| WRYK proteins are a family of transcription factors(TFs)endemic to plants that are widely involved in plant development and anti-stress responses.WRKY transcription factors are found in 74 in Arabidopsis thaliana and more than 90 in rice.The name of the WRKY family derives from the most prominent feature of these proteins,the WRKY domain,a highly conserved region of 60 amino acids among family members.The WRKY domain consists of the N-terminal conserved amino acid sequence WRKYGQK and a new zinc finger motif.These proteins are regulatory transcription factors that specifically recognize W-box(TTCACC/T),but differ in regulating the expression of multiple target genes.The WRKY protein is highly conserved.On the one hand,the WRKY domain sequence is highly similar,on the other hand,the homologous cis-acting element W-box is highly conservative.These(T)(T)TGAC(C/T)sequence elements contain a constant TGAC core that is necessary for WRKY bonding and functioning.Different WRKY subgroup family members within the same plant species may also differ in their DNA binding requirements.This raises the question,how do different WRKY members achieve functional specificity?This may be related to the fact that the WRKY transcription factor is also affected by additional nucleotide sequences on both sides of the W-box and that the WRKY transcription factor can form complexes with different proteins.We mainly use nuclear magnetic resonance(NMR)methods to study the regulation mechanism of WRKY transcription factors by adjacent nucleotides near the 5’ end of the W-box.Arabidopsis WRKY11(AtWRKY11)belongs to the Group Ⅱ family of WRKY TFs and plays an important role in regulating plant defenses against abiotic stress.At present,there have been many studies on the structure of WRKY TFs in Group Ⅰ and the mechanism of their interaction with DNA,but there is little research on the structural characteristics of WRKY TFs in Group Ⅱ and their mechanisms regulated by DNA.In this paper,the structure of the DNA-binding domain(DBD)of AtWRKY11 and its interaction with DNA were studied using nuclear magnetic resonance technology.Firstly,the atWRKY11-DBD was mainchain attributed using triple resonance experiments,and on the basis of chemical displacement,the secondary structure prediction was made by CSI,and the results showed that AtWRKY11-DBD may have five β folds like the C-terminal proteins in other WRKY transcription factor Group I members.AtWRKY11-DBD is homologously modeled based on the crystal structure of AtWRKY1-C,and there may be local differences between the β1 and β5 folded pieces of At WRKY 11-DBD compared with At WRKY 1-C.The β1 chain(TVRV fragment)of at WRKY11-DBD in the manually assigned prediction structure based on CSI secondary structure analysis appears to move toward the N-terminal of the sequence,and the loop connecting β1 and β2 in AtWRKY11-DBD may be longer and more flexible.The structure of the NMR solution of AtWRKY11-DBD was then analyzed,and the structure of the β2-β4 folded piece of AtWRKY11-DBD was basically the same as that of At WRKY1-C,but the β piece near the N terminal could not be calculated due to the NOE too weak to perform structural calculations,and the folded sheet may not be very stable.On the basis of free-state structure,the interaction of At WRKY 11-DBD with DN A was studied.Firstly,the specificity of protein-DNA binding was studied,and the atWRKY11-DBD specific binding to W-box was demonstrated by observing the change in the NMR spectrogram chemical shift of AtWRKY11-DBD bound to the 12 bp W-box sequence and the random sequence,respectively.To find the critical interface between AtWRKY11-DBD binding to DNA,the complexes of AtWRKY11-DBD and W-box were analyzed for backbone attribution and chemical displacement perturbations.The results show that the residues with relatively large chemical displacement disturbance are mainly located in the conserved β2 chain(W251RKYGQK257),the β1-β2 loop region(K240IADI244)and the K260 β2-β3 loop region.To reveal the mechanism by which WRKY TFs are regulated by DNA,the Wbox sequence of AtWRKY11-DBD on different 5’-terminal adjacent nucleotides was further investigated(XTTGACC;X=binding selectivity of A,C,T).The results show that the binding strength of TTTGACC is comparable to that of GTTGACC.And AtWRKY11-DBD has high selectivity for W-box sequences with G adjacent nucleotides at the 5’ end.Our study lays the foundation for revealing the mechanism by which WRKY TFs are regulated by DNA. |
