Snapshots of the Signaling Complex DesK:DesR in Different Functional States Using Rational Mutagenesis and X-ray Crystallography

Juan A Imelio (1) , Nicole Larrieux (1) , Ariel E Mechaly (1) , Felipe Trajtenberg (1) , Alejandro Buschiazzo (1, 2)

1 Molecular and structural microbiology / Microbiología Molecular y Estructural [Montevideo]
2 Département de Microbiologie - Department of Microbiology

We have developed protocols to generate site-specific variants of the histidine-kinase DesK and its cognate response regulator DesR, conducive to trapping different signaling states of the proteins. Co-expression of both partners in E. coli, ensuring an excess of the regulator, was essential for soluble production of the DesK:DesR complexes and further purification. The 3D structures of the complex trapped in the phosphotransferase and in the phosphatase reaction steps, were solved by X-ray crystallography using molecular replacement. The solution was not trivial, and we found that in silico-generated models used as search probes, were instrumental to succeeding in placing a large portion of the complex in the asymmetric unit. Electron density maps were then clear enough to allow for manual model building attaining complete atomic models. These methods contribute to tackling a major challenge in the bacterial signaling field, namely obtaining stable kinase:regulator complexes, in distinct conformational states, amenable for high-resolution crystallographic studies.

Mots clés: Signaling proteins; Protein phosphorylation; Trapping conformational rearrangements; Structure-based mutagenesis; X-ray crystallography; Protein engineering

Domaines: Sciences du Vivant [q-bio]

Juan A Imelio, Nicole Larrieux, Ariel E Mechaly, Felipe Trajtenberg, Alejandro Buschiazzo. Snapshots of the Signaling Complex DesK:DesR in Different Functional States Using Rational Mutagenesis and X-ray Crystallography. Bio-protocol , 2017, 7 (16), ⟨10.21769/BioProtoc.2510⟩. ⟨pasteur-02872312⟩