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Oxidative Unfolding of the Rubredoxin Domain and the Natively Disordered N-terminal Region Regulate the Catalytic Activity of Mycobacterium tuberculosis Protein Kinase G*

  • Matthias Wittwer
    Affiliations
    From the Biomolecular NMR Spectroscopy and
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  • Qi Luo
    Footnotes
    Affiliations
    Computational Biocatalysis, Department of Chemistry, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany,

    the Soft Matter Research Center and Department of Chemistry, Zhejiang University, Hangzhou 310027, China, and
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  • Ville R.I. Kaila
    Affiliations
    Computational Biocatalysis, Department of Chemistry, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany,
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  • Sonja A. Dames
    Correspondence
    Supported by the Technische Universität München (TUM) Diversity and Talent Management Office and the Helmholtz portfolio theme “Metabolic Dysfunction and Common Disease” of the Helmholtz Zentrum München. To whom correspondence should be addressed: E-mail: .
    Affiliations
    From the Biomolecular NMR Spectroscopy and

    the Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany
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  • Author Footnotes
    * This work was supported by grants from the German Research Foundation (Deutsche Forschungsgemeinschaft) (SFB1035, Project B04 and SFB1035, Project B12) (to S. A. D. and V. R. I. K., respectively). Computer resources were provided in part by the Gauss Centre for Supercomputing/Leibniz Supercomputing Centre (Grant pr84pa to V. R. I. K.). The authors declare that they have no conflicts of interest with the contents of this article.
    This article contains supplemental Figures S1–S12, Results, and References.
    1 Supported by the China Scholarship Council.
Open AccessPublished:November 03, 2016DOI:https://doi.org/10.1074/jbc.M116.747089
      Mycobacterium tuberculosis escapes killing in human macrophages by secreting protein kinase G (PknG). PknG intercepts host signaling to prevent fusion of the phagosome engulfing the mycobacteria with the lysosome and, thus, their degradation. The N-terminal NORS (no regulatory secondary structure) region of PknG (approximately residues 1–75) has been shown to play a role in PknG regulation by (auto)phosphorylation, whereas the following rubredoxin-like metal-binding motif (RD, residues ∼74–147) has been shown to interact tightly with the subsequent catalytic domain (approximately residues 148–420) to mediate its redox regulation. Deletions or mutations in NORS or the redox-sensitive RD significantly decrease PknG survival function. Based on combined NMR spectroscopy, in vitro kinase assay, and molecular dynamics simulation data, we provide novel insights into the regulatory roles of the N-terminal regions. The NORS region is indeed natively disordered and rather dynamic. Consistent with most earlier data, autophosphorylation occurs in our assays only when the NORS region is present and, thus, in the NORS region. Phosphorylation of it results only in local conformational changes and does not induce interactions with the subsequent RD. Although the reduced, metal-bound RD makes tight interactions with the following catalytic domain in the published crystal structures, it can also fold in its absence. Our data further suggest that oxidation-induced unfolding of the RD regulates substrate access to the catalytic domain and, thereby, PknG function under different redox conditions, e.g. when exposed to increased levels of reactive oxidative species in host macrophages.

      Introduction

      Mycobacterium tuberculosis (Mtb),
      The abbreviations used are: Mtb, Mycobacterium tuberculosis; MWCO, molecular weight cut-off; PknG, protein kinase G; NORS, no regulatory secondary structure; RD, rubredoxin-like domain; TPRD, tetratricopeptide repeat domain; ROS, reactive oxidative species; MD, molecular dynamics; IDP, intrinsically disordered protein; RDC, residual dipolar coupling; SASA, solvent-accessible surface area; FHA, forkhead-associated; ADP, adenosine 5⁁-diphosphate; ATP-⁁S, adenosine 5⁁-[⁁-thio] triphosphate; HSQC, heteronuclear single quantum coherence.
      the causative agent of tuberculosis, has evolved different mechanisms to monitor redox signals. The capability of Mtb to sense redox stress and to maintain redox homeostasis is important for the survival of the pathogen in the human host (
      • Bhat S.A.
      • Singh N.
      • Trivedi A.
      • Kansal P.
      • Gupta P.
      • Kumar A.
      The mechanism of redox sensing in Mycobacterium tuberculosis.
      ,
      • Chim N.
      • Johnson P.M.
      • Goulding C.W.
      Insights into redox sensing metalloproteins in Mycobacterium tuberculosis.
      • Kumar A.
      • Farhana A.
      • Guidry L.
      • Saini V.
      • Hondalus M.
      • Steyn A.J.
      Redox homeostasis in mycobacteria: the key to tuberculosis control?.
      ). Recent studies have shown that redox stress responses of mycobacteria are linked to the phosphorylation of several proteins by eukaryote-like serine/threonine kinases (
      • Sureka K.
      • Hossain T.
      • Mukherjee P.
      • Chatterjee P.
      • Datta P.
      • Kundu M.
      • Basu J.
      Novel role of phosphorylation-dependent interaction between FtsZ and FipA in mycobacterial cell division.
      ,
      • Park S.T.
      • Kang C.M.
      • Husson R.N.
      Regulation of the SigH stress response regulon by an essential protein kinase in Mycobacterium tuberculosis.
      ). Of the 11 eukaryote-like serine/threonine kinases encoded by the Mtb genome (
      • Av-Gay Y.
      • Everett M.
      The eukaryotic-like Ser/Thr protein kinases of Mycobacterium tuberculosis.
      ), protein kinase G (PknG) and E (PknE) harbor specific redox-sensitive motifs (
      • Jayakumar D.
      • Jacobs Jr, W.R.
      • Narayanan S.
      Protein kinase E of Mycobacterium tuberculosis has a role in the nitric oxide stress response and apoptosis in a human macrophage model of infection.
      ,
      • Scherr N.
      • Honnappa S.
      • Kunz G.
      • Mueller P.
      • Jayachandran R.
      • Winkler F.
      • Pieters J.
      • Steinmetz M.O.
      Structural basis for the specific inhibition of protein kinase G, a virulence factor of Mycobacterium tuberculosis.
      ). The soluble PknG has been proposed to promote cellular survival of mycobacteria in host macrophages by blocking their lysosomal delivery and, thus, degradation (
      • Walburger A.
      • Koul A.
      • Ferrari G.
      • Nguyen L.
      • Prescianotto-Baschong C.
      • Huygen K.
      • Klebl B.
      • Thompson C.
      • Bacher G.
      • Pieters J.
      Protein kinase G from pathogenic mycobacteria promotes survival within macrophages.
      ). Moreover, because PknG is secreted into the cytosol of host macrophages, it is a promising drug target (
      • Walburger A.
      • Koul A.
      • Ferrari G.
      • Nguyen L.
      • Prescianotto-Baschong C.
      • Huygen K.
      • Klebl B.
      • Thompson C.
      • Bacher G.
      • Pieters J.
      Protein kinase G from pathogenic mycobacteria promotes survival within macrophages.
      ). PknG is a multidomain protein consisting of four functional regions (Fig. 1A). The N-terminal ∼75 residues of the no regulatory secondary structure (NORS) region have been suggested to be intrinsically disordered and to harbor a major in vivo phosphorylation site at Thr63 (
      • Scherr N.
      • Honnappa S.
      • Kunz G.
      • Mueller P.
      • Jayachandran R.
      • Winkler F.
      • Pieters J.
      • Steinmetz M.O.
      Structural basis for the specific inhibition of protein kinase G, a virulence factor of Mycobacterium tuberculosis.
      ,
      • Scherr N.
      • Müller P.
      • Perisa D.
      • Combaluzier B.
      • Jenö P.
      • Pieters J.
      Survival of pathogenic mycobacteria in macrophages is mediated through autophosphorylation of protein kinase G.
      ,
      • Tiwari D.
      • Singh R.K.
      • Goswami K.
      • Verma S.K.
      • Prakash B.
      • Nandicoori V.K.
      Key residues in Mycobacterium tuberculosis protein kinase G play a role in regulating kinase activity and survival in the host.
      ). Based on the crystal structure of an N-terminal truncated variant (PknG74–750) in complex with a newly detected inhibitor (AX20017, Fig. 1B), the following redox-sensitive rubredoxin-like metal binding domain (RD) makes tight interactions with the catalytic domain (
      • Scherr N.
      • Honnappa S.
      • Kunz G.
      • Mueller P.
      • Jayachandran R.
      • Winkler F.
      • Pieters J.
      • Steinmetz M.O.
      Structural basis for the specific inhibition of protein kinase G, a virulence factor of Mycobacterium tuberculosis.
      ). C-terminally, the kinase domain is flanked by a tetratricopeptide repeat domain (TPRD), a structural motif typically involved in protein-protein interactions (
      • Scherr N.
      • Honnappa S.
      • Kunz G.
      • Mueller P.
      • Jayachandran R.
      • Winkler F.
      • Pieters J.
      • Steinmetz M.O.
      Structural basis for the specific inhibition of protein kinase G, a virulence factor of Mycobacterium tuberculosis.
      ).
      Figure thumbnail gr1
      FIGURE 1.The NORS region of PknG shows local structural order, and the RD can also fold in the absence of the catalytic domain. A, schematic of the domain structure of PknG. PknG consists of the N-terminal NORS region (blue), the subsequent RD (red), the catalytic KD (yellow), and the TPRD (green). B, ribbon representation of the crystal structure of PknG74–750 (RD-KD-TPRD) in complex with the inhibitor AX20017 (stick representation in green, PDB code 2PZI) (
      • Scherr N.
      • Honnappa S.
      • Kunz G.
      • Mueller P.
      • Jayachandran R.
      • Winkler F.
      • Pieters J.
      • Steinmetz M.O.
      Structural basis for the specific inhibition of protein kinase G, a virulence factor of Mycobacterium tuberculosis.
      ). The domain color coding is the same as in A. C and D, analysis of the secondary structure content and backbone dynamics of the N-terminal regions of PknG by NMR spectroscopy. C, 13Cα secondary shifts for His-PknG1–75 (NORS, blue) and PknG74–147 (RD - reduced, metal-bound, red) plotted as function of the amino acid sequence (
      • Wishart D.S.
      • Sykes B.D.
      The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data.
      ). Chemical shift values significantly higher than the random coil value indicate the presence of α-helical and those significantly lower of β-sheet secondary structure. The secondary structure elements for the RD presented above the sequence were extracted from the crystal structure of PknG74–750 in complex with AX20017 (PDB code 2PZI, see B) (
      • Scherr N.
      • Honnappa S.
      • Kunz G.
      • Mueller P.
      • Jayachandran R.
      • Winkler F.
      • Pieters J.
      • Steinmetz M.O.
      Structural basis for the specific inhibition of protein kinase G, a virulence factor of Mycobacterium tuberculosis.
      ). show the 3JHNHα coupling constants and 1Hα secondary shifts for both isolated regions, which are both also sensitive to the secondary structure content, and shows the 13Cα secondary shifts for His-PknG1–147. D, {1H}-15N NOE data for His-PknG1–75 (NORS, red), PknG 74–147 (RD, reduced, metal-bound, blue), and His-PknG1–147 (NORS-RD, reduced, metal-bound, black) plotted as a function of the residue sequence position. Negative to slightly positive values indicate flexible, unstructured regions, whereas positive values around 0.4–0.8 indicate well structured regions. The respective 15N-T1 as well as 15N-T2 values are shown in . E, comparison of the structures of the isolated metal-bound rubredoxin-like domain in solution and in the crystal structures of PknG fragments containing, additionally, the kinase domain. The experimental residual dipolar couplings (RDCs) for the Zn2+-bound RD (black symbols) and those back-calculated based on the three published crystal structures of PknG using the software PALES (
      • Zweckstetter M.
      NMR: prediction of molecular alignment from structure using the PALES software.
      ) were plotted as a function of the residue sequence position. The back-calculated RDCs based on the Cd2+-bound RD in PknG74–750 in complex with the inhibitor AX20017 (PDB code 2PZI) (
      • Scherr N.
      • Honnappa S.
      • Kunz G.
      • Mueller P.
      • Jayachandran R.
      • Winkler F.
      • Pieters J.
      • Steinmetz M.O.
      Structural basis for the specific inhibition of protein kinase G, a virulence factor of Mycobacterium tuberculosis.
      ) are represented as green symbols, those of the Zn2+-bound RD of PknG74–405 in complex with an ATP analogue (PDB code 4Y12) (
      • Lisa M.N.
      • Gil M.
      • André-Leroux G.
      • Barilone N.
      • Durán R.
      • Biondi R.M.
      • Alzari P.M.
      Molecular basis of the activity and the regulation of the eukaryotic-like S/T protein kinase PknG from Mycobacterium tuberculosis.
      ) as red symbols, and those in complex with ADP (PDB code 4Y0X) (
      • Lisa M.N.
      • Gil M.
      • André-Leroux G.
      • Barilone N.
      • Durán R.
      • Biondi R.M.
      • Alzari P.M.
      Molecular basis of the activity and the regulation of the eukaryotic-like S/T protein kinase PknG from Mycobacterium tuberculosis.
      ) as blue symbols. A comparison of the experimental 13Cα secondary shift chemical shift values for the metal-bound RD with those back-calculated based on the published crystal structure data can be found in . Plots of the experimental versus calculated RDC values for each crystal structure and a larger representation of the RD structure as well as ribbon representations of the RD-KD part of each crystal structure are shown in . A superposition of the 1H-15N HSQC spectra of PknG74–147 and His-PknG74–420 in complex with ATP is shown in .
      PknG can autophosphorylate itself in trans (
      • Scherr N.
      • Müller P.
      • Perisa D.
      • Combaluzier B.
      • Jenö P.
      • Pieters J.
      Survival of pathogenic mycobacteria in macrophages is mediated through autophosphorylation of protein kinase G.
      ,
      • Tiwari D.
      • Singh R.K.
      • Goswami K.
      • Verma S.K.
      • Prakash B.
      • Nandicoori V.K.
      Key residues in Mycobacterium tuberculosis protein kinase G play a role in regulating kinase activity and survival in the host.
      ). However, in contrast to other (mycobacterial) kinases, the autophosphorylation does not affect the kinase activity but is required for the survival of pathogenic mycobacteria within host macrophages (
      • Scherr N.
      • Müller P.
      • Perisa D.
      • Combaluzier B.
      • Jenö P.
      • Pieters J.
      Survival of pathogenic mycobacteria in macrophages is mediated through autophosphorylation of protein kinase G.
      ). In rubredoxins, an iron atom is typically tetrahedrally coordinated by four cysteine residues (
      • Sieker L.C.
      • Stenkamp R.E.
      • LeGall J.
      Rubredoxin in crystalline state.
      ), but other metal ions, such as cobalt, nickel, and zinc, can replace the iron (
      • Dauter Z.
      • Wilson K.S.
      • Sieker L.C.
      • Moulis J.M.
      • Meyer J.
      Zinc- and iron-rubredoxins from Clostridium pasteurianum at atomic resolution: a high-precision model of a ZnS4 coordination unit in a protein.
      ). The redox-sensitive RD of PknG contains two CXXCG motifs (Fig. 1A) that can, in vitro, coordinate a divalent metal ion, such as zinc, iron, or cadmium, in the reduced state (
      • Scherr N.
      • Honnappa S.
      • Kunz G.
      • Mueller P.
      • Jayachandran R.
      • Winkler F.
      • Pieters J.
      • Steinmetz M.O.
      Structural basis for the specific inhibition of protein kinase G, a virulence factor of Mycobacterium tuberculosis.
      ,
      • Lisa M.N.
      • Gil M.
      • André-Leroux G.
      • Barilone N.
      • Durán R.
      • Biondi R.M.
      • Alzari P.M.
      Molecular basis of the activity and the regulation of the eukaryotic-like S/T protein kinase PknG from Mycobacterium tuberculosis.
      ,
      • Wittwer M.
      • Dames S.A.
      Expression and purification of the natively disordered and redox sensitive metal binding regions of Mycobacterium tuberculosis protein kinase G.
      • Gil M.
      • Graña M.
      • Schopfer F.J.
      • Wagner T.
      • Denicola A.
      • Freeman B.A.
      • Alzari P.M.
      • Batthyány C.
      • Durán R.
      Inhibition of Mycobacterium tuberculosis PknG by non-catalytic rubredoxin domain specific modification: reaction of an electrophilic nitro-fatty acid with the Fe-S center.
      ). However, it is currently unknown which metal ion is coordinated under in vivo conditions. Three crystal structures of PknG have been published, one of PknG74–750 (RD-KD-TPRD), with the RD coordinating Cd2+ and the KD in complex with the small molecule inhibitor AX20017 (PDB code 2PZI, Fig. 1B), and two of PknG74–405 (RD-KD), with the RD coordinating Zn2+ and the KD in complex with either an ATP analogue (ATP-γS) or ADP as well as Mg2+ (PDB codes 4Y12 and 4Y0X) (
      • Scherr N.
      • Honnappa S.
      • Kunz G.
      • Mueller P.
      • Jayachandran R.
      • Winkler F.
      • Pieters J.
      • Steinmetz M.O.
      Structural basis for the specific inhibition of protein kinase G, a virulence factor of Mycobacterium tuberculosis.
      ,
      • Lisa M.N.
      • Gil M.
      • André-Leroux G.
      • Barilone N.
      • Durán R.
      • Biondi R.M.
      • Alzari P.M.
      Molecular basis of the activity and the regulation of the eukaryotic-like S/T protein kinase PknG from Mycobacterium tuberculosis.
      ). The structure of the RD is very similar in all three solved structures, but the orientation of the RD with respect to the kinase domain is slightly different. In the inhibitor-bound structure, the RD interacts with the N-terminal and C-terminal lobes of the kinase domain, whereas, in the ATP analogue-bound form, the RD makes contact only with the N-terminal lobe (
      • Scherr N.
      • Honnappa S.
      • Kunz G.
      • Mueller P.
      • Jayachandran R.
      • Winkler F.
      • Pieters J.
      • Steinmetz M.O.
      Structural basis for the specific inhibition of protein kinase G, a virulence factor of Mycobacterium tuberculosis.
      ,
      • Lisa M.N.
      • Gil M.
      • André-Leroux G.
      • Barilone N.
      • Durán R.
      • Biondi R.M.
      • Alzari P.M.
      Molecular basis of the activity and the regulation of the eukaryotic-like S/T protein kinase PknG from Mycobacterium tuberculosis.
      ). It was proposed that the RD regulates the intrinsic kinase activity by restricting the accessibility of the active site (
      • Scherr N.
      • Honnappa S.
      • Kunz G.
      • Mueller P.
      • Jayachandran R.
      • Winkler F.
      • Pieters J.
      • Steinmetz M.O.
      Structural basis for the specific inhibition of protein kinase G, a virulence factor of Mycobacterium tuberculosis.
      ,
      • Lisa M.N.
      • Gil M.
      • André-Leroux G.
      • Barilone N.
      • Durán R.
      • Biondi R.M.
      • Alzari P.M.
      Molecular basis of the activity and the regulation of the eukaryotic-like S/T protein kinase PknG from Mycobacterium tuberculosis.
      ). Mutation of all four cysteines in the two conserved CXXCG motifs to alanines or serines impairs the kinase activity and renders PknG insensitive to regulation by redox changes (
      • Scherr N.
      • Honnappa S.
      • Kunz G.
      • Mueller P.
      • Jayachandran R.
      • Winkler F.
      • Pieters J.
      • Steinmetz M.O.
      Structural basis for the specific inhibition of protein kinase G, a virulence factor of Mycobacterium tuberculosis.
      ,
      • Tiwari D.
      • Singh R.K.
      • Goswami K.
      • Verma S.K.
      • Prakash B.
      • Nandicoori V.K.
      Key residues in Mycobacterium tuberculosis protein kinase G play a role in regulating kinase activity and survival in the host.
      ).
      Cells of the innate immune system, such as macrophages, release high concentrations of reactive oxidative species (ROS) to kill engulfed pathogenic bacteria (
      • Miller R.A.
      • Britigan B.E.
      Role of oxidants in microbial pathophysiology.
      ). However, based on the published crystal structures and functional data for wild-type and mutant PknG proteins, the exact mechanism of the redox regulation of the kinase activity under oxidative stress conditions remains elusive. Here we present combined NMR spectroscopy, in vitro kinase assay, and molecular dynamics (MD) simulation data that show how the dynamics as well as the local and global structure of PknG change upon oxidation of the RD and that the so far uncharacterized NORS region is indeed natively unfolded and the target region for PknG autophosphorylation in trans.

      Author Contributions

      S. A. D. designed and coordinated the study, helped acquire and analyze the NMR data, and wrote the paper. M. W. designed, performed, and analyzed the NMR and kinase assay data shown in FIGURE 1., FIGURE 2., FIGURE 3. and supplemental Figs. S1–S9 and helped write the manuscript. Q. L. designed, performed, and analyzed the molecular dynamics simulation data shown in Fig. 3 and supplemental Figs. S10 –S12. V. R. I. K. designed and coordinated the molecular dynamics part of the study and wrote the corresponding part of the paper. All authors reviewed the results and approved the final version of the manuscript.

      Acknowledgments

      We thank Dr. Nicole Scherr, who did her Ph.D. thesis in the group of Prof. Dr. Jean Pieters, Hélène Rossez, and Prof. Dr. Jean Pieters from the Biozentrum of the University of Basel, for providing the expression plasmids for His-tagged PknG1–147, PknG74–750, and PknG1–750 as well as purification protocols. We thank Tobias Bauer, Eugen Dornstauder, and Milica Vunjak for contributions while they were doing practical work and/or a thesis project in our group. We also thank Prof. Dr. Michael Sattler and Prof. Dr. Bernd Reif from the Technische Universität München and Helmholtz Zentrum München for hosting our group and for continuous support and for sharing their facilities with us.

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