Oxidative Unfolding of the Rubredoxin Domain and the Natively Disordered N-terminal Region Regulate the Catalytic Activity of Mycobacterium tuberculosis Protein Kinase G.
Mycobacterium tuberculosis escapes killing in human macrophages by secreting protein kinase G (PknG). PknG intercepts host signaling to prevent fusion of the phagosome engulfing the mycobacteria with the lysosome and, thus, their degradation. The N-terminal NORS (no regulatory secondary structure) region of PknG (approximately residues 1-75) has been shown to play a role in PknG regulation by (auto)phosphorylation, whereas the following rubredoxin-like metal-binding motif (RD, residues ∼74-147) has been shown to interact tightly with the subsequent catalytic domain (approximately residues 148-420) to mediate its redox regulation. Deletions or mutations in NORS or the redox-sensitive RD significantly decrease PknG survival function. Based on combined NMR spectroscopy, in vitro kinase assay, and molecular dynamics simulation data, we provide novel insights into the regulatory roles of the N-terminal regions. The NORS region is indeed natively disordered and rather dynamic. Consistent with most earlier data, autophosphorylation occurs in our assays only when the NORS region is present and, thus, in the NORS region. Phosphorylation of it results only in local conformational changes and does not induce interactions with the subsequent RD. Although the reduced, metal-bound RD makes tight interactions with the following catalytic domain in the published crystal structures, it can also fold in its absence. Our data further suggest that oxidation-induced unfolding of the RD regulates substrate access to the catalytic domain and, thereby, PknG function under different redox conditions, e.g. when exposed to increased levels of reactive oxidative species in host macrophages.
From the Biomolecular NMR Spectroscopy and.
Matthias
M
Wittwer
Computational Biocatalysis, Department of Chemistry, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany.|the Soft Matter Research Center and Department of Chemistry, Zhejiang University, Hangzhou 310027, China, and.
Qi
Q
Luo
Computational Biocatalysis, Department of Chemistry, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany.
Ville R I
VR
Kaila
From the Biomolecular NMR Spectroscopy and sonja.dames@tum.de.|the Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany.
Sonja A
0000-0001-7495-6943
SA
Dames
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D056004:Molecular Dynamics Simulation; D009154:Mutation / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D012416:Rubredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D012416:Rubredoxins; D017869:Cyclic GMP-Dependent Protein Kinases
Mycobacterium tuberculosis; NMR; autophosphorylation; intrinsically disordered protein; metal ion-protein interaction; molecular dynamics; oxidative stress; protein kinase G (PKG); protein phosphorylation; redox regulation