Crystal Structure of Phosphotransmitter AHP2 and Modeling of its Interaction with Sensor Histidine Kinase CKI1 - Towards Elucidating Molecular Recognition in the Multistep Phosphorelay Signaling in Plants

Oksana Degtjarik^1,2*, Radka Dopitová¹*, David Řeha², Sandra Puehringer³, Blanka Pekarová¹, Olga Otrusinová¹, Michal Kutý², Manfred S. Weiss³, Lukáš Žídek¹, Ivana Kutá-Smatanová², Lubomír Janda¹ and Jan Hejátko¹

¹Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic

²Academy of Sciences of the Czech Republic, Inst. of Nanobiology and Structural Biology GCRC, Nové Hrady, Czech Republic

³ Helmholtz-Zentrum Berlin for Materials und Energy BESSY-II, Berlin, Germany

In multistep phosphorelay (MSP) pathway, the His-containing phosphotransmitters (HPt) encoded by AHP genes in Arabidopsis act as signaling intermediates integrating signaling inputs from diverse sensor histidine kinases. In order to identify the molecular determinants of the previously reported specificity of the interaction between receiver domain of sensor histidine kinase CKI1 (CKI1_RD) and AHPs at the atomic resolution, we determined the structure of AHP2 via experimental phasing after lutetium soaking of AHP2 crystals at 2.53 Å resolution. The key residues responsible for the AHP2-CKI1_RD interaction revealing strong protein-protein binding were identified via molecular dynamic simulations for 100 ns. The AHP2-CKI1_RD interaction was confirmed via NMR measurements and resulting chemical shifts partially overlap with the model. Comparison of the AHP2-CKI1_RD model with recently published structure of AHP1-AHK5_RD suggests that the interacting surfaces differ significantly between both complexes, particularly in the amino acid residues mediating hydrophilic interactions. Thus, in spite the vast majority of interacting residues in the AHP proteins is recruited from highly conserved residues, small structural differences of both AHPs and AHK_RDs seem to be sufficient for determination of specific molecular recognition as could be seen by our bioinformatical and structural comparisons.

Supported by CZ.1.05/1.1.00/02.0068 and P305/11/0756.