Abstract
Recent efforts have underlined the role of serine/threonine protein kinases in growth, pathogenesis, and cell wall metabolism in Mycobacterium tuberculosis. Although most kinases have been investigated for their physiological roles, little information is available regarding how serine/threonine protein kinase-dependent phosphorylation regulates the activity of kinase substrates. Herein, we focused on M. tuberculosis Rv2175c, a protein of unknown function, conserved in actinomycetes, and recently identified as a substrate of the PknL kinase. We solved the solution structure of Rv2175c by multidimensional NMR and demonstrated that it possesses an original winged helix-turn-helix motif, indicative of a DNA-binding protein. The DNA-binding activity of Rv2175c was subsequently confirmed by fluorescence anisotropy, as well as in electrophoretic mobility shift assays. Mass spectrometry analyses using a combination of MALDI-TOF and LC-ESI/MS/MS identified Thr(9) as the unique phosphoacceptor. This was further supported by complete loss of PknL-dependent phosphorylation of an Rv2175c_T9A mutant. Importantly, the DNA-binding activity was completely abrogated in a Rv2175c_T9D mutant, designed to mimic constitutive phosphorylation, but not in a mutant lacking the first 13 residues. This implies that the function of the N-terminal extension is to provide a phosphoacceptor (Thr(9)), which, following phosphorylation, negatively regulates the Rv2175c DNA-binding activity. Interestingly, the N-terminal disordered extension, which bears the phosphoacceptor, was found to be restricted to members of the M. tuberculosis complex, thus suggesting the existence of an original mechanism that appears to be unique to the M. tuberculosis complex.
Highlights
The existence of an original mechanism that appears to be unique to the M. tuberculosis complex
M. tb contains eleven serine/threonine and tyrosine protein kinases (STPKs) [4, 5], and most are being investigated for their physiological roles and potential application for future drug development to combat tuberculosis [6]
The cell wall of M. tb plays a critical role in the defense of this pathogen in the host, and changes in cell wall composition in response to various environmental stimuli are critical to M. tb adaptation during infection
Summary
M. tb, M. tuberculosis; EMSA, electrophoretic mobility shift assay; STPK, Ser/Thr protein kinase; TEV, tobacco etch virus; HSQC, heteronuclear single quantum coherence; HTH, helix-turn-helix; wHTH, winged HTH; MS, mass spectrometry. A further characterization of STPKs substrates is critical to unraveling the mechanisms by which STPK-dependent phosphorylation induces modifications, regulating their activity, conditioning biological responses in mycobacteria. Such studies may provide the key to designing new inhibitors that target signal transduction pathways specific to M. tb. We provide strong evidence that Rv2175c is a DNA-binding protein and investigated how phosphorylation of a unique Thr residue in the N-terminal domain of the protein affects its DNA-binding activity
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