Biophysical Society Thematic Meeting - November 16-20, 2015

Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Poster Abstracts

11-POS

Board 11

NMR Characterization of the Natively Unfolded N-Terminus and the Redox-Sensitive Rubredoxin Motif of Mycobacterial Protein Kinase C–A Tuberculosis Drug Target Sonja Alexandra Dames 1,2 , Matthias Wittwer 1 . 1 Technische Universtiät München, Garching, Bavaria, Germany, 2 Helmholtz Zentrum München, Neuherberg, Bavaria, Germany. Protein kinase G (PknG) is a eukaryotic-like ser/thr kinase blocking the degradation of pathogenic organisms such as Mycobacterium tuberculosis within host macrophages. Because PknG is secreted into the host cytosol, it is an ideal target for tuberculosis drugs since they do not have to pass the only low permeable mycobacterial cell wall. The catalytic ser/thr kinase domain ( ∼ 147-390) is N-terminally flanked by a rubredoxin-like metal-binding motif (RD, ∼ 74 –147) and C-terminally by a tetratricopeptide repeat domain (TPRD). The N-terminal 73 residues are intrinsically disordered (NORS) and harbor the only site (T63) phosphorylated in vivo 1 . Both, deletions or mutations in the NORS or the redox-sensitive RD significantly decrease PknG-mediated mycobacterial survival in the host 1,2 . It has been shown that PknG is more active in the presence of an oxidizing agent 1 . Here, we present the NMR characterization of the NORS region and its chemical shift assignment as well as of the redox-regulated un- and refolding of the RD and its ability to interact with membrane mimetics. The NMR data is complemented by standard kinase assays. Based on our data controlled unfolding of the RD by oxidization may regulate the kinase activity. The detected membrane mimetic interactions may play a role for PknG localization. References: 1. Tiwari, D., Singh, R. K., Goswami, K., Verma, S. K., Prakash, B., and Nandicoori, V. K. (2009). J Biol. Chem. 284, 27467-27479. 2. Scherr, N., Honnappa, …, Pieters, J., and Steinmetz, M. O. (2007). Proc. Natl. Acad. Sci. U S A 104, 12151-12156. Acknowledgments: We thank the group of Prof. Dr. J. Pieters from the Biozentrum Basel for fruitful collaboration activities. The project is funded by the German Research Foundation (collaborative research center 1035, project B04).

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