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Title

Structure of a chimeric EnvZ, visualizing histidine kinase autophosphorylation and deciphering cis-trans directionality

AuthorsCasino, Patricia ; Miguel-Romero, Laura ; Marina, Alberto
Issue Date19-Jun-2013
CitationXIII Congress SBE (2013)
AbstractReversible protein phosphorylation is the most widespread regulatory mechanism in signal transduction. The autophosphorylation in a dimeric sensor histidine kinase (HK) is the first step in two-component signaling, the predominant signal transduction mechanism in bacteria [1]. HK autophosphorylation can proceeds in cis or trans within the dimer, but why the course of the reaction is different is still unknown. We proposed that the alternative ways were imposed by the different handedness and length of the loop which connects the two helices in the dimerization domain (DHp) and that both ways followed a similar conserved phosphorylation mechanism [2]. We have demonstrated our hypothesis by solving the crystal structure of a chimeric EnvZ which demonstrates how a HK which autophosphorylates in trans (EnvZ) reverts its autophosphorylation into cis by exchanging the apex of the DHp from a HK which autophosphorylates in cis (HK853). Furthermore, the structure of the chimeric EnvZ has shown the enzyme cis-autophosphorylating in an asymmetric conformation. We have performed sitedirected mutagenesis of residues in HK853 and EnvZ that are involved in the catalytic center according to the structure of chimeric EnvZ. In vitro kinase assays, using radiolabeled ATP, and isothermal calorimetry to measure ATP binding for wt and mutant proteins have confirmed the catalytic residues for the autophosphorylation reaction and have allowed us to propose a mechanism for HK autophosphorylation that seems to be independent of the reaction directionality cis or trans. We have also corroborated asymmetry in the autophosphorylation reaction of HK853, caused by accumulation of ADP product. Symmetric autophosphorylation can be reached by removing ADP with a coupled assay using phoshoenolpyruvate and pyruvate kinase. Our functional and structural results have helped to stablish the molecular basis of the autophosphorylation reaction.
DescriptionTrabajo presentado en el XIII Congress SBE/International Congress of SBE, celebrado en Valencia, España, del 19 al 21 de junio de 2013
URIhttp://hdl.handle.net/10261/158359
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