Histidine-containing phosphotransfer proteins from Arabidopsis thaliana (AHP1-5) act as intermediates between sensor histidine kinases and response regulators in a signalling system called multi-step phosphorelay (MSP). AHP proteins mediate and potentially integrate various MSP-based signalling pathways (e.g. cytokinin or osmosensing). However, structural information about AHP proteins and their importance in MSP signalling is still lacking. To obtain a deeper insight into the structural basis of AHP-mediated signal transduction, the three-dimensional structure of AHP2 was determined. The AHP2 coding sequence was cloned into pRSET B expression vector, enabling production of AHP2 fused to an N-terminal His tag. AHP2 was expressed in soluble form in Escherichia coli strain BL21 (DE3) pLysS and then purified to homogeneity using metal chelate affinity chromatography and anion-exchange chromatography under reducing conditions. Successful crystallization in a buffer which was optimized for thermal stability yielded crystals that diffracted to 2.5 Å resolution.

• MeSH
• Arabidopsis metabolismus   MeSH
• difrakce rentgenového záření MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• fosfotransferasy chemie   izolace a purifikace   MeSH
• krystalizace MeSH
• proteiny huseníčku chemie   izolace a purifikace   MeSH
• signální transdukce * MeSH
• tranzitní teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Multistep phosphorelay (MSP) signaling mediates responses to a variety of important stimuli in plants. In Arabidopsis MSP, the signal is transferred from sensor histidine kinase (HK) via histidine phosphotransfer proteins (AHP1-AHP5) to nuclear response regulators. In contrast to ancestral two-component signaling in bacteria, protein interactions in plant MSP are supposed to be rather nonspecific. Here, we show that the C-terminal receiver domain of HK CKI1 (CKI1(RD) ) is responsible for the recognition of CKI1 downstream signaling partners, and specifically interacts with AHP2, AHP3 and AHP5 with different affinities. We studied the effects of Mg²⁺, the co-factor necessary for signal transduction via MSP, and phosphorylation-mimicking BeF₃⁻ on CKI1(RD) in solution, and determined the crystal structure of free CKI1(RD) and CKI1(RD) in a complex with Mg²⁺. We found that the structure of CKI1(RD) shares similarities with the only known structure of plant HK, ETR1(RD) , with the main differences being in loop L3. Magnesium binding induces the rearrangement of some residues around the active site of CKI1(RD) , as was determined by both X-ray crystallography and NMR spectroscopy. Collectively, these results provide initial insights into the nature of molecular mechanisms determining the specificity of MSP signaling and MSP catalysis in plants.

• MeSH
• Arabidopsis enzymologie   genetika   fyziologie   MeSH
• fosforylace MeSH
• fosfotransferasy genetika   metabolismus   MeSH
• histidin metabolismus   MeSH
• krystalografie rentgenová MeSH
• mapování interakce mezi proteiny MeSH
• molekulární modely MeSH
• mutace MeSH
• proteinkinasy chemie   genetika   izolace a purifikace   metabolismus   MeSH
• proteiny huseníčku chemie   genetika   izolace a purifikace   metabolismus   MeSH
• rekombinantní fúzní proteiny MeSH
• senzitivita a specificita MeSH
• signální transdukce fyziologie   MeSH
• terciární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH