Identification of Ser/Thr kinase and Forkhead Associated Domains in <i>Mycobacterium ulcerans:</i> Characterization of Novel Association between Protein Kinase Q and MupFHA

<div><p>Background</p><p><i>Mycobacterium ulcerans</i>, the causative agent of Buruli ulcer in humans, is unique among the members of <i>Mycobacterium</i> genus due to the presence of the virulence determinant megaplasmid pMUM001. This plasmid encodes multiple virulence-associated genes, including <i>mup011</i>, which is an uncharacterized Ser/Thr protein kinase (STPK) PknQ.</p><p>Methodology/Principal Findings</p><p>In this study, we have characterized PknQ and explored its interaction with MupFHA (Mup018c), a FHA domain containing protein also encoded by pMUM001. MupFHA was found to interact with PknQ and suppress its autophosphorylation. Subsequent protein-protein docking and molecular dynamic simulation analyses showed that this interaction involves the FHA domain of MupFHA and PknQ activation loop residues Ser¹⁷⁰ and Thr¹⁷⁴. FHA domains are known to recognize phosphothreonine residues, and therefore, MupFHA may be acting as one of the few unusual FHA-domain having overlapping specificity. Additionally, we elucidated the PknQ-dependent regulation of MupDivIVA (Mup012c), which is a DivIVA domain containing protein encoded by pMUM001. MupDivIVA interacts with MupFHA and this interaction may also involve phospho-threonine/serine residues of MupDivIVA.</p><p>Conclusions/Significance</p><p>Together, these results describe novel signaling mechanisms in <i>M. ulcerans</i> and show a three-way regulation of PknQ, MupFHA, and MupDivIVA. FHA domains have been considered to be only pThr specific and our results indicate a novel mechanism of pSer as well as pThr interaction exhibited by MupFHA. These results signify the need of further re-evaluating the FHA domain –pThr/pSer interaction model. MupFHA may serve as the ideal candidate for structural studies on this unique class of modular enzymes.</p></div

Conserved FHA-domains in <i>M. ulcerans</i>.

<p>Conserved FHA-domains in <i>M. ulcerans</i>.</p

Docking analysis of PknQ with MupFHA domain.

<p>(<b>A</b>) Homology modeling derived structural models showing docking of wild-type PknQ (stick diagram) with the wild-type FHA domain variable loop region of MupFHA (green ribbon diagram). Phosphate group (orange) has been added to Ser¹⁷⁰ and Thr¹⁷⁴ of PknQ and the phosphorylated residues have been renamed as Sep170 and Tpo174, respectively. The red encircled region of interaction has been enlarged in (<b>B</b>). The residues Arg⁴¹, Arg⁵³, Ser⁵⁵, Arg⁵⁶ and Ser⁷⁵ of MupFHA show stable interactions with the PknQ activation loop and form H-bonds with the negatively charged pSer¹⁷⁰ (Sep170) and pThr¹⁷⁴ (Tpo174). (<b>C</b>) Enlarged region of interaction between PknQ-pThr¹⁷⁴ and MupFHA. Canonical interaction of pThr¹⁷⁴ is observed showing H-bonds with Arg⁵³, Ser⁵⁵ and Ser⁷⁵ of MupFHA (see text). (<b>D</b>) Enlarged region of interaction between PknQ-pSer¹⁷⁰ and MupFHA. PknQ-pSer¹⁷⁰ is shown to be anchored by the residues Arg⁴¹ and Arg⁵⁶ of MupFHA. (<b>E</b>) Region of interaction between PknQ-pThr¹⁷⁴ and MupFHA^S55A (in red stick). (<b>F</b>) Region of interaction between PknQ-pSer¹⁷⁰ and MupFHA^R41A (in red stick). Both (E) and (F) show the loss of H-bond network and thus destabilized interaction between PknQ and MupFHA.</p

<i>In silico</i> analysis of <i>M. ulcerans</i> STPKs.

<p>(<b>A</b>) Genomic alignment of <i>M. ulcerans</i> STPKs showing conserved patterns (NCBI). Genetic patterns show twelve STPKs present in the chromosome in addition to one STPK, PknQ, which is encoded by the virulence-associated plasmid pMUM001. (<b>B</b>) Phylogenetic analysis of all <i>M. ulcerans</i> STPKs. The phylogenetic tree was generated using protein FASTA sequences of <i>M. ulcerans</i> STPKs in Phylip. PknQ clearly belongs to the PknF/PknI/Mul_2200 clade.</p

Phosphorylation of MupFHA by PknQ.

<p>(<b>A</b>) <i>In vitro</i> kinase assay showing phosphorylation of MupFHA by PknQ and PknQ^K41M. The upper panel shows a coomassie-stained SDS-PAGE and the lower panel shows the corresponding autoradiogram. MupFHA is phosphorylated by PknQ while no phosphorylation was observed with PknQ^K41M. Surprisingly, in the presence of MupFHA, the level of PknQ phosphorylation was reduced (lanes 1 and 3). (<b>B</b>) Phosphorylation status of MupFHA, co-expressed with PknQ or PknQ^K41M in <i>E. coli</i>, was estimated using ProQ Diamond phosphoprotein staining (upper panel). MupFHA co-expressed with PknQ was found to be phosphorylated (MupFHA-P), while no phosphorylation was observed when it was co-expressed with PknQ^K41M (MupFHA-UP). The same gel was stained with Sypro Ruby stain (lower panel) to show equal loading of both samples. (<b>C</b>) The mutants of PknQ, which showed loss in autophosphorylation potential, were used to assess their phosphotransfer ability on MupFHA. Phosphorylation by wild-type PknQ was taken as 100% and relative phosphorylation was calculated. The experiments were repeated three times and error bars show S.D. of three values. Representative autoradiograms with MupFHA bands are shown above the histograms (left and right panels).</p

Phosphorylation sites of MupFHA and role of FHA domain residues.

<p>(<b>A</b>) PAA analysis of MupFHA phosphorylated by PknQ. The left panel shows ninhydrin-stained phosphoamino acid spots and the right panel shows the corresponding autoradiogram. Phosphorylation was detected on the spot corresponding to Thr(P). (<b>B</b>) Domain architecture of MupFHA analyzed by SMART software. The four threonine residues (Thr⁸, Thr¹²³, Thr²¹⁰, and Thr²¹⁴) that were phosphorylated are marked. The two conserved residues in the FHA domain (Arg⁴¹ and Ser⁵⁵) are also marked. (<b>C</b>) Multiple mutants of MupFHA phosphorylation sites were generated and the loss in phosphorylation by PknQ was assessed. Phosphorylation on wild-type MupFHA was taken as 100% and relative phosphorylation was calculated. As shown in the histogram, maximum loss was observed when Thr²¹⁰ of MupFHA was mutated. The experiment was repeated three times and error bars show S.D. of three individual values. A representative autoradiogram with MupFHA bands is shown above the histogram. (<b>D</b>) Histogram showing phosphorylation of MupFHA and its FHA domain mutants by PknQ. Phosphorylation on wild-type MupFHA was taken as 100% and relative phosphorylation was calculated. A significant loss was observed for the MupFHA^S55A mutant compared to MupFHA^R41A. The experiment was repeated three times and error bars show S.D. of three values. A representative autoradiogram with MupFHA bands is shown above the histogram.</p

Phosphorylated residues of MupDivIVA identified by <i>in vitro</i> kinase assay.

<p>Sequences of the phosphorylated peptides identified in MupDivIVA phosphorylated by PknQ are indicated, as determined by mass spectrometry. Phosphorylated residues (pT/pS) are shown in bold.</p><p>Phosphorylated residues of MupDivIVA identified by <i>in vitro</i> kinase assay.</p

Phosphorylated residues of PknQ identified by <i>in vitro</i> kinase assays.

<p>Sequences of the phosphorylated peptides identified in autophosphorylated PknQ in the presence of MupFHA are indicated, as determined by mass spectrometry. Phosphorylated residues (pT or pS) are shown in bold.</p><p>Phosphorylated residues of PknQ identified by <i>in vitro</i> kinase assays.</p

Phosphorylation of MupDivIVA by PknQ and interaction with MupFHA.

<p>(<b>A</b>) An <i>in vitro</i> kinase assay showing the phosphorylation of MupDivIVA by PknQ and PknQ^K41M. MupDivIVA was phosphorylated by PknQ, while no phosphorylation was observed by PknQ^K41M. The upper panel shows coomassie-stained SDS-PAGE and the lower panel shows the corresponding autoradiogram. (<b>B</b>) MupDivIVA was co-expressed with PknQ or PknQ^K41M in <i>E. coli</i> and the phosphorylation status of MupDivIVA was estimated using ProQ Diamond phosphoprotein staining. As shown in the upper panel, MupDivIVA co-expressed with PknQ was phosphorylated (MupDivIVA-P), while no phosphorylation was observed when it was co-expressed with PknQ^K41M (MupDivIVA-UP). The same gel was stained with Sypro Ruby stain (lower panel) to show equal loading of both samples. (<b>C</b>) PAA analysis of MupDivIVA phosphorylated by PknQ. The left panel shows ninhydrin-stained phosphoamino acid spots and the right panel shows the corresponding autoradiogram. Phosphorylation was detected on the spot corresponding to Thr(P), while minor phosphorylation was also seen on Ser(P). (<b>D</b>) Domain architecture of MupDivIVA analyzed by SMART domain prediction software. The phosphorylated residues (Ser⁴³, Ser⁴⁵, Ser⁴⁹, Thr⁵⁹, Thr⁶⁴, and Thr⁷⁷) are marked. (<b>E</b>) Relative phosphorylation of MupDivIVA phospho-site mutants using PknQ. Multiple mutants of MupDivIVA phosphorylation sites were generated and the loss in phosphorylation by PknQ was assessed. Phosphorylation on wild-type MupDivIVA was taken as 100% and relative phosphorylation was calculated. As shown in the histogram, maximum loss was observed in MupDivIVA^S43/45/49A triple mutant. The experiment was repeated three times and error bars show S.D. of three values.</p

Phosphorylation sites of PknQ.

<p>(<b>A</b>) Phosphoamino acid (PAA) analysis of PknQ_kd autophosphorylation using 2D-TLE. The left panel shows ninhydrin-stained phospho-amino acid spots and the right panel shows the corresponding autoradiogram. Phosphorylation was detected on the spots corresponding to Ser(P) and Thr(P). (<b>B</b>) Domain architecture of PknQ showing catalytic domain (cytosolic) and extracellular FepB-like transporter domain. Four of the phosphorylated residues that are present in the activation loop are marked. (<b>C</b>) Single and double site mutants of PknQ were generated for the two most conserved activation loop residues (Thr¹⁶⁴ and Thr¹⁶⁶) (left histogram). Single site mutants of PknQ were subsequently generated for multiple residues (Ser¹⁷⁰, Thr¹⁷⁴ and Thr²⁶⁰) as identified by mass spectrometry (right histogram). Phosphorylation of PknQ was taken as 100% and relative phosphorylation was estimated. The experiment was repeated three times and error bars show S.D. of three values. A representative autoradiogram is shown above each histogram. PknQ^S170A exhibited the maximum loss in phosphorylation.</p