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

Abstract

Background Mycobacterium ulcerans, the causative agent of Buruli ulcer in humans, is unique among the members of Mycobacterium genus due to the presence of the virulence determinant megaplasmid pMUM001. This plasmid encodes multiple virulence-associated genes, including mup011, which is an uncharacterized Ser/Thr protein kinase (STPK) PknQ.Methodology/Principal FindingsIn 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.Conclusions/SignificanceTogether, these results describe novel signaling mechanisms in M. ulcerans 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.

Systematic Analysis of Mycobacterial Acylation Reveals First Example of Acylation-mediated Regulation of Enzyme Activity of a Bacterial Phosphatase

“…For other proteins, homology modeling was performed as described earlier (53,54). The templates were chosen for each protein, Rv1436 (PDB code 1VC2), Rv1437 (PDB code 1V6S), and Rv2889c (PDB code 1EFU), based on the sequence identities of 61, 49, and 39%, respectively.…”

“…Similar co-expression system has been efficiently used to assess the phosphorylation in previous studies (54,57,62,69). The purified proteins were analyzed for the presence of acetylation by mass spectrometry.…”

The Ser/Thr protein kinase PrkC imprints phenotypic memory in Bacillus anthracis spores by phosphorylating the glycolytic enzyme enolase

“…An indirect ELISA was performed as described earlier with some modifications (47). Briefly, His 6 -tagged Eno (100 ng/well) was dissolved in a coating buffer (carbonate-bicarbonate buffer, pH 9.6) and adsorbed on the surface of a 96-well ELISA plate (Maxisorb, Nunc) for 16 h at 4°C.…”

Buruli Ulcer in Japan

“…Despite the deficiency of a functional STPK gene in Japanese clinical isolates, no apparent bacteriological differences were observed, indicating the possibility that some signaling pathways compensate for STPK function in M. ulcerans subsp. shinshuense [14]. Intriguingly, a Chinese M. ulcerans strain has a 16S rRNA gene identical in sequence to that in M. ulcerans subsp.…”