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Highly conserved protein Rv1211 in Mycobacterium tuberculosis is a natively unfolded protein that binds to a calmodulin antagonist, trifluoperazine

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Choo, Munki; Oh, Sehyun; Jo, Sihyang; Jin, Xing; Song, Yonghyun; Wen, He; Park, Sunghyouk; Kang, Sunmi

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Academic Press
Biochemical and Biophysical Research Communications, Vol.610, pp.182-187
Rv1211 is a conserved hypothetical protein in Mycobacterium tuberculosis and is required for the growth and pathogenesis of the bacteria. The protein has been suggested as a calmodulin-like calcium-binding protein with an EF-hand motif and as a target of trifluoperazine, a calmodulin antagonist in eukaryotes that inhibits mycobacterial growth. Here, we expressed the recombinant protein of Rv1211 and per -formed structural and biochemical studies of Rv1211 and its interaction with Ca2 thorn or trifluoperazine. Surprisingly, Rv1211 exhibited an elution property typical of a natively unfolded protein. Subsequent circular dichroism experiments with temperature elevation and trifluoroethanol treatment showed that Rv1211 has unfolded structure. Additional NMR experiment confirmed the unfolded state of the protein and further showed that it does not bind to Ca2 + . Still, Rv1211 did bind to trifluoperazine, as evidenced by the two-dimensional NMR spectra of N-15-labeled Rv1211. However, there were no peak shifts upon binding, showing that Rv1211 retained its unfolded state even after the trifluoperazine binding. The residues involved in the binding were clustered in the C-terminal region, as identified by the sequence assignment. Isothermal titration calorimetry showed that the Kd of trifluoperazine-Rv1211 binding is 41 mM and that the stoichiometry is 1 : 2 (Rv1211: trifluoperazine). Our results argue against the sug-gestion of Rv1211 as a Ca2+ -binding calmodulin-like protein, and show that Rv1211 isa natively unfolded protein that binds to trifluoperazine. In addition, our results suggest the evidence of the "Fuzziness " in the Rv1211-trifluoperazine interaction that differs from the conventional binding-induced folding of natively unfolded proteins. (C) 2022 Elsevier Inc. All rights reserved.
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