Structure–Function Analysis of Arabidopsis thaliana Histidine Kinase AHK5 Bound to Its Cognate Phosphotransfer Protein AHP1

ABSTRACTThe multi-step phosphorelay (MSP) system defines a key signal transduction pathway in plants and many eukaryotes. In this system, external stimuli first lead to the activation of a histidine kinase, followed by transfer of a phosphoryl group from the receiver domain of the kinase (HKRD) to downstream, cytosolic phosphotransfer proteins (HPs). In order to establish the determinants of specificity for this signaling relay system, we have solved the first crystal structure of a plant HKRD, AHK5RD, in complex with one of its cognate HPs, AHP1. AHP1 binds AHK5RD via a prominent hydrogen bond docking ridge and a hydrophobic patch. These features are conserved among all AHP proteins, but differ significantly from other structurally characterized prokaryotic and eukaryotic HPs. Surface plasmon resonance experiments show that AHK5RD binds to AHP1-3 with similar, micromolar affinity, consistent with the transient nature of this signaling complex. Our correlation of structural and functional data provide the first insight, at the atomic level as well as with quantitative affinity data, into the molecular recognition events governing the MSP in plants.SUMMARYThe crystal structure of the AHK5RD–AHP1 complex reveals the determinants of specificity in plant two-component signaling. Our correlation of structural and quantitative affinity data provide the first insight into the molecular recognition events governing the multi-step phosphorelay system in plants.