Functional Analyses Of The Conserved Residues In The Extracellular Domain Of Leucine-rich-repeat Receptor-like Kinase BRI1

The Leucine-rich-repeat receptor-like-kinases(LRR-RLKs)represent a class of receptors that can sense various environmental signals and regulate plant development.As a well-studied LRR-RLK in Arabidopsis,brassinosteroid insensitive 1(BRI1)functions as a cell surface receptor for brassinosteroids(BRs),the smallest ligands known thus far.BRI1 extracellular domain consists of 25 LRRs in forms of superhelical assembly and a 70-amino acid island inserted between the 21st and 22nd LRRs.The crystal analysis shows that the inner surface of the superhelix together with ID domain is involved in ligand binding and muiltiple asparagine(Asn,N)residues are modified with N-glycans.Previous studies have shown that the mutation of Ser to Phe(S662F,bril-9)at the inner surface of BRI1 superhelix leads to the endoplasmic reticulum(ER)retention.The sequence alignment reveals that the Ser662 residue is highly conserved among BRI1 LRRs,indicating that these Ser residues(forming the S*-chain)might have crucial roles.In addition,BRI1 harbores multiple N-glycans at its extracelluar domain and undergoes an N-glycan dependent assistant folding process.Yet little is known about the the biological functions of the redundant N-glycans on BRI1 folding.In the current study,we analyzed the evolutional significance and functional roles of these Ser residues and those N-glycans in BRI1.In this study,we first examined the importance of the highly conserved Ser residues at BRI1 extracellular domain in the protein conformations.The results showed that the change of conserved Ser*residues to Phe would result in BRI1 structural defects and thus be trapped in ER,especially those at N-termini of AtBRI1 LRRs as 7 out of 11 Ser*residues that caused protein ER retention lied in LRRs at N-termini.The replacement of smaller apolar residue Ala for Ser*at a single site had little impact on protein escaping form ER,however,successive S?A mutantion could result in protein ER localization,indicating that size of these Ser*residue and their successive polar contact/polarity along the S*-chain makes the existence of the Ser*residues an inevitable result in AtBRI1.Evolutionary analyses further indicated that the Ser*residues were also conservatively presented in LRRs of many BRI1 homologs.In addition,the continuous presence of Ser*is mainly disrupted at the LRR-island domain interface,and the changes of these four non-serine residues to serine greatly decreased the protein ability to complement bri1-301 compact phenotype and the BR signaling activation.The sequence alignment revealed that other known LRR-RLK also harbors the S*-chain and the non-Ser*residues at the ligand-binding region along the S*-chain,which confirms the evolutionary significance of residues at these sites in plant LRRRLKs.We also examined the function of site-specific N-glycans on BRI1 folding through site-directed mutagenesis.By deleting a single N-glycosylation site(N?Q),we found that only a single mutant of N154Q could lead to bril ER retention,indicating an important role of N154 in BRI1 proper folding.We also noticed that higher conserved N-glycan played more important roles in BRI1 proper folding:the deletion of two highly conserved N-glycan would cause protein ER retention while the deletion of five low conserved N-glycan could escape ER like wild type BRI 1.However,for structural defective BRI1 S*?F mutant proteins,the remove of a single N-glycan promoted ER retention of bril harboring structural defect spatially near the N-glycan deleted and a proper addition of an N-glycan nearby could to some extent suppress the local defect.Moreover,since bri 1-9 was reported to be retained in an N-glycan dependent way,the systematic characterization of N-glycan functionality for bril-9 folding process in ER was investigated by stepwise mutagenesis of N-glycosylation sites based on the distance between the potential acceptor N-glycan and local defect position.We found that the retention of misfolded bril-9 by lectins and chaperones in the ER relied on the presence of multiple N-glycans distal to the local defects.Our findings revealed that the N-glycans might play a protective or monitoring role according to the folding state of associated protein or the distance from structural defects.Overall,this study demonstrated the crucial roles of highly conserved serine residues of BRI1 LRRs and its evolutional significance,and also provided a valuable addition to the limited information available on the contribution of site-specific Nglycosylation plant receptor kinases folding process.