Much of plant development depends on cell-to-cell redistribution of the plant hormone auxin, which is facilitated by the plasma membrane (PM) localized PIN FORMED (PIN) proteins. Auxin export activity, developmental roles, subcellular trafficking, and polarity of PINs have been well studied, but their structure remains elusive besides a rough outline that they contain two groups of 5 alpha-helices connected by a large hydrophilic loop (HL). Here, we focus on the PIN1 HL as we could produce it in sufficient quantities for biochemical investigations to provide insights into its secondary structure. Circular dichroism (CD) studies revealed its nature as an intrinsically disordered protein (IDP), manifested by the increase of structure content upon thermal melting. Consistent with IDPs serving as interaction platforms, PIN1 loops homodimerize. PIN1 HL cytoplasmic overexpression in Arabidopsis disrupts early endocytic trafficking of PIN1 and PIN2 and causes defects in the cotyledon vasculature formation. In summary, we demonstrate that PIN1 HL has an intrinsically disordered nature, which must be considered to gain further structural insights. Some secondary structures may form transiently during pairing with known and yet-to-be-discovered interactors.

• Keywords
• PIN1, dimerization, hydrophilic hoop, intrinsic disorder, subcellular trafficking,
• MeSH
• Arabidopsis * metabolism   MeSH
• Biological Transport MeSH
• Plant Roots metabolism   MeSH
• Indoleacetic Acids metabolism   MeSH
• Membrane Transport Proteins genetics   metabolism   MeSH
• Arabidopsis Proteins * genetics   metabolism   MeSH
• Intrinsically Disordered Proteins * genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Indoleacetic Acids MeSH
• Membrane Transport Proteins MeSH
• PIN1 protein, Arabidopsis MeSH Browser
• Arabidopsis Proteins * MeSH
• Intrinsically Disordered Proteins * MeSH

Multistep phosphorelay (MSP) cascades mediate responses to a wide spectrum of stimuli, including plant hormonal signaling, but several aspects of MSP await elucidation. Here, we provide first insight into the key step of MSP-mediated phosphotransfer in a eukaryotic system, the phosphorylation of the receiver domain of the histidine kinase CYTOKININ-INDEPENDENT 1 (CKI1RD) from Arabidopsis thaliana We observed that the crystal structures of free, Mg2+-bound, and beryllofluoridated CKI1RD (a stable analogue of the labile phosphorylated form) were identical and similar to the active state of receiver domains of bacterial response regulators. However, the three CKI1RD variants exhibited different conformational dynamics in solution. NMR studies revealed that Mg2+ binding and beryllofluoridation alter the conformational equilibrium of the β3-α3 loop close to the phosphorylation site. Mutations that perturbed the conformational behavior of the β3-α3 loop while keeping the active-site aspartate intact resulted in suppression of CKI1 function. Mechanistically, homology modeling indicated that the β3-α3 loop directly interacts with the ATP-binding site of the CKI1 histidine kinase domain. The functional relevance of the conformational dynamics observed in the β3-α3 loop of CKI1RD was supported by a comparison with another A. thaliana histidine kinase, ETR1. In contrast to the highly dynamic β3-α3 loop of CKI1RD, the corresponding loop of the ETR1 receiver domain (ETR1RD) exhibited little conformational exchange and adopted a different orientation in crystals. Biochemical data indicated that ETR1RD is involved in phosphorylation-independent signaling, implying a direct link between conformational behavior and the ability of eukaryotic receiver domains to participate in MSP.

• Keywords
• X-ray crystallography, histidine kinase, nuclear magnetic resonance (NMR), protein dynamic, protein phosphorylation, receiver domain, relaxation dispersion,
• MeSH
• Arabidopsis enzymology   genetics   MeSH
• Crystallography, X-Ray MeSH
• Nuclear Magnetic Resonance, Biomolecular MeSH
• Protein Kinases chemistry   genetics   MeSH
• Protein Domains MeSH
• Arabidopsis Proteins chemistry   genetics   MeSH
• Receptors, Cell Surface chemistry   genetics   MeSH
• Protein Structure, Secondary MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CKI1 protein, Arabidopsis MeSH Browser
• ETR1 protein, Arabidopsis MeSH Browser
• Protein Kinases MeSH
• Arabidopsis Proteins MeSH
• Receptors, Cell Surface MeSH