Article

PknB kinase activity is regulated by phosphorylation in two Thr residues and dephosphorylation by PstP, the cognate phospho-Ser/Thr phosphatase, in Mycobacterium tuberculosis.

Unité de Biochimie Structurale, URA 2185 CNRS, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, cedex 15, France.
Molecular Microbiology (Impact Factor: 5.03). 10/2003; 49(6):1493-508. DOI: 10.1046/j.1365-2958.2003.03657.x
Source: PubMed

ABSTRACT Bacterial genomics revealed the widespread presence of eukaryotic-like protein kinases and phosphatases in prokaryotes, but little is known on their biochemical properties, regulation mechanisms and physiological roles. Here we focus on the catalytic domains of two trans-membrane enzymes, the Ser/Thr protein kinase PknB and the protein phosphatase PstP from Mycobacterium tuberculosis. PstP was found to specifically dephosphorylate model phospho-Ser/Thr substrates in a Mn2+-dependent manner. Autophosphorylated PknB was shown to be a substrate for Pstp and its kinase activity was affected by PstP-mediated dephosphorylation. Two threonine residues in the PknB activation loop, found to be mostly disordered in the crystal structure of this kinase, namely Thr171 and Thr173, were identified as the target for PknB autophosphorylation and PstP dephosphorylation. Replacement of these threonine residues by alanine significantly decreased the kinase activity, confirming their direct regulatory role. These results indicate that, as for eukaryotic homologues, phosphorylation of the activation loop provides a regulation mechanism of mycobacterial kinases and strongly suggest that PknB and PstP could work as a functional pair in vivo to control mycobacterial cell growth.

Download full-text

Full-text

Available from: Frederique Pompeo, Nov 20, 2014
0 Followers
 · 
64 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Ser/Thr Kinase is one of the four M. tuberculosis kinases that are conserved in the downsized genome of Mycobacterium leprae and are therefore presumed to play an important role in the processes that regulate the complex life cycle of mycobacteria. It is known that there are two main superfamilies of protein kinases, one including STPKs1 and PTKs and that of His kinases. For a long time, the former were only found in eukaryotes, and the latter were only found in prokaryotes. In this paradigm, proteins from each superfamily were supposed to play analogous roles in the essentially different organization of signal transduction in both phyla. In this study we report the binding mode of Ser/Thr kinase with derivatives of Betulin ((lup-20(29)-ene-3β,28-diol) on the basis of structural similarity, substructure, isomers & conformers. Molecular docking approach using Lamarckian Genetic Algorithm was carried out to find out the potent inhibitors for Ser/Thr Kinase on the basis of calculated ligand-protein pairwise interaction energies. Study was carried out on 3000 molecules which were virtually screened from different databases on the basis of the structural similarity of Betulin. The grid maps representing the protein were calculated using auto grid and grid size was set to 60*60*60 points with grid spacing of 0.375 Ǻ. Docking was carried out with standard docking protocol on the basis of a population size of 150 randomly placed individuals; a maximum number of 2.5 *107 energy evaluations, a mutation rate of 0.02, a crossover rate of 0.80 and an elitism value of 1. Fifteen independent docking runs were carried out for each ligand and results were clustered according to the 1.0 Ǻ rmsd criteria. The docking result of the study of 3000 molecules demonstrated that the binding energies were in the range of -12.72 kcal/mol to -1.71 kcal/mol, with the minimum binding energy of –12.72 kcal/mol. 6 molecules showing hydrogen bonds with the active site residue VAL 95. Further in-vitro and in-vivo study is required on these molecules as the binding mode provided hints for the future design of new derivatives with higher potency and specificity.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The essential Mycobacterium tuberculosis Ser/Thr protein kinase (STPK), PknB, plays a key role in regulating growth and division, but the structural basis of activation has not been defined. Here, we provide biochemical and structural evidence that dimerization through the kinase-domain (KD) N-lobe activates PknB by an allosteric mechanism. Promoting KD pairing using a small-molecule dimerizer stimulates the unphosphorylated kinase, and substitutions that disrupt N-lobe pairing decrease phosphorylation activity in vitro and in vivo. Multiple crystal structures of two monomeric PknB KD mutants in complex with nucleotide reveal diverse inactive conformations that contain large active-site distortions that propagate > 30 Å from the mutation site. These results define flexible, inactive structures of a monomeric bacterial receptor KD and show how "back-to-back" N-lobe dimerization stabilizes the active KD conformation. This general mechanism of bacterial receptor STPK activation affords insights into the regulation of homologous eukaryotic kinases that form structurally similar dimers.
    Structure 12/2010; 18(12):1667-77. DOI:10.1016/j.str.2010.09.019 · 6.79 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Mycobacterium tuberculosis (M. tb) has a complex lifestyle in different environments and involving several developmental stages. The success of M. tb results from its remarkable capacity to survive within the infected host, where it can persist in a non-replicating state for several decades. The survival strategies developed by M. tb are linked to the presence of an unusual cell envelope. However, little is known regarding its capacity to modulate and adapt production of cell wall components in response to environmental conditions or to changes in cell shape and cell division. Signal sensing leading to cellular responses must be tightly regulated to allow survival under variable conditions. Although prokaryotes generally control their signal transduction processes through two-component systems, signalling through Ser/Thr phosphorylation has recently emerged as a critical regulatory mechanism in bacteria. The genome of M. tb possesses a large family of eukaryotic-like Ser/Thr protein kinases (STPKs). The physiological roles of several mycobacterial STPK substrates are connected to cell shape/division and cell envelope biosynthesis. Although these regulatory mechanisms have mostly been studied in Mycobacterium, Ser/Thr phosphorylation appears also to regulate cell division and peptidoglycan synthesis in Corynebacterium and Streptomyces. This review focuses on the proteins which have been identified as STPK substrates and involved in the synthesis of major cell envelope components and cell shape/division in actinomycetes. It is also intended to describe how phosphorylation affects the activity of peptidoglycan biosynthetic enzymes or cell division proteins.
    Molecular Microbiology 03/2010; 75(5):1064-77. DOI:10.1111/j.1365-2958.2009.07041.x · 5.03 Impact Factor