, respectively. The double substitution to PknB allows KT5720 to bind at 10 μM. (B) Ribbon diagram showing the KT5720–PknD surrogate KD structure. Monomers A (yellow) and B (blue) form an asymmetric dimer stabilized by contacts of the G helices (highlighted in transparent surface) and the activation loop (red) of monomer B. (C) Superposition of the active site of the KT5720–PknD surrogate KD (blue) with wild-type PknB (grey). The Met145Leu and Met155Val substitutions derived from PknD create room in the active site for the inhibitor. (D) Superposition of the two independent PknD surrogate monomers. The two monomers adopt similar overall structures (0.43 Å r.m.s.d.). The activation loop (residues 161–179) of monomer A was disordered, but this segment (red surface) except for residues 174–177 could be placed in the electron density in monomer B. (E) View of G-helix interface. The dimer is not two-fold symmetric, putting identical residues in the monomers in distinct environments. Van der Waals contacts include Val222 (B, blue)-Val229 (A, yellow), Ala225 (A)-Val222 (B), Leu183 (B)-Val222 (A) (not shown). (F) Interactions in the G-helix interface. Several backbone interactions occur between the EF loop (monomer A) and the activation loop (monomer B). Arg161 and Asp219 form an ion pair, and Leu183 also contacts the opposing G helix. The only phosphorylated residue with clear density, pThr171 (B), is exposed and contacts Thr217 (A) in the EF loop. (G) Sequence alignment of 90 PknB orthologues mapped onto surface of PknB. Conserved regions (red >95% identical, blue least conserved) include the ATP site, the putative protein–substrate-binding site, the N-lobe interface (not shown) and the G helix.

Figure 2

Helix G substitutions reduced autophosphorylation of the PknB KD. (A) Autoradiogram and plot showing autophosphorylation activities of dephosphorylated PknB KD variants. The Val222Asp, Ala225Glu, Ala225Leu and Val229Asp substitutions (cyan) were made in the G helix. The acidic substitutions of residues 222, 225 and 229 individually reduced autophosphorylation 3.7-, 4.3- and 4.5-fold after 15 min, respectively (values are tabulated in Supplementary Figure 3). The Ala225Leu PknB KD showed 2.4-fold increased autophosphorylation activity at 15 min compared with the wild-type KD. The Leu33Asp substitution (orange) in the conserved regulatory N-lobe interface on the opposite side of the KD and the Val222Asp G-helix substitution showed synergistic effects. (B) Relative activities of the PknB 1–308 KD variant populations purified from E. coli. Activities were determined by measuring in vitro phosphorylation of MyBP. The relative transphosphorylation activities of each variant correlated well with the degree of intrinsic autophosphorylation (Table II) and the autophosphorylation activity observed in vitro.

Figure 3

Tandem mass spectrum (MS/MS) showing modifications of the 28-residue activation-loop peptide. (A) Mass spectrum showing ions formed by electrospray ionization. The inset shows detail for the (M+2H)²⁺ ions of the activation-loop fragment of PknB (residues 162–189) with two, three and four modifications. The asterisks denote ions that are 18 Da lower in mass, presumably due to losses of water molecules. (B) Quadrupole isolation mass spectrum of the (M+2H)²⁺ ion with three modifications. (C) MS/MS spectrum of the (M+2H)²⁺ ion with three modifications showing fragment ions formed by collisionally activated dissociation.

Figure 4

MS/MS spectra and cleavage maps of the (M+2H)²⁺ ions of the activation-loop fragment of PknB (residues 162–189) with (A) two, (B) three and (C) four modifications. Chemical modification increases the mass of each phospho-residue by 60 Da. The asterisks in the spectra denote residual precursor ion and the arrowheads in the cleavage maps denote modification sites (bold). The MS/MS spectrum of the ion with three modifications (B) indicates a mixture of isoforms with ∼80% having the third modification on Ser169 and ∼20% on Thr179.

Figure 5

The Val222Asp substitution causes minimal changes in the PknB KD structure. Leu33Asp PknB KD bound to ATPγS (light pink) superimposed on the Leu33Asp/Val222Asp PknB KD bound to ADP (dark blue). Spheres show the locations of Leu33Asp in the N-lobe and Val222 in the G-helix.

Figure 6

Intermolecular PknB autophosphorylation involves G-helix contacts. Autoradiograms showing intermolecular phosphorylation of inactive Asp138Asn PknB (1–279) KD substrates by active PknB (1–308) KD variants. Corresponding SDS–PAGE gels are shown as loading controls. (A) The Val229Asp mutation in helix G of both the active kinase (lanes 6–8) and inactive substrate KD (lanes 5 and 8) reduced trans-autophosphorylation of the Asp138Asn KD substrate. (B) The Leu33Asp mutation reduced intermolecular autophosphorylation (by 60%) when engineered into the active kinase domain. However, PknB (1–308) efficiently phosphorylated both the PknB (1–279) Asp138Asn KD substrate with and without the Leu33Asp mutation.

Figure 7

Model of the PknB intermolecular autophosphorylation complex. Active, wild-type PknB KD dimer (grey) superimposed on the KT5720–PknD surrogate complex (monomers A and B represented by yellow and blue, respectively). This ‘two-on-one' model is consistent with mutational effects and the structural organization of the N-lobe and G-helix (C-lobe) interfaces. The wild-type N-lobe dimer superimposes on each monomer in the asymmetric C-lobe dimer with an average r.m.s.d. of 1.59 Å with no overlapping contacts. For the putative substrate monomer (yellow), the disordered activation loop is modelled (grey surface). The ordered activation loop of the putative active monomer (blue) is highlighted in red. A KD monomer is phosphorylated efficiently, but the substrate KD may also be phosphorylated as a dimer. Because the monomeric KD is not directly activated by an extracellular cue and the phosphorylated monomer is active, phosphorylation can amplify the signal.