Structural And Functional Studies Of The Plant Cyclic Nucleotide-Gated Lon Channel CNGC1

Calcium ion and cyclic nucleotide monophosphates(cNMPs)are important signaling molecules in plants,which play key roles in plant growth,development and stress responses,such as root hair growth,pollen tube development,pathogen infection and heavy metal stress.Located in plasma membrane and tonoplast,cyclic nucleotide-gated channels(CNGCs)are a group of non-selective cation channels widely expressed in plants,which can be activated by intracellular cNMPs and improve the concentration of cytosolic calcium ion,thus regulating the physiological processes.The channel activity of CNGCs is regulated by many factors,such as the aggregation states,the phosphorylation and the binding of cNMPs or calmodulins(CaMs).In order to understand the regulation mechanisms of CNGCs,we carried out the structural and biochemical studies on the representative members.Using the eukaryotic cell expression systems,we tried to express and purify the representative members of CNGCs,and chose the highly expressed CNGC1 with good behaviors as our main research target.After optimizations,we obtained high quality protein samples of CNGC1.Using the cryo-electron microscope(cryo-EM)single particle analysis,we successfully determined its structure with an overall resolution of 2.9 ?.Based on the structural analysis,CNGC1 forms a homo-tetramer which is composed of the extracellular domain,the transmembrane domain,the intracellular C-linker,and the cyclic nucleotide binding domain(CNBD).Each subunit contains 6 transmembrane helices(S1-S6),S1-S4 form the voltage-sensor-like domain(VSLD)and S5-S6 form the pore domain.The ion conduction pore can be divided into four layers,the outer pore,the selectivity filter,the central gate and the inner pore.Residues L369-N372 consist the selectivity filter,and the narrowest constricting site of the selectivity filter is formed by the side chains of Q371.The C-linkers of two neighbouring subunits interact with each other in an elbow-on-shoulder manner.The CNBD exhibits a conserved fold,consisting of helices A,P,B,C and a β-roll.We verified the calcium ion channel activity of CNGC1 by the calcium imaging assays,and confirmed that the cNMPs and CaMs can enhance the channel activity by affecting the CNBD and the isoleucine glutamine motif(IQ motif)of CNGC1,respectively.Furthermore,we tried to measure the binding affinity between cNMP,or CaM and CNGC1 by microscale thermophoresis and isothermal titration calorimetry.In conclusion,we determined the cryo-EM structure of the Arabidopsis CNGC1.Based on the biochemical analysis,we examined the functions of the key residues and domains to the channel activity.These structural and functional studies enrich our understanding of the regulation mechanisms of CNGC1,and provide valuable insights to the plant cyclic nucleotide-gated channel research.