Structural And Biochemical Insights Into The Recognition Of RNA Helicase CGH-1 By CAR-1 And EDC3 In C.elegans

'Germ granule' termed P granule in Caenorhabditis elegans,which is a kind of RNP granule specific to the germline.Previous studies have shown that RNA helicase CGH-1 and RNA binding protein CAR-1 are co-located in P granule.A complex formed by CGH-1 and CAR-1 is involved in various aspects of function in C.elegans,including apoptosis,cytokinesis and regulation of phase transition.However,the structural basis for the assembly of this protein complex remains unclear.Here,we elucidate the molecular basis of the recognition of CGH-1 by CAR-1.In this study,we determined the structures of the two RecA-like domains of CGH-1,RecAl and RecA2,by X-ray crystallography at resolutions of 1.85 and 2.40 A,respectively(PDB ID:7DTK,7DTJ).CGH-1 and CAR-1 can form a complex in vitro,as indicated by size-exclusion chromatography,and further verified by biochemical experiments.Unfortunately,we failed to co-crystalize the complex of CGH-1 RecA2 with CAR-1,probally due to a weak binding affinity with a dissociation equilibrium constant(KD)of?3?M as measured by ITC.Next,we used NMR methods,a very powerful tool,to identify a potential interface(s)consisting of 11 residues of CGH-1 RecA2 with chemical shift perturbation(CSP)more than 0.05,by which CAR-1 was recognized.Additionally,structural and biochemical approaches revealed a bipartite interface between CGH-1 RecA2 and the FDF-TFG motif of CAR-1.NMR and structure-based mutations in CGH-1 RecA2 or CAR-1 attenuated or disrupted CGH-1 binding to CAR-1,assessed by ITC and GST-pulldown in vitro.Furthermore,sequence and structural alignments indicated that the recognition mechanism is highly conserved.These findings provide insights into a conserved mechanism in the recognition of CGH-1 by CAR-1.Finally,we also demonstrated that CGH-1 shows lower ATPase activity in the absence of RNA or NTL-laMIF4G in vitro,but the activity is robustly stimulated by poly U RNA and NTL-laMIF4G.Together,our data provide the missing physical links in understanding the assembly and function of CGH-1 and CAR-1 in C.elegans.H.sapiens DDX6 or Saccharomyces cerevisiae Dhhlp(CGH-1 orthologs)can form complexes with EDC3(mRNA decapping enhancer 3),respectively,which is of importance for translation inhibition and mRNA decay.However,it's currently unknown whether CGH-1 and EDC3 can form a functional complex in C.elegans.ITC titration revealed that EDC3 FDF motif directly binds CGH-1 RecA2 with a KD of?0.34?M.Combined with homology modeling and mutation analysis,we found that EDC3 is mainly anchored to CGH-1 RecA2 through a conserved hydrophobic surface.Our studies provide a basis for the functional research of the complex of CGH-1 and interactors in C.elegans.