Structural and Functional Characterization of Autophosphorylation in Bacterial Histidine Kinases

  • Laura Miguel-Romero
  • Cristina Mideros-Mora
  • Alberto MarinaEmail author
  • Patricia CasinoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2077)


Autophosphorylation of histidine kinases (HK) is the first step for signal transduction in bacterial two-component signalling systems. As HKs dimerize, the His residue is phosphorylated in cis or trans depending on whether the ATP molecule used in the reaction is bound to the same or the neighboring subunit, respectively. The cis or trans autophosphorylation results from an alternative directionality in the connection between helices α1 and α2 in the HK DHp domain, in such a way that α2 could be oriented almost 90° counterclockwise or clockwise with respect to α1. Sequence and length variability of this connection appears to lie behind the different directionality and is implicated in partner recognition with the response regulator (RR), highlighting its importance in signal transduction. Despite this mechanistic difference, HK autophosphorylation appears to be universal, involving conserved residues neighboring the phosphoacceptor His residue. Herein, we describe a simple protocol to determine both autophosphorylation directionality of HKs and the roles of the catalytic residues in these protein kinases.

Key words

Histidine kinases Two-component systems Cis–trans autophosphorylation Signal transduction Heterodimer production and purification X-ray crystallography 



This work was supported by Spanish Government (Ministry of Economy and Competitiveness) grants BIO2016-78571-P to A.M. and BFU2016-78606-P to P.C. P.C. is the recipient of a Ramón y Cajal contract, from the Ministry of Economy and Competitiveness. C.M.-M. is the recipient of a Ph.D. fellowship from the Progama de becas, Secretaría de Educación Superior, Ciencia, Tecnología e Innovación of Ecuador Government (2015-AR2Q9228).


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© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas (IBV-CSIC)ValenciaSpain
  2. 2.Facultad de Ciencias de la Salud Eugenio EspejoUniversidad Tecnológica EquinoccialQuitoEcuador
  3. 3.CIBER de Enfermedades Raras (CIBERER-ISCIII)MadridSpain
  4. 4.Departament de Bioquímica i Biología MolecularUniversitat de ValènciaBurjassotSpain
  5. 5.Estructura de Recerca Interdisciplinar en Biotecnologia i Biomedicina (ERI BIOTECMED)Universitat de ValènciaBurjassotSpain

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