Regulatory Mechanisms Of Various Domains Of CYP38 On Its Structure And Function

Arabidopsis cyclophilin38(CYP38),a thylakoid lumen protein,is critial for PS? assembly and maintenance.CYP38 protein contains three distinct domains: an N-terminal ?-helical bundle(amino acid 92-215),a C-terminal ?-sheet PPIase barrel(amino acid 239-437),and an intermediate acidic loop(amino acid 216-238).CYP38 is in a closely packed state with its N-terminal domain binding its C-terminal domain,blocking an access to the target protein,the CP47 E-loop.In aim to explore the working mechanism of CYP38,we examined the structural and functional significance of its C-terminus,N-terminus and the intermediate loop in vitro and in planta.We proposed that the C-terminal domain of CYP38 of Arabidopsis serves as the target binding domain,and its conserved residues(R290,F294,Q372,and F374)are important for the structure and function.In yeast two-hybrid and protein pull-down assays,CYP38 s with single-sited mutations(R290A,F294 A,Q372A,or F374A)did not interact with the CP47E-loop as the wild-type CYP38.In contrast,CYP38 with the R290A/F294A/Q372A/F374 A quadruple mutation could bind the CP47 E-loop.Gene transformation analysis showed that the quadruple mutation prevented CYP38 to efficiently complement the mutant phenotype of cyp38.The cyp38 plants expressing CYP38 with the quadruple mutation showed a similar BN-PAGE profile as cyp38,but distinct from the wild type.The CYP38 protein with the quadruple mutation associated with the thylakoid membrane less efficiently than the wild-type CYP38.We concluded that these four conserved residues are indispensable as changes of all these residues together resulted in a subtle conformational change of CYP38 and reduced its intramolecular N-C interaction and the ability to associate with the thylakoid membrane,thus impairing its function in chloroplasts.Then we examined the structural and functional significance of CYP38 N-terminal domain.Yeast two-hybrid and protein pull-down assays showed that a truncated form of CYP38 missing the N-terminal 92-124 fragment,CYP38(125-437),interacted with the CP47 E-loop similar to the C-domain protein,CYP38(239-437).Expressing CYP38(125-437)or the C-domain protein in cyp38 failed to rescue the abnormal phenotype of the mutant.On the other hand,the truncated CYP38 isoform,CYP38(125-437),associated with the thylakoid membrane less efficiently than the wild-type CYP38,while the C-domain protein did not bind to the thylakoid membrane at all.We concluded that the N-terminal bundle of CYP38 is required for the intramolecular interaction and the association of CYP38 with the thylakoid membrane.The intermediate loop of CYP38,which connects the N-terminal helical domain and the C-terminal bundle domain,harbors several charged residues.We examined the structural and functional significance of these charged residues.Yeast two-hybrid and protein pull-down assays showed mutations at three basic residues in the intermediate loop could alter the structure of CYP38 and enable it to interact with its target,the CP47 E-loop.Transgenic cyp38 plants expressing CYP38 s with triple mutations at those residues showed a stunted growth similar to the mutant,demonstrating that these basic residues were critical for the function of CYP38.CYP38 s with triple mutations at those three basic residues in intermediate loop showed a stronger thylakoid membrane association than the normal CYP38.We proposed the basic residues in the middle loop could control its N-C interaction via mediating the configuration of CYP38 and the dynamic regulation of the N-C interaction play vital roles for the function of CYP38.In addition,we confirmed the interaction between CYP38 and PS? core proteins,CP43 and CP47.In summary,we analyze the roles of CYP38 C-terminal domain,N-terminal domain and loop region in function of CYP38.Based on this analysis,we propose a working mechanism of CYP38 as follow: CYP38 docks onto the thylakoid membrane via the N-terminal domain,and basic residues in the intermediate loop lead to a configuration change of CYP38 and turn the packed CYP38 into an open state.Then,the C-terminal domain is free from the inhibition of the N-terminus,and binds its targets.After CYP38 completes its function,the C terminal domian interact with the the N-terminal bundle.Finally,CYP38 will disassociate from the thylakoid membrane and back to the thylakoid lumen.In our minds,this study sets a guidance for studying the function of other immunophilins in chloroplasts.