Structural insights into the catalytic regulation of a thermosensor histidine kinase

TRAJTENBERG, FELIPE; RUETALO, NATALIA; ALBANESI, DANIELA; LARRIEUX, NICOLE; BOTTI, HORACIO; ALZARI, PEDRO; DE MENDOZA, DIEGO; BUSCHIAZZO, ALEJANDRO

Salta

Congreso; 3rd Latin American Protein Society Meeting; 2010

Latin American Protein Society

The molecular mechanisms of detection and transmission of external signals to the interior of the cell are poorly understood and of great biological relevance. We are interested in elucidating the mechanisms of biological signal transduction pathways widely used by bacteria, essential for triggering adaptive responses, denominated two-component system. In Bacillus subtilis, the membrane-bound histidine kinase DesK, together with the response regulator DesR, constitutes a canonical two-component system able to sense and respond to changes in the membrane fluidity. We recently reported the crystallographic characterization of DesK’s catalytic core, in different functional states along the signaling pathway1. The biochemical and structural studies we performed allowed us to propose a model of signal transmission and catalytic regulation of the three activities of DesK: phosphatase, autokinase and phosphotransferase. The high plasticity of the central dimerization and phosphotransfer domain and the changes in the ATP binding domain mobility are key determinants in the regulation of the autokinase activity and the ability to form the complex with DesR. We also modeled the autophosphorylation conformation by bioinformatics means and were able to trap this intermediate state by protein engineering at the predicted domain-domain interaction surface. The putative structure of this transient state and the characterization of autophosphorylation activity allowed us to propose an asymmetric trans-autophosphorylation mechanism2. Finally, structure-guided proteinengineering experiments aimed at stabilizing the phosphatase or kinase state in vitro are in full agreement with the proposed regulation model for the DesK-DesR system.1. Albanesi D., Martín M., Trajtenberg F., Mansilla M.C., Haouz A., Alzari P.M., de Mendoza D. and Buschiazzo A. Proc Natl Acad Sci USA, 106,16185-16190 (2009)2. Trajtenberg F., Graña M., Ruétalo N., Botti H. and Buschiazzo A., J Biol Chem, (2010), Epub ahead of printWe acknowledge the support of ANR, ANII, Institut Pasteur, Institut Pasteur de Montevideo.