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The domain architecture of PtkA, the first tyrosine kinase from Mycobacterium tuberculosis, differs from the conventional kinase architecture

  • Anna Niesteruk
    Affiliations
    Institute for Organic Chemistry and Chemical Biology, Centre for Biomolecular Magnetic Resonance (BMRZ), Goethe-University Frankfurt am Main, Max-von-Laue-Strasse 7, D-60438 Frankfurt am Main, Germany
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  • Hendrik R.A. Jonker
    Affiliations
    Institute for Organic Chemistry and Chemical Biology, Centre for Biomolecular Magnetic Resonance (BMRZ), Goethe-University Frankfurt am Main, Max-von-Laue-Strasse 7, D-60438 Frankfurt am Main, Germany
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  • Christian Richter
    Affiliations
    Institute for Organic Chemistry and Chemical Biology, Centre for Biomolecular Magnetic Resonance (BMRZ), Goethe-University Frankfurt am Main, Max-von-Laue-Strasse 7, D-60438 Frankfurt am Main, Germany
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  • Verena Linhard
    Affiliations
    Institute for Organic Chemistry and Chemical Biology, Centre for Biomolecular Magnetic Resonance (BMRZ), Goethe-University Frankfurt am Main, Max-von-Laue-Strasse 7, D-60438 Frankfurt am Main, Germany
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  • Sridhar Sreeramulu
    Affiliations
    Institute for Organic Chemistry and Chemical Biology, Centre for Biomolecular Magnetic Resonance (BMRZ), Goethe-University Frankfurt am Main, Max-von-Laue-Strasse 7, D-60438 Frankfurt am Main, Germany
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  • Harald Schwalbe
    Correspondence
    Member of the DFG-funded cluster of excellence: macromolecular complexes. To whom correspondence should be addressed. Tel.:49-69-79829737; Fax:49-69-79829515;.
    Affiliations
    Institute for Organic Chemistry and Chemical Biology, Centre for Biomolecular Magnetic Resonance (BMRZ), Goethe-University Frankfurt am Main, Max-von-Laue-Strasse 7, D-60438 Frankfurt am Main, Germany
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  • Author Footnotes
    2 The abbreviations used are: MtbMycobacterium tuberculosisPTMpost-translational modificationTBtuberculosisSTPKserine-threonine protein kinaseVPS33Bvacuolar protein sorting 33BHADhaloacid dehydrogenase-like hydrolaseIDDintrinsically disordered domainKCDkinase core domain1D2D, and 3D, one-, two-, and three-dimensional, respectivelyPREparamagnetic relaxation enhancementSDSLsite-directed spin labelMTSL1-oxy-2,2,5,5-tetramethyl-d-pyrroline-3-methyl-methane-thiosulfonateTROSYtransverse relaxation optimized spectroscopyHSQCheteronuclear single quantum coherence.
Open AccessPublished:June 08, 2018DOI:https://doi.org/10.1074/jbc.RA117.000120
      The discovery that MptpA (low-molecular-weight protein tyrosine phosphatase A) from Mycobacterium tuberculosis (Mtb) has an essential role for Mtb virulence has motivated research of tyrosine-specific phosphorylation in Mtb and other pathogenic bacteria. The phosphatase activity of MptpA is regulated via phosphorylation on Tyr128 and Tyr129. Thus far, only a single tyrosine-specific kinase, protein-tyrosine kinase A (PtkA), encoded by the Rv2232 gene has been identified within the Mtb genome. MptpA undergoes phosphorylation by PtkA. PtkA is an atypical bacterial tyrosine kinase, as its sequence differs from the sequence consensus within this family. The lack of structural information on PtkA hampers the detailed characterization of the MptpA–PtkA interaction. Here, using NMR spectroscopy, we provide a detailed structural characterization of the PtkA architecture and describe its intra- and intermolecular interactions with MptpA. We found that PtkA's domain architecture differs from the conventional kinase architecture and is composed of two domains, the N-terminal highly flexible intrinsically disordered domain (IDDPtkA) and the C-terminal rigid kinase core domain (KCDPtkA). The interaction between the two domains, together with the structural model of the complex proposed in this study, reveal that the IDDPtkA is unstructured and highly dynamic, allowing for a “fly-casting-like” mechanism of transient interactions with the rigid KCDPtkA. This interaction modulates the accessibility of the KCDPtkA active site. In general, the structural and functional knowledge of PtkA gained in this study is crucial for understanding the MptpA–PtkA interactions, the catalytic mechanism, and the role of the kinase–phosphatase regulatory system in Mtb virulence.

      Introduction

      Mycobacterium tuberculosis (Mtb)
      The abbreviations used are: Mtb
      Mycobacterium tuberculosis
      PTM
      post-translational modification
      TB
      tuberculosis
      STPK
      serine-threonine protein kinase
      VPS33B
      vacuolar protein sorting 33B
      HAD
      haloacid dehydrogenase-like hydrolase
      IDD
      intrinsically disordered domain
      KCD
      kinase core domain
      1D
      2D, and 3D, one-, two-, and three-dimensional, respectively
      PRE
      paramagnetic relaxation enhancement
      SDSL
      site-directed spin label
      MTSL
      1-oxy-2,2,5,5-tetramethyl-d-pyrroline-3-methyl-methane-thiosulfonate
      TROSY
      transverse relaxation optimized spectroscopy
      HSQC
      heteronuclear single quantum coherence.
      (
      • Cole S.T.
      • Brosch R.
      • Parkhill J.
      • Garnier T.
      • Churcher C.
      • Harris D.
      • Gordon S.V.
      • Eiglmeier K.
      • Gas S.
      • Barry 3rd, C.E.
      • Tekaia F.
      • Badcock K.
      • Basham D.
      • Brown D.
      • Chillingworth T.
      • et al.
      Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence.
      ) increasingly evolves multidrug-resistant (
      • Pablos-Mendez A.
      • Gowda D.K.
      • Frieden T.R.
      Controlling multidrug-resistant tuberculosis and access to expensive drugs: a rational framework.
      ) and extensive drug resistant (
      • Shah N.S.
      • Wright A.
      • Bai G.-H.
      • Barrera L.
      • Boulahbal F.
      • Martín-Casabona N.
      • Drobniewski F.
      • Gilpin C.
      • Havelková M.
      • Lepe R.
      • Lumb R.
      • Metchock B.
      • Portaels F.
      • Rodrigues M.F.
      • Rüsch-Gerdes S.
      • et al.
      Worldwide emergence of extensively drug-resistant tuberculosis.
      ) strains, causing epidemic problems on a global scale (
      World Health Organization
      ). New therapeutics against tuberculosis (TB) are thus urgently needed. The development of new inhibitors is challenging, especially if one considers that the requirements of anti-TB drugs to exhibit bactericidal and sterilizing activity are complex and that the molecular mechanisms responsible for the Mtb pathogenic regulation are not yet fully understood (
      • van den Boogaard J.
      • Kibiki G.S.
      • Kisanga E.R.
      • Boeree M.J.
      • Aarnoutse R.E.
      New drugs against tuberculosis: problems, progress, and evaluation of agents in clinical development.
      ). Cellular pathways that trigger virulence (such as enzymes that support the mycobacterial dormancy, persistency, and drug resistance) are potential targets for the implementation of new anti-tubercular agents. In particular, proteins involved in the Mtb signaling network appear interesting.
      Reversible phosphorylation is co-regulated by kinases and phosphatases and provides a basis for the signal transmission in multiple organisms (
      • Grangeasse C.
      • Nessler S.
      • Mijakovic I.
      Bacterial tyrosine kinases: evolution, biological function and structural insights.
      ). The genome of Mtb encodes 27 enzymes involved in the cellular cross-talk; 12 two-component systems (
      • Parish T.
      Two-component regulatory systems of mycobacteria.
      ), 11 eukaryotic-like serine-threonine protein kinases (STPKs, PknA–PknL), one serine-threonine phosphatase (
      • Av-Gay Y.
      • Everett M.
      The eukaryotic-like Ser/Thr protein kinases of Mycobacterium tuberculosis.
      ), and two protein-tyrosine phosphatases (MptpA and MptpB) (
      • Koul A.
      • Choidas A.
      • Treder M.
      • Tyagi A.K.
      • Drlica K.
      • Singh Y.
      • Ullrich A.
      Cloning and characterization of secretory tyrosine phosphatases of Mycobacterium tuberculosis.
      ) were identified that adjust the phosphorylation status of specific amino acids (His/Asp and Ser/Thr/Tyr) during diverse cellular processes. Despite the well-established significance of the protein phosphomodification in mycobacteria, its functional role in the pathogenicity has remained poorly understood (
      • Greenstein A.E.
      • Grundner C.
      • Echols N.
      • Gay L.M.
      • Lombana T.N.
      • Miecskowski C.A.
      • Pullen K.E.
      • Sung P.Y.
      • Alber T.
      Structure/function studies of Ser/Thr and Tyr protein phosphorylation in Mycobacterium tuberculosis.
      ,
      • Kusebauch U.
      • Ortega C.
      • Ollodart A.
      • Rogers R.S.
      • Sherman D.R.
      • Moritz R.L.
      • Grundner C.
      Mycobacterium tuberculosis supports protein tyrosine phosphorylation.
      ). Several STPKs for post-translational modifications of other endogenous kinases have been identified in Mtb. In Mtb, phosphorylation of threonine residues is highly abundant compared with serine phosphorylation (
      • Prisic S.
      • Dankwa S.
      • Schwartz D.
      • Chou M.F.
      • Locasale J.W.
      • Kang C.-M.
      • Bemis G.
      • Church G.M.
      • Steen H.
      • Husson R.N.
      Extensive phosphorylation with overlapping specificity by Mycobacterium tuberculosis serine/threonine protein kinases.
      ). The first strong indication regarding the importance of pTyr modifications for Mtb pathogenicity followed from the discovery of the low-molecular weight MptpA (17.5 kDa) and its critical role for Mtb virulence (
      • Wong D.
      • Chao J.D.
      • Av-Gay Y.
      Mycobacterium tuberculosis-secreted phosphatases: from pathogenesis to targets for TB drug development.
      ). During infection, MptpA is secreted from the pathogenic mycobacteria into the cytosol of host macrophages and interferes with the host endogenous signaling pathway. MptpA catalyzes dephosphorylation of the human vacuolar protein sorting 33B (VPS33B) complex, which leads to suppression of the phagosome-lysosome fusion (
      • Chow K.
      • Ng D.
      • Stokes R.
      • Johnson P.
      Protein tyrosine phosphorylation in Mycobacterium tuberculosis.
      ,
      • Wong D.
      • Bach H.
      • Sun J.
      • Hmama Z.
      • Av-Gay Y.
      Mycobacterium tuberculosis protein tyrosine phosphatase (PtpA) excludes host vacuolar-H+-ATPase to inhibit phagosome acidification.
      ). The virulence factor MptpA escapes the complex structure of the Mtb cell wall, which is otherwise impermeable to many drugs, thus making it an attractive target for inhibitor development. However, the exact mechanism that modulates the activity of MptpA and its translocation from the bacterial cytosol into the cytosol of the infected macrophages is still unknown.
      On the molecular level, our detailed structural characterization of MptpA indicated a conformational rearrangement from the open to a closed state, induced by ligand binding (
      • Stehle T.
      • Sreeramulu S.
      • Löhr F.
      • Richter C.
      • Saxena K.
      • Jonker H.R.A.
      • Schwalbe H.
      The apo-structure of the low molecular weight protein-tyrosine phosphatase A (MptpA) from Mycobacterium tuberculosis allows for better target-specific drug development.
      ). One of the proposed mechanisms of MptpA regulation involves phosphorylation of the vicinally located tyrosine residues (Tyr128/Tyr129) (
      • Zhou P.
      • Li W.
      • Wong D.
      • Xie J.
      • Av-Gay Y.
      Phosphorylation control of protein tyrosine phosphatase A activity in Mycobacterium tuberculosis.
      ). To the best of our knowledge, there is no information available about the phosphorylation status of each tyrosine or on the regulation of the tyrosine phosphorylation. Until now, only one protein-tyrosine kinase (PtkA, 30.6 kDa) has been found in Mtb (
      • Bach H.
      • Wong D.
      • Av-Gay Y.
      Mycobacterium tuberculosis PtkA is a novel protein tyrosine kinase whose substrate is PtpA.
      ). PtkA was classified as an atypical bacterial kinase, or so-called “odd” tyrosine kinase (
      • Chao J.D.
      • Wong D.
      • Av-Gay Y.
      Microbial protein-tyrosine kinases.
      ), due to the lack of the sequential consensus with any members of the known bacterial kinase families (bacterial tyrosine kinases, BY-kinases (
      • Grangeasse C.
      • Nessler S.
      • Mijakovic I.
      Bacterial tyrosine kinases: evolution, biological function and structural insights.
      )) or eukaryotic-like protein kinases. PtkA is considered as the cognate kinase of MptpA and was proposed to modulate its activity by tyrosine-specific phosphorylation (
      • Zhou P.
      • Li W.
      • Wong D.
      • Xie J.
      • Av-Gay Y.
      Phosphorylation control of protein tyrosine phosphatase A activity in Mycobacterium tuberculosis.
      ). Biochemical analysis annotates PtkA as a member of the haloacid dehydrogenase-like hydrolase (HAD) superfamily, due to the presence of the active site motif D85XD, which is essential for the catalytic and autophosphorylation activity (
      • Bach H.
      • Wong D.
      • Av-Gay Y.
      Mycobacterium tuberculosis PtkA is a novel protein tyrosine kinase whose substrate is PtpA.
      ). Furthermore, PtkA acts as substrate for Mtb endogenous eukaryotic-like STPKs (
      • Zhou P.
      • Wong D.
      • Li W.
      • Xie J.
      • Av-Gay Y.
      Phosphorylation of Mycobacterium tuberculosis protein tyrosine kinase A PtkA by Ser/Thr protein kinases.
      ), which might represent an additional level of regulation of PtkA activity. MptpA and PtkA, even if they do not represent typical on/off switches of signal transduction (
      • Chao J.D.
      • Wong D.
      • Av-Gay Y.
      Microbial protein-tyrosine kinases.
      ), are part of the Mtb regulatory system and therefore important target candidates for anti-TB therapy. Moreover, recent studies show the essential role of PtkA for Mtb growth in macrophages, establishing a central role for the PtkA–MptpA operon in Mtb pathogenesis (
      • Wong D.
      • Li W.
      • Chao J.D.
      • Zhou P.
      • Narula G.
      • Tsui C.
      • Ko M.
      • Xie J.
      • Martinez-Frailes C.
      • Av-Gay Y.
      Protein tyrosine kinase, PtkA, is required for Mycobacterium tuberculosis growth in macrophages.
      ). In this context, the lack of structural information of PtkA hampers a general understanding of the interactions between MptpA and PtkA, impairing the development of strategies for rational design of TB inhibitors.
      Here, we present an extensive study on the structure and dynamics of the WT PtkA using NMR spectroscopy, describing the intrinsic structural architecture of PtkA at atomic resolution. We characterize the two domains that build up the PtkA structure, an N-terminal intrinsically disordered domain (IDDPtkA) and C-terminal kinase core domain (KCDPtkA). Additionally, we solved the three-dimensional NMR structure of KCDPtkA. Further, using a broad spectrum of NMR techniques, we characterize inter- and intramolecular interactions of PtkA, giving important insights into the key regulatory catalytic processes of tyrosine phosphorylation-dephosphorylation mediated by PtkA–MptpA.

      Results

      Structural study of PtkA

      The sequence-based analysis of PtkA (30.6 kDa, 291 amino acids) predicts a high disorder tendency for the N-terminal part comprising 80 amino acids (Fig. S1). Our NMR data confirm the prediction, showing that PtkA consists of a well-folded KCD and an unstructured IDD. The 2D 1H,15N TROSY spectrum of the full-length PtkA shows in total 231 of 270 expected amide resonances (Fig. 1A). Two subsets of signals can be clearly observed: (i) a set of well-dispersed peaks, typical for a well-folded structure, and (ii) another set of signals with high intensity, which are clustered in the center of the spectrum (1H: 7.6–8.6 ppm), indicative of unstructured regions within the full-length PtkA. The 2D 1H,15N HSQC spectrum of PtkA shows only minor changes of the chemical shift in the presence of ATP (Fig. S8). Unfortunately, incubation of PtkA with ATP for 24 h leads to precipitation of the protein, which excludes phosphorylated PtkA to be used for long-term NMR experiments.
      Figure thumbnail gr1
      Figure 1A, backbone assignment of the full-length PtkA. Shown is the 2D 1H,15N TROSY spectrum of the full-length PtkA, acquired at 950 MHz, 298 K, and pH 7.5. The spectral region with a large signal overlap (between 7.6 and 8.6 ppm in the 1H dimension and between 107 and 129 ppm in the 15N dimension) is highlighted with a box. B, PtkA construct optimization. Right, 2D 1H,15N HSQC spectra of KCDPtkA (residues 76–291, 23 kDa); left, IDDPtkA (residues 1–81, 8.5 kDa) acquired at 600 MHz, 298 K, and pH 7.5. C, primary amino acid sequence of the full-length PtkA (Rv2234, Met1–Val291). The region of the IDD is indicated by a blue dotted line. Residues with missing backbone amide assignment are highlighted with a gray background.

      Sequence-specific NMR resonance assignment of full-length PtkA

      The assignment of the 1H, 15N, and 13C backbone resonances of the full-length PtkA was obtained using a combination of standard triple-resonance NMR experiments on triple-labeled (2H, 15N, 13C) and/or double-labeled (15N, 13C) PtkA samples. We observed that the number of visible cross-peaks increases to 20% in the presence of Mg2+, leading to better quality of the NMR spectra. Therefore, all assignment experiments were performed in the presence of 10 mm MgCl2. In addition, the selectively 15N-Val– and 15N-Phe–labeled PtkA samples were prepared to confirm ambiguous assignments. We were able to assign 93.5% of the observable backbone amides (216 of 231 detected amide resonance in the 2D 1H,15N TROSY spectrum), which is 80% of the expected amide cross-peaks. Most of the missing chemical shift assignments were restricted to three regions: Thr25–Ser29, Thr210–Asp219, and Val257–His278 (Fig. 1C), presumably due to intermediate exchange on the NMR time scale. To complete and verify the assignment of the full-length PtkA, we undertook a protein construct optimization. The assignment of the full-length PtkA was thus aided by using two PtkA truncation mutants (Fig. 1B) containing either the N-terminal 81 amino acids (Met1–Pro81) representing the intrinsically disordered domain (IDDPtkA, 8.5 kDa) or the C-terminal (Val75–Val291) region representing the kinase core domain (KCDPtkA, 23 kDa). The structural properties of both constructs were investigated by NMR spectroscopy.

      Backbone assignment of KCDPtkA

      The 2D 1H,15N HSQC spectrum of KCDPtkA revealed a substantial reduction of the signal overlap, and the remaining 172 well-resolved backbone amide signals fit almost perfectly with those observed in the spectrum of the full-length PtkA (with the exception of those residues that are located close the truncation site: Gly76, Glu77, and Ser78), indicating proper folding of the KCDPtkA. Using the KCDPtkA in separation, we were able to obtain 96.5% of the backbone assignment (166 of 172 observable amide cross-peaks; Fig. S2A), which is 76.9% of the expected amide cross-peaks. Hence, the KCDPtkA was used for the three-dimensional NMR-based structure determination.

      Backbone assignment of IDDPtkA

      The presence of intrinsically disordered domain hampers the unambiguous assignment in the overcrowded spectral region of the full-length PtkA. The 2D 1H,15N HSQC spectrum of IDDPtkA at pH 7.5 revealed poor signal dispersion, similar to the observation made in the spectrum of the full-length PtkA. To facilitate the backbone assignment of the IDDPtkA, we investigated the IDDPtkA also at low pH. In the 2D 1H,15N HSQC spectrum of IDDPtkA measured at pH 2.0, we could detect 68 well-resolved signals and were able to assign all of the amide backbone using a set of 3D NMR experiments (HNCACB, (H)N(CA)NNH, HNHA, and HNCO) (Fig. S2C). A pH titration series of 2D 1H,15N HSQC spectra were recorded, ranging from pH 2.0 to 7.5 with a 1.5-pH unit increment, to enable the backbone assignment of the 2D 1H,15N HSQC spectrum of IDDPtkA at pH 7.5 (Fig. S2B). The detailed characterization of the IDDPtkA was recently published by our group (
      • Niesteruk A.
      • Hutchison M.
      • Sreeramulu S.
      • Jonker H.R.A.
      • Richter C.
      • Abele R.
      • Bock C.
      • Schwalbe H.
      Structural characterization of the intrinsically disordered domain of Mycobacterium tuberculosis protein tyrosine kinase A.
      ).
      The resonance assignment from the KCDPtkA (residues 76–291) and IDDPtkA (residues 1–81) was used to validate the assignment of the full-length PtkA (residues 1–291). We were able to assign 69% of the IDD (47 signals of 68 expected) and 84% of the KCD (169 of 202 expected) backbone amide signals in the full-length PtkA at pH 7.5 (Fig. 1A). The backbone assignment of PtkA has been deposited in the Biological Magnetic Resonance Data Bank (BMRB) with entry number 34204.

      3D structure determination of the KCDPtkA

      The NMR solution structure of the PtkA kinase core domain was determined (Protein Data Bank entry 6F2X). Due to the limited completeness of the resonance assignment of residues located in or near to the center of the protein, the conventional structure calculation with fully automated NOESY cross-peak assignment by CYANA was unsuccessful. Missing assignments within and around two central regions (e.g. Thr210–Asp219 and Val257–His278) indicate dynamics or exchange on an intermediate NMR time scale for this part of the molecule, complicating the structural characterization of the KCDPtkA. Both regions are particularly interesting, as they contain residues involved in the catalytic regulation of PtkA. To be able to elucidate the three-dimensional structure of PtkA, homology models were used to assist the NOESY cross-peak assignment by CYANA, thereby just avoiding NOE misassignments between atoms that are actually not close to each other (assigned resonances that coincide with those of the undefined region), which would otherwise disrupt the structure and cause the convergence to fail. The subsequent structure refinement was done without the additional global distance restraints (see “Experimental procedures” for more details). This allowed us to characterize the structural propensities of the PtkA core domain (Fig. 2). The regions with missing assignments (Thr210–Val216 and Tyr262–Val275, indicated in gray) are undefined due to the lack of restraints (thus merely force field–based), and their spread does not necessarily indicate dynamics. Six parallel β-sheets located in lobe 1 (Fig. 2B) of the KCDPtkA build up the hydrophobic core of the KCDPtkA. Lobe 1 is the essential part of the PtkA-containing catalytic loop (D85LD motif), the lysine residues essential for ATP binding (Lys184, Lys217, and Lys270), and the autophosphorylation site (Tyr262). Lobe 2 of the core domain consists of three long α-helices (designated α1–α3) and a short α-helical section that is located between α-helix α1 and α-helix α2. Lobe 2 contains two additional tyrosine residues (Tyr146 and Tyr150) with an unknown role. However, the description of the catalytic site of PtkA by NMR spectroscopy in any detail is difficult because only a subset of the residues involved in the catalysis is amenable for interpretation, largely due to missing amide cross-peaks at native-like pH (pH 7.5). However, mapping of the position of residues involved in the catalytic regulation of PtkA (
      • Bach H.
      • Wong D.
      • Av-Gay Y.
      Mycobacterium tuberculosis PtkA is a novel protein tyrosine kinase whose substrate is PtpA.
      ) onto the structure reveals that all of the three lysine residues (Lys184, Lys217, and Lys270), the tyrosines (Tyr146, Tyr150, and Tyr262), and the Asp85 located in the conserved DXD motif are surrounding the autophosphorylation site (Fig. S3A). In addition, primary sequence alignment of PtkA and two putative phosphatases (from Clostridium difficile and phosphatase from Clostridium acetobutylicum) revealed no sequence analogy for the N-terminal IDD, whereas four conserved HAD signature motifs were found to be conserved within the C-terminal domain (Fig. S3B).
      Figure thumbnail gr2
      Figure 2NMR solution structure of KCDPtkA. A, structural statistics for the ensemble of the 20 best NMR structures of PtkA. B, ribbon representation of the lowest-energy structure (top) and the bundle of the 20 lowest-energy structures (bottom) rotated by 180°. Lobes 1 and 2, the catalytic motif (D85LD), and the autophosphorylation site (circle) are highlighted. The overall structure of KCDPtkA consists of six parallel folded β-sheets and nine α-helix secondary structures. Regions with the missing assignment (Thr210–Val216 and Tyr262–Val275) are colored in gray. The figure was generated by PyMOL.

      Study of PtkA dynamics: Heteronuclear 15N relaxation studies

      PtkA dynamics were studied using standard heteronuclear 15N relaxation experiments. The experiments were performed on the full-length PtkA as well as on the KCDPtkA and the IDDPtkA to investigate whether the domains influence the dynamics of each other. The experimentally obtained relaxation rates for 15N-T1, 15N-T2, and 15N{1H}-heteronuclear NOE were used to determine the Lipari–Szabo order parameter, S2 (Fig. 3A). The relaxation study of the internal motions in the full-length PtkA shows a rigid KCD and a higher flexibility of the N-terminal domain (Met1–Glu77; indicated by an increase of the R1 rate, a decreased R2 rate, and a lower 15N{1H}-heteronuclear NOE value). The relaxation data obtained for the KCDPtkA construct are highly similar to those for the full-length PtkA, which indicates that there is no major effect on the local backbone dynamics of the KCD in the absence of the IDD. For the IDDPtkA, we determine 15N-R1 relaxation rates of 1.78 ± 0.13 s−1 and 15N-R2 rates of 3.44 ± 0.33 s−1. The relaxation rates thus obtained for the IDDPtkA and the full-length PtkA are comparable and confirm high flexibility and disordered state at native-like conditions for the N-terminal domain of PtkA.
      Figure thumbnail gr3
      Figure 3A, PtkA dynamics before and after truncation of one of the domains. Top, cylinders, α-helices; arrows, β-sheet. Shown is a schematic representation of an NMR chemical shift-based TALOS+ prediction of the secondary structure elements of KCDPtkA. Shown is a plot of the determined order parameter (S2) for the full-length PtkA and KCDPtkA construct as a function of residue number (S2 was generated using TENSOR2 software (
      • Dosset P.
      • Hus J.-C.
      • Blackledge M.
      • Marion D.
      Efficient analysis of macromolecular rotational diffusion from heteronuclear relaxation data.
      ) based on the three experimentally measured relaxation parameters: T1, T2, and 15N heteronuclear NOE). Shown is a plot of the R1 and R2 relaxation rates and 15N{1H}-heteronuclear NOE as a function of residue number of the full-length PtkA (black) and KCDPtkA (red) determined at 700 MHz and IDDPtkA (blue) measured at 600 MHz, 298 K, in 50 mm HEPES-NaOH buffer (pH 7.5) containing 300 μm protein, 300 mm NaCl, 10 mm DTT, 10 mm MgCl2, 10% D2O/90% H2O. B, hydrogen–deuterium exchange rate (Kex, s−1) of full-length PtkA (black) and KCDPtkA (red) as a function of residue number. The missing exchange rates could not be determined due to rapidly exchanging amide hydrogens (directly after solvation in D2O), for unassigned or overlapping cross-peaks, and for proline residues. C, determined temperature coefficient (ppb/K) of full-length PtkA (black), KCDPtkA (red), and IDDPtkA (blue) as a function of residue number. The error estimate is derived from the Gaussian noise in the peak intensity.

      Hydrogen–deuterium exchange studies

      Hydrogen–deuterium exchange studies were used to study the solvent accessibility of the amide protons of PtkA in the presence and absence of the IDD. The exchange rates (kex) of the backbone amides of the full-length PtkA and the KCDPtkA were determined (Fig. 3B). Fifteen minutes after reconstitution of the freeze-dried full-length PtkA sample into D2O, only 33% of the backbone amide cross-peaks were observed compared with the signals in H2O. All amide signals corresponding to the residues of IDD disappeared, which is typical for very flexible dynamic regions and thus well in agreement with the results from our heteronuclear relaxation studies. In contrast, 10% of the amide signals of residues located in the KCD were still visible even after 19 h. These very slowly exchanging backbone amide protons correspond to the residues Val82–Asp85, Leu167–Arg172, Val176–Thr182, Leu193–Phe196, Ala206, Leu225, Val236–Asp240, and Leu285–Val291, suggesting involvement of those residues in the hydrogen bond network of highly protected secondary structure elements in the KCD. Furthermore, the correlation between the secondary structure elements and hydrogen–deuterium exchange rate reveals that the KCD is inherently stable and rigid. At the same time, different dynamic properties of the α-helices in the KCD were observed. Slow exchanging amide protons from residues in the loop region between β-sheet β1 and α-helix α1 and residues in the α-helix α4–6 suggest potential exposure to the hydrophobic part of the protein, whereas α-helix α1–3 and α6–7 fluctuate in solution. Moreover, for PtkA without the IDD, slight differences in the backbone amide hydrogen exchange behaviors were observed after 3 h of reconstitution in D2O (Fig. S4). Signals corresponding to the residues Val144 and Ala152, located in the α-helix (α2) containing Tyr146 and Tyr150; Phe161, located in the α23 loop; and Val256, located near the Tyr262, were missing in the full-length PtkA but still detectable in the spectrum of KCDPtkA. We speculate that these minor differences observed may play an important role during the autocatalytic regulation of PtkA, involving tyrosine phosphorylation (
      • Parish T.
      Two-component regulatory systems of mycobacteria.
      ). Hence, the dynamic nature of the intrinsically disordered N-terminal domain may play a role in modulating the behavior and arrangement of the important helices involved in catalysis.

      Intramolecular hydrogen bonding

      A temperature series was performed to study intramolecular hydrogen bonding based on the amide proton temperature dependence. We determined the temperature coefficient (Tcoeff., in ppb/K; Fig. 3C) from the amide chemical shift values by acquiring a series of 2D 1H,15N HSQC spectra measured at different temperatures, from 301 to 283 K with 3 K increments (Fig. S5). We analyzed the full-length PtkA as well as the KCDPtkA and IDDPtkA, to examine the involvement of the amide protons in the formation of rigid or transient hydrogen-bonded structure elements. Amide protons with a temperature coefficient more negative than −4.5 ppb/K (indicated by the dotted line in Fig. 3C) are generally not hydrogen-bonded (
      • Baxter N.J.
      • Williamson M.P.
      Temperature dependence of 1H chemical shifts in proteins.
      ). Many values obtained for amide protons of residues located in the KCD are larger than −4.5 ppb/K, which clearly suggests their involvement in the hydrogen bond formation, which is in agreement with the presence of abundant secondary structural elements in this domain. On the other hand, for the IDD, nearly all values are below −4.5 ppb/K, indicating that the residues in this domain are generally not hydrogen-bonded. Only Asn48 (−3.7 ppb/K), Gly49 (−3.6 ppb/K), and Asn60 (−4.0 ppb/K), which show a temperature coefficient larger than −4.5 ppb/K, were observed, indicating a potential involvement of those residues in transient hydrogen bonding.

      PtkA intramolecular IDD–KCD interaction

      The intramolecular domain–domain interactions were examined by comparing the chemical shifts observed in 2D 1H,15N HSQC spectra of the full-length PtkA, IDDPtkA, and KCDPtkA (Fig. S6, A and B). The spectra of the full-length PtkA and the KCDPtkA indicate that the folding of the KCD is not affected by truncation of the IDD. The comparison of the amide chemical shift of the IDD in the full-length PtkA and the IDDPtkA spectra shows the same chemical shift for most of the cross-peaks, except for the residues located proximate to the truncation site as well for Asn48 (Fig. S6A), suggesting weak domain–domain interactions. To study whether such interactions actually occur, we performed NMR titration experiments between 15N-labeled KCDPtkA and unlabeled IDDPtkA (up to molar ratio 1:2) and vice versa (up to molar ratio 1:3) at 298 K and pH 7.5 in the presence and absence of ATP (10 mm). The 2D 1H,15N HSQC spectra recorded during the titration series revealed no significant CSPs (Fig. S6, C and D). Based on these results, we assume that interactions between the IDDPtkA and KCDPtkA in trans (IDD and KCD are separated by mutational deletion), if present, are transient and rather weak. Nevertheless, information based on the amide chemical shift is primarily limited to the protein backbone and may thus be insufficient to detect weak interactions. Paramagnetic relaxation enhancement (PRE) NMR spectroscopy has been efficiently used to study weak and transient interactions. To detect these potential weak interdomain interactions of PtkA, we used the site-directed spin label (SDSL) approach. With SDSL, we analyzed long-range PtkA domain–domain interactions in cis (native state in full-length PtkA). The nitroxide radical, 1-oxy-2,2,5,5-tetramethyl-d-pyrroline-3-methyl-methane-thiosulfonate (MTSL), was covalently attached to the conserved cysteine (Cys61) residue located on the IDD. Further, to investigate the presence of residual structure in the IDD (Fig. S1), two single-cysteine PtkA mutants were designed, including mutations on alanine 10 (PtkA/C61A/A10C) and serine 41 (PtkA/C61A/A10C). The point mutations were introduced far away from the potential predicted secondary structure element, such that it did not disturb the fold (A10C), and a second mutant (S41C) was introduced within the folded region to interrupt the formation of the secondary structural element. After spin labeling with MTSL, 2D 1H,15N HSQC spectra of the paramagnetic and diamagnetic protein (after the addition of ascorbic acid) were acquired. In all spectra of the protein containing a paramagnetic center, PRE-induced line-broadening effects (Fig. 4A) were observed. The intensity ratio analysis of paramagnetic and diamagnetic (Ipara/Idia) PtkA resolved the most affected regions (Fig. 4B). For all three of the investigated PtkA constructs, the same regions in KCD were affected (Ipara/Idia ≤ 0.4, Glu114–Gly134, Asp162, Thr188–Ile192, Ile205–Gly212, Leu231–Met237, Trp260, and Ile282), suggesting a preferential position of the IDD relative to the core domain and the possibility of transient long-range domain–domain interactions. To visualize the IDD–KCD interaction, a set of experimental restraints from the spin-labeled complexes was used for the calculation of the model shown in Fig. 4C. Analysis of this model shows the presence of multiple orientations for the IDD within the IDD–KCD complex, suggesting a dynamic nature for this interaction. Further, the ensemble of similar orientation indicates that the IDD transiently interacts with the KCD and masks the ATP-binding site (Fig. 4C, right).
      Figure thumbnail gr4
      Figure 4Structural model for the IDD–KCD interaction in PtkA. A, overlay of the 2D 1H,15N HSQC spectra of paramagnetic (orange) and diamagnetic (black) full-length PtkA: PtkA C61A/A10C, MTSL-labeled residue Cys10 (left); PtkA C61A/S41C, MTSL-labeled residue Cys41 (middle); WT PtkA, MTSL-labeled residue Cys61 (right). Spectra were measured at 900 MHz, 298 K in 50 mm HEPES-NaOH buffer (pH 7.5) containing 300 mm NaCl, 10 mm MgCl2, 10% D2O/90% H2O. MTSL was reduced with ascorbic acid. The signals broadened out beyond detection due to the paramagnetic center are labeled. The C61A mutation is highlighted with a circle. B, normalized intensity ratios (Ipara/Idia) of amide cross-peaks versus amino acid sequence, determined for WT PtkA (C61-MTSL), PtkA C61A/A10C (C10-MTSL), and PtkA C61A/S41C (C41-MTSL). Missing data points correspond to peak overlap or unassigned residues. The error estimate is derived from the Gaussian noise in the peak intensity. C, PRE-based model representing the transient interaction between IDD and KCD. The blue part corresponds to the N-terminal IDD, and gray indicates KCD. Top, schematic representation; bottom, cartoon representation showing spin-labeled position (orange, A10C, S41C, and Cys61) and the catalytic site (red, D85LD, Tyr262).

      PtkA autophosphorylation and regulation of its catalytic activity

      The catalytic activity of PtkA is regulated via autophosphorylation of tyrosine residues in the presence of phosphate donor ATP or GTP (
      • Bach H.
      • Wong D.
      • Av-Gay Y.
      Mycobacterium tuberculosis PtkA is a novel protein tyrosine kinase whose substrate is PtpA.
      ). For the transfer of the phosphoryl group (PO32−) from ATP to the target protein and the formation of the physiological substrate XATP1−, the kinase requires divalent cations (e.g. X = Mn2+, Mg2+). Three tyrosine residues (Tyr146, Tyr150, and Tyr262) are present in the sequence of PtkA and represent potential sites for autophosphorylation. In particular, Tyr262 was considered as the target for phosphorylation during autocatalysis (
      • Bach H.
      • Wong D.
      • Av-Gay Y.
      Mycobacterium tuberculosis PtkA is a novel protein tyrosine kinase whose substrate is PtpA.
      ). PtkA itself represents a target protein for phosphorylation by the Mtb endogenous STPK (
      • Zhou P.
      • Wong D.
      • Li W.
      • Xie J.
      • Av-Gay Y.
      Phosphorylation of Mycobacterium tuberculosis protein tyrosine kinase A PtkA by Ser/Thr protein kinases.
      ). We investigated (i) the autophosphorylation of PtkA, (ii) the effect of Mg2+, (iii) interactions between PtkA in the nonphosphorylated and phosphorylated state with MptpA, (iv) dephosphorylation of PtkA by MptpA, and (v) PtkA phosphorylation by serine/threonine kinase (Fig. S11) (
      • Niesteruk A.
      • Hutchison M.
      • Sreeramulu S.
      • Jonker H.R.A.
      • Richter C.
      • Abele R.
      • Bock C.
      • Schwalbe H.
      Structural characterization of the intrinsically disordered domain of Mycobacterium tuberculosis protein tyrosine kinase A.
      ).

      PtkA autophosphorylation

      The autophosphorylation activity of full-length PtkA was previously measured and confirmed using a luciferase assay (
      • Stehle T.
      • Sreeramulu S.
      • Löhr F.
      • Richter C.
      • Saxena K.
      • Jonker H.R.A.
      • Schwalbe H.
      The apo-structure of the low molecular weight protein-tyrosine phosphatase A (MptpA) from Mycobacterium tuberculosis allows for better target-specific drug development.
      ). Furthermore, the autophosphorylation site in PtkA resides on the KCD and not on the IDD. To test the influence of the IDD on the autophosphorylation of PtkA, we performed a luminescence assay using a KCDPtkA construct alone and in the presence of the IDDPtkA and normalized the activity to that of the PtkA, which was set to 100% (Fig. 5C and Fig. S12). The results thus obtained suggest that the KCDPtkA is 5 times more active than the full-length PtkA. Activity of the KCDPtkA measured together with isolated IDD (IDDPtkA) shows a decrease compared with the KCDPtkA alone, suggesting that the IDD has an inhibitory effect on the kinase activity. The IDD in PtkA presents several potential PTM (phosphorylation) sites. Surprisingly, the activity of the PtkA in which the IDD was phosphorylated (PtkA*-P) showed 4 times more activity than that of its nonphosphorylated form (PtkA*) (Fig. 5C). These results strongly suggest that the IDD transiently binds to the KCD and masks the substrate-binding site and inhibits the kinase activity. Using 1D 31P NMR spectroscopy, we monitored the autophosphorylation reaction of PtkA in the presence and absence of the IDD after overnight incubation of PtkA (100 μm) with ATP (10 mm) at pH 7.5, 25 °C (Fig. 5D). In both cases, an additional signal at −0.98 ppm, indicative of the phosphorylated state of PtkA, was observed. MALDI-MS confirmed that PtkA was phosphorylated one time (Fig. S10).
      Figure thumbnail gr5
      Figure 5PtkA autophosphorylation and regulation of its catalytic activity. A, schematic representation of the PtkA regulatory system: autophosphorylation of tyrosine residues in PtkA and dephosphorylation by MptpA. B, model depicting the role of IDD in regulating the catalytic activity of PtkA. The dynamic character of IDD modulates the accessibility of Tyr262 generating an open (active protein) and closed (inactive protein) state of PtkA. Multisite PTM of IDD promotes the open state of PtkA, due to the phosphorylation-induced conformational changes of the IDD, increasing the accessibility of Tyr262 for autophosphorylation. C, luciferase assay of PtkA. The activity of the full-length PtkA was set to 100%. *, autophosphorylated protein. Error bars, S.D. D, 1D 31P NMR spectra of ATP in buffer (bottom), full-length PtkA (middle, blue), and KCDPtkA construct (top, black) after overnight incubation with ATP at room temperature. Signal indicating phosphorylation of PtkA is marked with a black arrow and red box. Signal from Pi results from the hydrolysis of ATP. E, 1D 31P NMR spectra of PtkA phosphorylated peptide Asp264–Lys270/pTyr262 (blue) and after incubation with MptpA (black).

      PtkA interactions with Mg2+

      To explore the effect of Mg2+ on the structural propensities of PtkA, we performed NMR studies in the presence and absence of MgCl2. The addition of MgCl2 (10 mm) into NMR buffer greatly improves the quality of the 2D 1H,15N HSQC spectrum of PtkA, resulting in 20% more detectable amide cross-peaks. Comparison of the chemical shift of the full-length PtkA obtained in the presence and absence of MgCl2 allowed us to delineate strong Mg2+-induced CSPs. The most affected resonances come from residues of the KCD. Additional experiments using KCDPtkA showed a similar effect of Mg2+ (Fig. S7).

      PtkA–MptpA interface

      In addition to previously reported MptpA–PtkA interaction studies (
      • Stehle T.
      • Sreeramulu S.
      • Löhr F.
      • Richter C.
      • Saxena K.
      • Jonker H.R.A.
      • Schwalbe H.
      The apo-structure of the low molecular weight protein-tyrosine phosphatase A (MptpA) from Mycobacterium tuberculosis allows for better target-specific drug development.
      ), for which the interaction was only mapped on the MptpA protein, we performed a set of NMR titrations for mapping the interaction interface on PtkA. For these studies, 15N-labeled full-length PtkA as well as 15N-labeled KCDPtkA were titrated with unlabeled MptpA (up to molar ratio 1:4) in the presence or absence of 5 mm ATP. Similar to the interaction studies from the MptpA, we obtained a number of small but evident CSPs, which were consistent for both of the constructs (PtkA and KCDPtkA) (Fig. S8, A and B). The largest number of chemical shift changes were observed for the residues surrounding the catalytic loop (D85LD), located between β-sheet (β1) and α-helix (α1). Additionally, residues located in the α-helix (α5) and β-sheet (β3) that envelops the autophosphorylation site are also affected, indicating their involvement in the binding interfaces with MptpA. Furthermore, mapping of the CSPs onto the structure of PtkA shows slight differences of the affected KCD area in the presence and absence of IDD. The α2-α3 loop located in lobe 2 of KCD seems to be more involved in the interactions in the absence of IDD. Interestingly, the α3-helix, which contains the two tyrosine residues Tyr146 and Tyr150 also showed similar differences in the hydrogen–deuterium exchange experiments measured in the presence and absence of IDD.

      Dephosphorylation of PtkA by MptpA

      To study the dephosphorylation of PtkA by MptpA, we synthesized peptides derived from PtkA, representing the autophosphorylation site in (i) nonphosphorylated (Asp264–Lys270/Tyr262) and (ii) phosphorylated Tyr262 (Asp264–Lys270/pTyr262) state. 1D 31P NMR of the PtkA (Asp264–Lys270/pTyr262) peptide shows a signal at −0.17 ppm, indicating phosphorylation of Tyr262 (Fig. 5E). We monitored this signal of the PtkA peptide (Asp264–Lys270/pTyr262) both upon the addition of and in the absence of the phosphatase MptpA (Fig. S9). Dephosphorylation of PtkA peptide (Asp264–Lys270/pTyr262) is indicated by the disappearance of the signal in the 1D 31P NMR spectra at −0.17 ppm and the appearance of one additional signal at 2.29 ppm corresponding to the Pi. The aromatic signals (Hδ and Hϵ) of Tyr262 reappear at a different chemical shift upon dephosphorylation by MptpA. In addition, the 2D 1H,13C HSQC spectrum of the aromatic region of PtkA peptide (Asp264–Lys270/pTyr262) acquired in the absence and presence of MptpA shows clear CSPs corresponding to the Tyr262 side-chain aromatic hydrogens (Hδ and Hϵ). These studies confirm that MptpA can potentially dephosphorylate isolated phosphorylated peptide derived from PtkA.

      Discussion

      Noncanonical bacterial kinases possessing tyrosine kinase activity play an important role in the cellular regulation of bacteria. Those kinases possibly belong to various protein families with multiple functions, as there is no sequential consensus between them. Besides PtkA from M. tuberculosis (
      • Bach H.
      • Wong D.
      • Av-Gay Y.
      Mycobacterium tuberculosis PtkA is a novel protein tyrosine kinase whose substrate is PtpA.
      ), other kinases, such as DivL of Caulobactercerescentus (
      • Wu J.
      • Ohta N.
      • Zhao J.-L.
      • Newton A.
      A novel bacterial tyrosine kinase essential for cell division and differentiation.
      ), WaaP of Pseudomonas aeruginosa (
      • Zhao X.
      • Lam J.S.
      WaaP of Pseudomonas aeruginosa is a novel eukaryotic type protein-tyrosine kinase as well as a sugar kinase essential for the biosynthesis of core lipopolysaccharide.
      ), or PutA of Salmonella typhimurium (
      • Ostrovsky P.C.
      • Maloy S.
      Protein phosphorylation on serine, threonine, and tyrosine residues modulates membrane-protein interactions and transcriptional regulation in Salmonella typhimurium.
      ), were found and termed as odd PTKs (
      • Chao J.D.
      • Wong D.
      • Av-Gay Y.
      Microbial protein-tyrosine kinases.
      ). How widespread such tyrosine kinases are in prokaryotes is still unclear, but the existence of atypical tyrosine kinases within one species suggests the existence of additional members of the same subfamily. The presence of homologs in the entire operon (similar to that found for the protein-tyrosine kinase PtkA of Mtb) in multiple Actinomycetes species like Rhodococcus, Corynebacterium, Gordonia, or Amycolicicoccus suggests that these proteins, even with limited homology, are protein-tyrosine kinases. Furthermore, as many as 924 unique PtkA orthologs were identified in all of the domains of life (
      • Chao J.D.
      • Wong D.
      • Av-Gay Y.
      Microbial protein-tyrosine kinases.
      ).
      The tyrosine phosphorylation and dephosphorylation represent a significant part of the survival regulation of Mtb (

      Bach, H., Papavinasasundaram, K. G., Wong, D., Hmama, Z., and Av-Gay, Y., Mycobacterium tuberculosis virulence is mediated by PtpA dephosphorylation of human vacuolar protein sorting 33B. Cell Host Microbe 3, 316–322

      ). The interaction of Mtb protein-tyrosine phosphatase MptpA with the host defense machinery plays an important role in the mycobacterial virulence (
      • Zhou P.
      • Li W.
      • Wong D.
      • Xie J.
      • Av-Gay Y.
      Phosphorylation control of protein tyrosine phosphatase A activity in Mycobacterium tuberculosis.
      ). Moreover, PtkA was recently shown to play a central role in promoting the growth of Mtb in macrophages (
      • Wong D.
      • Li W.
      • Chao J.D.
      • Zhou P.
      • Narula G.
      • Tsui C.
      • Ko M.
      • Xie J.
      • Martinez-Frailes C.
      • Av-Gay Y.
      Protein tyrosine kinase, PtkA, is required for Mycobacterium tuberculosis growth in macrophages.
      ), further underlining the significance of the interaction studies between MptpA and its cognate kinase PtkA and its role in Mtb regulation. Both enzymes represent potential candidates for rational drug design, where inactivation of either one of the partners or their interaction may inhibit the pathogen survival. Furthermore, the PtkA–MptpA operon conserved among numerous Actinobacteria species with limited identity for PtkA (39–76%) is in contrast to the Mtb complex, where this operon is highly conserved, with 99.7–100% protein identity (
      • Chao J.D.
      • Wong D.
      • Av-Gay Y.
      Microbial protein-tyrosine kinases.
      ). This additionally indicates the important role of this protein in the Mtb physiology.
      We characterized the structure of PtkA and investigated its interactions with MptpA in detail using NMR spectroscopy. The structural architecture of PtkA deviates from other bacterial kinases, as it contains an N-terminal intrinsically disordered domain, which is linked to the well-folded kinase core domain. The KCD is the catalytic center of PtkA containing (i) a catalytic loop (D85LD motif) located between the β-sheet (β1) and α-helix (α1) secondary structure and (ii) the autophosphorylation site (Tyr262) located near the protein hydrophobic core. The PtkA conformation is stabilized by binding of a divalent cation Mg2+, which may regulate the substrate binding. Moreover, MptpA binds to the KCD of PtkA and dephosphorylates the kinase. The dynamic properties of both domains are very different and suggest the possibility of an interdomain controlled regulation of PtkA activity. The IDD is unstructured and highly dynamic, allowing for a “fly-casting-like” mechanism of transient interactions with the rigid KCD. This interaction thereby regulates the accessibility of the KCD active site (which is involved in autophosphorylation). The decrease of the PtkA activity in the presence of IDD suggests an inhibitory effect of the disordered domain during the autophosphorylation. Spin label studies indicated the existence of residual long-range transient interactions of the IDD with the KCD. Based on those results, we made a model of the IDD–KCD structure, which shows that the preferred position of the IDD is located near the catalytic site. Our studies show that the presence of the IDD does not affect the fold of the KCD and has only minor effects on the solvent accessibility of the KCD. The changes in dynamics are restricted to the upper lobe of the KCD (α-helix (α3)), which harbors two tyrosines whose role and phosphorylation status are unclear. The kinase activity was observed in the presence as well as in the absence of IDD, which is in agreement with the results obtained in 1D 31P NMR spectra, where in both cases the phosphorylation of PtkA was detected. However, our activity studies show that the IDD has an inhibitory effect on the PtkA enzymatic activity. This suggests that the IDD masks the region of Tyr262, thereby restricting the accessibility of this key residue involved in the autophosphorylation of PtkA. Based on the above observations, we propose two conformational states for PtkA: (i) open state, where the IDD is away from the autophosphorylation site, increasing the accessibility of Tyr262 for the phosphorylation, and (ii) closed state, where the Tyr262 is masked by the IDD. Multisite phosphorylation of IDD releases the IDD from the KCD, thereby uncovering Tyr262 and consequently increasing the PtkA activity. Phosphorylation of PtkA by serine/threonine kinase induces conformational changes of IDD, which abolish IDD movement and promote the open state of PtkA, where the Tyr262 is accessible to the phosphorylation. However, the exact catalytic mechanism behind the modulation of the PtkA activity can be a subject for further investigation.
      Based on our findings, we propose the following model to explain the potential function for this unusual IDD in PtkA, in the context of overall regulation of Mtb virulence (Fig. 6). MptpA is secreted from the mycobacterial cytosol to the cytosol of infected macrophages by an unknown translocation mechanism. During the phagosome–macrophage fusion, MptpA migrates into the macrophage cytosol. The phosphorylation state of MptpA at this point is unclear; however, we know that activated PtkA catalyzes the phosphorylation of MptpA, thereby increasing its activity (
      • Zhou P.
      • Li W.
      • Wong D.
      • Xie J.
      • Av-Gay Y.
      Phosphorylation control of protein tyrosine phosphatase A activity in Mycobacterium tuberculosis.
      ) which possibly plays a key role in the translocation of MptpA (Fig. 6, left inset) to the cytosol of infected macrophages. Moreover, multisite phosphorylation of IDD of PtkA shifts the balance between the open and closed states of the IDD toward a more open state. As a consequence of this PTM of the IDD, PtkA might now possess enhanced activity toward phosphorylating MptpA and might also become anchored to the Mtb cell wall (Fig. 6, right). Phosphorylation-driven membrane anchoring has been observed previously for protein kinase C (
      • Pepio A.M.
      • Sossin W.S.
      Membrane translocation of novel protein kinase Cs is regulated by phosphorylation of the C2 domain.
      ). In summary, the structural and functional knowledge of PtkA gained in this study is crucial for understanding the MptpA–PtkA interactions, catalytic mechanism, and in general the role of the kinase–phosphatase regulatory system in Mtb virulence.
      Figure thumbnail gr6
      Figure 6Potential role of PtkA and their unusual IDD in the M. tuberculosis virulence. Left, role of the MptpA in the Mtb virulence. MptpA interacts with the host endogenous signaling pathway upon binding to the H subunit of the vacuolar proton pump (V-H+ATPase) and dephosphorylation of human VPS33B, being a part of the homotypic fusion and protein-sorting (HOPS) complexes. Dephosphorylation of VPS33B suppresses the phagosome–lysosome fusion and promotes Mtb persistency. The mechanism of migration of MptpA from the cytosol of Mtb to the macrophage cytosol trough the mycobacterial cell wall is unknown. Right, schematic representation of Mtb, including its complex cell wall structure (according to Ref.
      • Bansal-Mutalik R.
      • Nikaido H.
      Mycobacterial outer membrane is a lipid bilayer and the inner membrane is unusually rich in diacyl phosphatidylinositol dimannosides.
      ) and the role of PtkA and its unusual IDD. IDD of PtkA regulates the accessibility of the autophosphorylation site, which is located in the KCD. Long-range transient interactions between those domains generate an open and closed state of PtkA, which control the protein autoactivity. PTM of IDD promotes the open state of PtkA, increasing its ability to autophosphorylate. High active PtkA regulates the phosphorylation of MptpA, thereby modulating MptpA activity (
      • Zhou P.
      • Li W.
      • Wong D.
      • Xie J.
      • Av-Gay Y.
      Phosphorylation control of protein tyrosine phosphatase A activity in Mycobacterium tuberculosis.
      ) and possibly contributing to its translocation trough the mycobacterial cell wall. On the other hand, the presence of IDD in the PtkA structure can play a role in the subcellular relocalization of PtkA to the cell membrane. The translocation of PtkA can be triggered by the autophosphorylation of the KCD or post-translational multisite phosphorylation of IDD. Right corner, the dynamic behavior of IDD in response to change in pH (
      • Niesteruk A.
      • Hutchison M.
      • Sreeramulu S.
      • Jonker H.R.A.
      • Richter C.
      • Abele R.
      • Bock C.
      • Schwalbe H.
      Structural characterization of the intrinsically disordered domain of Mycobacterium tuberculosis protein tyrosine kinase A.
      ).

      Experimental procedures

      Cloning, expression, and purification of PtkA

      The pET151/D-TOPO plasmid encoding WT PtkA (Rv2232, Met1–Val291) was transformed into Escherichia coli BL21 (DE3) cells for expression. The PtkA subdomains IDDPtkA (Met1–Leu81) and KCDPtkA (Gly76–Val291) were generated by PCR. WT PtkA DNA was used as a template for DNA replication. The PCR product was amplified into the pRS-45 vector using NedI and BamHI as restriction sites. The efficiency of the insertion was confirmed via nucleotide sequencing. The PtkA DNA plasmid generated via PCR was transformed into E. coli BL21 (DE3) for protein expression. The expression of recombinant PtkA was performed in LB medium for unlabeled protein and in M9 medium for 15N/13C uniformly labeled PtkA. Cell cultures (supplemented with 1 mm ampicillin) were grown at 37 °C with aeration (120 rpm) to A600 nm ∼0.7, incubated at 0 °C for 15 min, and induced using 1 mm isopropyl 1-thio-β-d-galactopyranoside for overexpression. Triple (2H,13C,15N)-labeled PtkA was expressed in rich growth medium solution labeled with stable isotopes (2H (>95%), 13C, 15N) (Silantes). After adaptation of the cells to 70 and 100% D2O at A600 nm ∼0.5, the cells were washed twice in PBS/D2O and resuspended in E. coli-OD2 CDN Silantes medium at A600 nm ∼0.7. The protein expression was induced at A600 nm ∼1.0 using 1 mm isopropyl 1-thio-β-d-galactopyranoside. After incubation overnight for unlabeled or uniformly 15N/13C-labeled PtkA and 12 h for 2H,13C,15N-labeled PtkA at 16 °C with aeration (120 rpm), the cells were centrifuged (4,000 × g, 45 min, 4 °C), and the cell pellet was resuspended in lysis buffer (300 mm NaCl, 50 mm Tris-HCl, pH 8.0, 10 mm 2-mercaptoethanol), supplemented with complete protease inhibitor (1 tablet/100 ml, EDTA-free, Roche Applied Science). The cells were disrupted for 15 min using M-110P microfluidizer (15,000 p.s.i.). The supernatant was separated from cell residues by centrifugation (18,000 × g, 45 min, 4 °C). The soluble protein fractions were applied to a nickel-nitrilotriacetic acid Fast Flow Column (GE Healthcare), following the manufacturer's recommendations. His6-tag cleavage was performed via dialysis (300 mm NaCl, 50 mm Tris-HCl, pH 8.0, 10 mm 2-mercaptoethanol) overnight at 4 °C using tobacco etch virus protease (1 mg/ml) and separated using the nickel-nitrilotriacetic acid Fast Flow Column. Subsequently, preparative size-exclusion chromatography was performed on a HiLoad 26/60 Superdex 75 column (GE Healthcare) in running buffer (50 mm HEPES-NaOH, pH 7.5, 300 mm NaCl, and 10 mm DTT) to increase protein purity. The eluted fractions were analyzed by SDS-PAGE. Fractions containing pure protein were pooled and stored at −80 °C or immediately used for further experimental procedures.

      Expression and purification of MptpA

      The plasmid pET16bTEV containing the MptpA sequence (Rv2234, Met1–Ser163) was transformed into BL21 (DE3) pLysS E. coli cells, expressed, and purified as described in detail previously (
      • Zhou P.
      • Li W.
      • Wong D.
      • Xie J.
      • Av-Gay Y.
      Phosphorylation control of protein tyrosine phosphatase A activity in Mycobacterium tuberculosis.
      ).

      Peptide synthesis

      The solid-phase peptide synthesis was carried out by standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. The C-terminal residue was manually attached to a 2-chlorotrityl chloride resin. Peptides were purified by reversed-phase HPLC and characterized using electrospray ionization MS and analytical HPLC.

      Luciferase assay

      The autophosphorylation activity of the protein was determined using a Kinase-Glo® luminescent kinase assay (Promega). 20 μm PtkA was added to the assay buffer (300 mm NaCl, 50 mm Tris-HCl, pH 8.0, 10 mm DTT) containing different amounts of ATP (1, 2.5, 5, 7.5, 10 μm; Fermentas) in the presence of 10 mm MgCl2. After overnight incubation at 25 °C, the assay mix (50 μl) was applied on to a 96-well plate (white; E&K Scientific EK-25075), and the luciferase mixture (50 μl) was added. After incubation for 2 h, the luminescence was measured using a VeritasTM microplate luminometer.

      Autophosphorylation reaction

      The autophosphorylation reaction of PtkA was performed using unlabeled 100 μm protein and 10 mm ATP in 50 mm HEPES-NaOH, pH 7.5, buffer containing 300 mm NaCl, 10 mm DTT, and 10 mm MgCl2. After incubation overnight at 25 °C, MALDI-MS as well as 1D 31P NMR were measured.

      Dephosphorylation/phosphorylation reaction

      The autophosphorylated PtkA (see above) was incubated overnight with MptpA at a concentration ratio of 1:0.5 and 1:1 and analyzed by 1D 31P NMR and MALDI-MS. The dephosphorylation assay with PtkA phosphorylated peptide (Asp264–Lys270/pTyr262) was performed in a 3-mm NMR tube containing 50 mm HEPES-NaOH, pH 7.5, buffer containing 300 mm NaCl, 10 mm MgCl2, 90% H2O/10% D2O, and 3 mm peptide. NMR spectra were acquired directly after the addition of 40 μm MptpA.

      NMR spectroscopy

      NMR experiments were performed at 298 K on Bruker spectrometers (600, 700, 800, 900, or 950 MHz) equipped with TXI-HCN cryogenic probes. The protein samples (0.1–0.3 mm) were measured in NMR buffer (50 mm HEPES-NaOH, pH 7.5, 300 mm NaCl, 10 mm DTT, 10 mm MgCl2, 90% H2O/10% D2O) using 3-mm NMR tubes. The spectrometer was locked on D2O.

      Resonance assignment experiments

      For the backbone assignment of PtkA, a set of 3D triple-resonance experiments were collected, including HNHA, HNCO, HNCA, HN(CO)CA, HNCACB, and HN(CO)CACB experiments on double- and/or triple-labeled protein. The side-chain assignment of the aliphatic resonances was obtained using HCCCONH and CCCONH experiments. 2D HBCBCGCDHD and HBCBCGCDCEHE spectra and an aromatic 13C-filtered TOCSY spectrum were used for the assignment of the aromatic side-chain resonances. For assignment and NOE-based distance restraints, 3D 1H,1H,15N NOESY-HSQC (mixing time, 120 ms) and aromatic and aliphatic 3D 1H,1H,13C NOESY-HSQC (mixing time, 75 ms and 120 ms) in H2O were collected.

      Heteronuclear relaxation experiments

      15N relaxation experiments were performed at 298 K on a 700-MHz spectrometer. The R1 longitudinal 15N relaxation rates were obtained from a series of experiments acquired with varying delays of 100, 200, 400, 600, 800, 1200, 1600, 2000, 2400, and 3000 ms. R2 transverse 15N relaxation rates were determined from a series of spectra using the following delays: 16.96, 33.92, 50.88, 67.84, 84.80, 101.76, 118.72, and 135.68 ms. The 15N{1H}-heteronuclear NOE values were obtained from the analysis of peak intensity ratio (INOE/INONOE) from the recorded spectra with and without saturation of amide protons. The order parameter, S2, was determined using TENSOR2 (
      • Dosset P.
      • Hus J.-C.
      • Blackledge M.
      • Marion D.
      Efficient analysis of macromolecular rotational diffusion from heteronuclear relaxation data.
      ).

      Hydrogen–deuterium exchange

      For the hydrogen–deuterium exchange experiment, 150 μl (200 μm) of protein in NMR buffer was lyophilized and dissolved in the equivalent volume of deuterium oxide (D2O). A series of 2D 1H,15N HSQC spectra were recorded immediately after the addition of D2O, for a duration of 15 min within the first hour and subsequently for 30 min, for a total duration of 19 h. As a reference for the evaluation of spectra, a 2D 1H,15N HSQC spectrum of the protein (200 μm) in NMR buffer (with 90% H2O/10% D2O) was measured before. The hydrogen–deuterium exchange rate for each residue was determined by fitting the amide peak intensities to exponential decays over the time for each residue (I(t) = I(0)·exp(−kex·t)), where I(t) and I(0) are the intensities at the given time t and t = 0, and kex is the rate constant of the hydrogen–deuterium exchange reaction.

      Temperature series

      The 2D 1H,15N HSQC spectra were recorded at 3 K intervals ranging from 283 to 301 K on a 700-MHz spectrometer.

      Paramagnetic spin labeling

      Single-cysteine mutations of PtkA (which contains one WT cysteine) were engineered using the QuikChange site-directed mutagenesis protocol (Invitrogen). The WT cysteine was mutated to alanine (C61A). This point-mutated DNA was used as a template for preparing single-cysteine mutations at Ala10 (C61A/A10C) and Ser41 (C61A/S41C). All mutations were confirmed by DNA sequencing of the entire ORF (Eurofins). Protein mutants were expressed in competent E. coli cells (BL21 (DE3), New England Biolabs), grown in M9 minimal medium enriched with 15N-ammonium chloride, and purified as described above. The SDSL was carried out using the MTSL spin label. Protein was dissolved in the DTT-free, 50 mm HEPES/NaOH (pH 7.5) labeling buffer, containing 300 mm NaCl, 10 mm MgCl2 and concentrated to 500 μm using Vivaspin concentrators (molecular weight cut-off of 10,000; GE Healthcare). For spin labeling, a 10-fold molar access of MTSL over cysteine was added. MTSL stock solution was prepared by dissolving 10 mg of MTSL in 50 μl of DMSO. After the addition of MTSL to the protein solution, the reaction mixture was incubated in the dark overnight at 25 °C (WT) and 4 °C (C61A/A10C and C61A/S41C). To remove excess spin label, the labeling buffer was exchanged (50 mm HEPES-NaOH, pH 7.5, 300 mm NaCl, 10 mm MgCl2) using a PD-10 desalting column (Sephadex G-25 medium, GE Healthcare). The modified protein was concentrated to 300–500 μm (Vivaspin concentrators, molecular weight cut-off of 10,000) for NMR. The spin label was reduced by the addition of 5 mm ascorbate from a freshly prepared stock solution of 500 mm, to yield an ∼10-fold molar excess of reducing agent over the spin label.

      NMR data analysis

      All spectra were processed using Topspin version 3.2 (Bruker Biospin) and analyzed using SPARKY version 3.114 (
      • Goddard T.D.
      • Kneller D.G.
      ).

      Structure calculation

      Structure calculations were performed using the software packages CYANA (
      • López-Méndez B.
      • Güntert P.
      Automated protein structure determination from NMR spectra.
      ,
      • Güntert P.
      Automated NMR structure calculation with CYANA.
      ,
      • Güntert P.
      Automated NMR protein structure calculation.
      ) and ARIA/CNS (
      • Linge J.P.
      • O'Donoghue S.I.
      • Nilges M.
      Automated assignment of ambiguous nuclear overhauser effects with ARIA.
      ,
      • Linge J.P.
      • Habeck M.
      • Rieping W.
      • Nilges M.
      ARIA: automated NOE assignment and NMR structure calculation.
      ). A conventional structure calculation with fully automated NOESY cross-peak assignment failed due to the limited completeness of the resonance assignment (about 75%), especially because the missing assignments reside in or near the center of the protein. Generally, a completeness of more than 90% (
      • Güntert P.
      Automated NMR protein structure calculation.
      ) would suffice for a reliable automated NOESY assignment. The NOESY cross-peaks originating from unassigned regions may be misinterpreted to distances between atoms with coinciding assigned resonances, consequently disrupting the structure and causing the convergence to fail. To circumvent this problem, we made use of initial homology models to assist the initial iterative structure calculation with automated NOESY cross-peak assignment by CYANA. These structures were built by Swiss-Model (
      • Benkert P.
      • Biasini M.
      • Schwede T.
      Toward the estimation of the absolute quality of individual protein structure models.
      ) using a selection of homologous structures (e.g. Protein Data Bank codes 3MC1, 3SD7, 4EX6, 2AH5, 2NYV, 2YY6, 2HCF, 2HI0, 2HDO, 2HDZ, 4EEL, 3L5K, 2FDR, and 3KLZ), which have a proper range (around residues 80–289) and sufficient sequence identity (above 20%). For the regions that are consistent with the TALOS-N secondary structure prediction (
      • Shen Y.
      • Bax A.
      Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks.
      ), very ample CA–CA distance restraints (up to 20 Å) were extracted, which merely define the global shape of the molecule, thereby just avoiding NOE misassignments between atoms that are actually not close to each other. The unambiguous assigned NOEs in the 3D 1H,1H,13C NOESY-HSQC (aliphatic and aromatic) and 3D 1H,1H,15N NOESY-HSQC spectra were validated and inspected by using of Sparky version 3.114 (
      • Goddard T.D.
      • Kneller D.G.
      ) and subsequently used for the structure refinement (without the additional global distance restraints). The chemical shift tolerances were set to 0.015 and 0.025 ppm for the bound protons and other protons, respectively, and 0.20 ppm for the heavy atoms. In addition to NOE data, hydrogen bond distances and dihedral angle restraints for the secondary structure elements (based on TALOS-N predictions and confirmed with the Swiss-Models and initial structure calculations), as well as 1D (HN) residual dipolar couplings and 3J(HN,Hα) coupling constant restraints were included in the structure calculation with CYANA (100 structures/iteration, 15,000 refinement steps). The final bundle of 20 best (lowest-energy) structures was used as input for a refinement in explicit water with CNS 1.1 (
      • Brünger A.T.
      • Adams P.D.
      • Clore G.M.
      • DeLano W.L.
      • Gros P.
      • Grosse-Kunstleve R.W.
      • Jiang J.S.
      • Kuszewski J.
      • Nilges M.
      • Pannu N.S.
      • Read R.J.
      • Rice L.M.
      • Simonson T.
      • Warren G.L.
      Crystallography & NMR system: a new software suite for macromolecular structure determination.
      ) using the ARIA 1.2 setup and protocols (
      • Linge J.P.
      • Williams M.A.
      • Spronk C.A.
      • Bonvin A.M.
      • Nilges M.
      Refinement of protein structures in explicit solvent.
      ). The 3J(HN,Hα) coupling constants were obtained via a 3D HN,Hα HMQC experiment (
      • Vuister G.W.
      • Bax A.
      Measurement of four-bond HN-Hα J-couplings in staphylococcal nuclease.
      ,
      • Vuister G.W.
      • Bax A.
      Quantitative J correlation: a new approach for measuring homonuclear three-bond J(HNHα) coupling constants in 15N-enriched proteins.
      ) and directly included in the structure calculation by using the Karplus relationship. 1D (HN) residual dipolar couplings were measured in Pf1 bacteriophages (6 mg/ml, strain LP11-92, ASLA Biotech) as alignment medium at 700 MHz and 298 K. The 1D (HN) were extracted from IPAP-15N,1H HSQC (
      • Ottiger M.
      • Delaglio F.
      • Bax A.
      Measurement of J and dipolar couplings from simplified two-dimensional NMR spectra.
      ) spectra. Residual dipolar couplings were included in the structure calculation as direct susceptibility anisotropy restraints (SANI) and examined using the program PALES (
      • Zweckstetter M.
      • Bax A.
      Prediction of sterically induced alignment in a dilute liquid crystalline phase: aid to protein structure determination by NMR.
      ). The rhombicity and axial coefficients for the final alignment tensor were as follows: R = 0.2 and D = 4.6 with a correlation coefficient r = 0.97. Moreover, 15N relaxation data (T1/T2 values) were included as diffusion anisotropy restraints in this final refinement stage. The values for the anisotropy (1.2) and rhombicity (0.3) of the rotational diffusion tensor from the final structure bundle were determined using the program Tensor 2 (
      • Dosset P.
      • Hus J.-C.
      • Blackledge M.
      • Marion D.
      Efficient analysis of macromolecular rotational diffusion from heteronuclear relaxation data.
      ). The impact of the diffusion restraints on the structure refinement is, however, negligible, as the anisotropy of the molecule turned out to be moderate and smaller than the suggested minimal value of 1.5 (
      • Tjandra N.
      • Garrett D.S.
      • Gronenborn A.M.
      • Bax A.
      • Clore G.M.
      Defining long range order in NMR structure determination from the dependence of heteronuclear relaxation times on rotational diffusion anisotropy.
      ). The overall rotational correlation time (tc = 12.9 ns) is in agreement with the value predicted for a monomeric protein (tc = 14.4 ns at 298 K) by the program HYDRONMR (
      • García de la Torre J.
      • Huertas M.L.
      • Carrasco B.
      HYDRONMR: prediction of NMR relaxation of globular proteins from atomic-level structures and hydrodynamic calculations.
      ).

      Author contributions

      A. N., H. R. A. J., S. S., and H. S. data curation; A. N. and H. R. A. J. formal analysis; A. N., H. R. A. J., and C. R. validation; A. N., H. R. A. J., S. S., and H. S. investigation; A. N., H. R. A. J., C. R., V. L., and S. S. methodology; A. N. writing-original draft; H. R. A. J., S. S., and H. S. writing-review and editing; V. L. resources; S. S. and H. S. conceptualization; S. S. and H. S. supervision; S. S. visualization; H. S. funding acquisition.

      Acknowledgments

      We thank Prof. Av-Gay for providing the PtkA plasmid vector and helpful discussions. We thank Dr. Tanja Stehle for participation in the early stages of this work.

      Supplementary Material

      References

        • Cole S.T.
        • Brosch R.
        • Parkhill J.
        • Garnier T.
        • Churcher C.
        • Harris D.
        • Gordon S.V.
        • Eiglmeier K.
        • Gas S.
        • Barry 3rd, C.E.
        • Tekaia F.
        • Badcock K.
        • Basham D.
        • Brown D.
        • Chillingworth T.
        • et al.
        Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence.
        Nature. 1998; 393 (9634230): 537-544
        • Pablos-Mendez A.
        • Gowda D.K.
        • Frieden T.R.
        Controlling multidrug-resistant tuberculosis and access to expensive drugs: a rational framework.
        Bull. World Health Organ. 2002; 80 (12132008 discussion 495–500): 489-495
        • Shah N.S.
        • Wright A.
        • Bai G.-H.
        • Barrera L.
        • Boulahbal F.
        • Martín-Casabona N.
        • Drobniewski F.
        • Gilpin C.
        • Havelková M.
        • Lepe R.
        • Lumb R.
        • Metchock B.
        • Portaels F.
        • Rodrigues M.F.
        • Rüsch-Gerdes S.
        • et al.
        Worldwide emergence of extensively drug-resistant tuberculosis.
        Emerg. Infect. Dis. 2007; 13 (17552090): 380-387
        • World Health Organization
        Global Tuberculosis Report. World Health Organization, Geneva2015
        • van den Boogaard J.
        • Kibiki G.S.
        • Kisanga E.R.
        • Boeree M.J.
        • Aarnoutse R.E.
        New drugs against tuberculosis: problems, progress, and evaluation of agents in clinical development.
        Antimicrob. Agents Chemother. 2009; 53 (19075046): 849-862
        • Grangeasse C.
        • Nessler S.
        • Mijakovic I.
        Bacterial tyrosine kinases: evolution, biological function and structural insights.
        Philos. Trans. R. Soc. Lond. B Biol. Sci. 2012; 367 (22889913): 2640-2655
        • Parish T.
        Two-component regulatory systems of mycobacteria.
        Microbiol. Spectrum. 2014; 2 (26082118 MGM2-0010-2013)
        • Av-Gay Y.
        • Everett M.
        The eukaryotic-like Ser/Thr protein kinases of Mycobacterium tuberculosis.
        Trends Microbiol. 2000; 8 (10785641): 238-244
        • Koul A.
        • Choidas A.
        • Treder M.
        • Tyagi A.K.
        • Drlica K.
        • Singh Y.
        • Ullrich A.
        Cloning and characterization of secretory tyrosine phosphatases of Mycobacterium tuberculosis.
        J. Bacteriol. 2000; 182 (10986245): 5425-5432
        • Greenstein A.E.
        • Grundner C.
        • Echols N.
        • Gay L.M.
        • Lombana T.N.
        • Miecskowski C.A.
        • Pullen K.E.
        • Sung P.Y.
        • Alber T.
        Structure/function studies of Ser/Thr and Tyr protein phosphorylation in Mycobacterium tuberculosis.
        J. Mol. Microbiol. Biotechnol. 2005; 9 (16415590): 167-181
        • Kusebauch U.
        • Ortega C.
        • Ollodart A.
        • Rogers R.S.
        • Sherman D.R.
        • Moritz R.L.
        • Grundner C.
        Mycobacterium tuberculosis supports protein tyrosine phosphorylation.
        Proc. Natl. Acad. Sci. 2014; 111 (24927537): 9265-9270
        • Prisic S.
        • Dankwa S.
        • Schwartz D.
        • Chou M.F.
        • Locasale J.W.
        • Kang C.-M.
        • Bemis G.
        • Church G.M.
        • Steen H.
        • Husson R.N.
        Extensive phosphorylation with overlapping specificity by Mycobacterium tuberculosis serine/threonine protein kinases.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107 (20368441): 7521-7526
        • Wong D.
        • Chao J.D.
        • Av-Gay Y.
        Mycobacterium tuberculosis-secreted phosphatases: from pathogenesis to targets for TB drug development.
        Trends Microbiol. 2013; 21 (23084287): 100-109
        • Chow K.
        • Ng D.
        • Stokes R.
        • Johnson P.
        Protein tyrosine phosphorylation in Mycobacterium tuberculosis.
        FEMS Microbiol. Lett. 1994; 124 (7529204): 203-207
        • Wong D.
        • Bach H.
        • Sun J.
        • Hmama Z.
        • Av-Gay Y.
        Mycobacterium tuberculosis protein tyrosine phosphatase (PtpA) excludes host vacuolar-H+-ATPase to inhibit phagosome acidification.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108 (22087003): 19371-19376
        • Stehle T.
        • Sreeramulu S.
        • Löhr F.
        • Richter C.
        • Saxena K.
        • Jonker H.R.A.
        • Schwalbe H.
        The apo-structure of the low molecular weight protein-tyrosine phosphatase A (MptpA) from Mycobacterium tuberculosis allows for better target-specific drug development.
        J. Biol. Chem. 2012; 287 (22888002): 34569-34582
        • Zhou P.
        • Li W.
        • Wong D.
        • Xie J.
        • Av-Gay Y.
        Phosphorylation control of protein tyrosine phosphatase A activity in Mycobacterium tuberculosis.
        FEBS Lett. 2015; 589 (25535696): 326-331
        • Bach H.
        • Wong D.
        • Av-Gay Y.
        Mycobacterium tuberculosis PtkA is a novel protein tyrosine kinase whose substrate is PtpA.
        Biochem. J. 2009; 420 (19366344): 155-160
        • Chao J.D.
        • Wong D.
        • Av-Gay Y.
        Microbial protein-tyrosine kinases.
        J. Biol. Chem. 2014; 289 (24554699): 9463-9472
        • Zhou P.
        • Wong D.
        • Li W.
        • Xie J.
        • Av-Gay Y.
        Phosphorylation of Mycobacterium tuberculosis protein tyrosine kinase A PtkA by Ser/Thr protein kinases.
        Biochem. Biophys. Res. Commun. 2015; 467 (26417687): 421-426
        • Wong D.
        • Li W.
        • Chao J.D.
        • Zhou P.
        • Narula G.
        • Tsui C.
        • Ko M.
        • Xie J.
        • Martinez-Frailes C.
        • Av-Gay Y.
        Protein tyrosine kinase, PtkA, is required for Mycobacterium tuberculosis growth in macrophages.
        Sci. Rep. 2018; 8 (29317718): 155
        • Niesteruk A.
        • Hutchison M.
        • Sreeramulu S.
        • Jonker H.R.A.
        • Richter C.
        • Abele R.
        • Bock C.
        • Schwalbe H.
        Structural characterization of the intrinsically disordered domain of Mycobacterium tuberculosis protein tyrosine kinase A.
        FEBS Lett. 2018; 592 (29494752): 1233-1245
        • Baxter N.J.
        • Williamson M.P.
        Temperature dependence of 1H chemical shifts in proteins.
        J. Biomol. NMR. 1997; 9 (9255942): 359-369
        • Wu J.
        • Ohta N.
        • Zhao J.-L.
        • Newton A.
        A novel bacterial tyrosine kinase essential for cell division and differentiation.
        Proc. Natl. Acad. Sci. 1999; 96 (10557274): 13068-13073
        • Zhao X.
        • Lam J.S.
        WaaP of Pseudomonas aeruginosa is a novel eukaryotic type protein-tyrosine kinase as well as a sugar kinase essential for the biosynthesis of core lipopolysaccharide.
        J. Biol. Chem. 2002; 277 (11741974): 4722-4730
        • Ostrovsky P.C.
        • Maloy S.
        Protein phosphorylation on serine, threonine, and tyrosine residues modulates membrane-protein interactions and transcriptional regulation in Salmonella typhimurium.
        Genes Dev. 1995; 9 (7544316): 2034-2041
      1. Bach, H., Papavinasasundaram, K. G., Wong, D., Hmama, Z., and Av-Gay, Y., Mycobacterium tuberculosis virulence is mediated by PtpA dephosphorylation of human vacuolar protein sorting 33B. Cell Host Microbe 3, 316–322

        • Pepio A.M.
        • Sossin W.S.
        Membrane translocation of novel protein kinase Cs is regulated by phosphorylation of the C2 domain.
        J. Biol. Chem. 2001; 276 (11073945): 3846-3855
        • Dosset P.
        • Hus J.-C.
        • Blackledge M.
        • Marion D.
        Efficient analysis of macromolecular rotational diffusion from heteronuclear relaxation data.
        J. Biomol. NMR. 2000; 16 (10718609): 23-28
        • Goddard T.D.
        • Kneller D.G.
        SPARKY version 3.105. University of California, San Francisco2002
        • López-Méndez B.
        • Güntert P.
        Automated protein structure determination from NMR spectra.
        J. Am. Chem. Soc. 2006; 128 (17017791): 13112-13122
        • Güntert P.
        Automated NMR structure calculation with CYANA.
        in: Downing A.K. Protein NMR Techniques. Humana Press, Totowa, NJ2004: 353-378
        • Güntert P.
        Automated NMR protein structure calculation.
        Prog. Nucl. Magn. Reson. Spectrosc. 2003; 43: 105-125
        • Linge J.P.
        • O'Donoghue S.I.
        • Nilges M.
        Automated assignment of ambiguous nuclear overhauser effects with ARIA.
        Methods Enzymol. 2001; 339 (11462826): 71-90
        • Linge J.P.
        • Habeck M.
        • Rieping W.
        • Nilges M.
        ARIA: automated NOE assignment and NMR structure calculation.
        Bioinformatics. 2003; 19 (12538267): 315-316
        • Bansal-Mutalik R.
        • Nikaido H.
        Mycobacterial outer membrane is a lipid bilayer and the inner membrane is unusually rich in diacyl phosphatidylinositol dimannosides.
        Proc. Natl. Acad. Sci. U.S.A. 2014; 111 (24639491): 4958-4963
        • Benkert P.
        • Biasini M.
        • Schwede T.
        Toward the estimation of the absolute quality of individual protein structure models.
        Bioinformatics. 2011; 27 (21134891): 343-350
        • Shen Y.
        • Bax A.
        Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks.
        J. Biomol. NMR. 2013; 56 (23728592): 227-241
        • Brünger A.T.
        • Adams P.D.
        • Clore G.M.
        • DeLano W.L.
        • Gros P.
        • Grosse-Kunstleve R.W.
        • Jiang J.S.
        • Kuszewski J.
        • Nilges M.
        • Pannu N.S.
        • Read R.J.
        • Rice L.M.
        • Simonson T.
        • Warren G.L.
        Crystallography & NMR system: a new software suite for macromolecular structure determination.
        Acta Crystallogr. D Biol. Crystallogr. 1998; 54 (9757107): 905-921
        • Linge J.P.
        • Williams M.A.
        • Spronk C.A.
        • Bonvin A.M.
        • Nilges M.
        Refinement of protein structures in explicit solvent.
        Proteins. 2003; 50 (12557191): 496-506
        • Vuister G.W.
        • Bax A.
        Measurement of four-bond HN-Hα J-couplings in staphylococcal nuclease.
        J. Biomol. NMR. 1994; 4 (8019134): 193-200
        • Vuister G.W.
        • Bax A.
        Quantitative J correlation: a new approach for measuring homonuclear three-bond J(HNHα) coupling constants in 15N-enriched proteins.
        J. Am. Chem. Soc. 1993; 115: 7772-7777
        • Ottiger M.
        • Delaglio F.
        • Bax A.
        Measurement of J and dipolar couplings from simplified two-dimensional NMR spectra.
        J. Magn. Reson. 1998; 131 (9571116): 373-378
        • Zweckstetter M.
        • Bax A.
        Prediction of sterically induced alignment in a dilute liquid crystalline phase: aid to protein structure determination by NMR.
        J. Am. Chem. Soc. 2000; 122: 3791-3792
        • Tjandra N.
        • Garrett D.S.
        • Gronenborn A.M.
        • Bax A.
        • Clore G.M.
        Defining long range order in NMR structure determination from the dependence of heteronuclear relaxation times on rotational diffusion anisotropy.
        Nat. Struct. Biol. 1997; 4 (9187651): 443-449
        • García de la Torre J.
        • Huertas M.L.
        • Carrasco B.
        HYDRONMR: prediction of NMR relaxation of globular proteins from atomic-level structures and hydrodynamic calculations.
        J. Magn. Reson. 2000; 147 (11042057): 138-146