Efficient Peptide Binding Domain Of The Combination Of The Features Of The New Strategy To Establish Its Application

●Protein interaction domain is a valuable starting point for high-efficiency protein-protein interaction researchCompiling protein-protein interaction network provides many new insights into protein functions and disease mechanisms.Some node proteins in the network usually play key roles in biological processes.A large set of proteins are composed of one or more domains and a substantial proportion of protein-protein interactions are mediated by families of protein interaction domains,such as PDZ,SH2,WW,SH3,etc.Therefore,the protein interaction domain of node proteins values as a simple and high efficient starting point for understanding the mechanisms and networks of protein interactions.In this study,with focus on PDZ domain,one of the most important protein interaction modules,we developed a high-efficiency strategy for binding property characterization of peptide-binding domains,and systematically characterized a number of PDZ domains and discovered a series of novel interactions.We found that the binding properties of the PDZ domains showed strong consensus as well as certain variability. Moreover,many of the identified ligand proteins of the PDZ domains were expressed extensively and had diverse functions.It provides the biochemical basis that PDZ proteins may play diverse roles in multiple systems.The complexity of protein functions exceeds our current understanding and expectation.We propose that proteins composed of common protein interaction domains might have many ligands and diverse functions in different biological systems.●Development of a high-efficiency strategy for binding property characterization of peptide-binding domainsIn recent years,several powerful methods,including oriented peptide library,SPOT synthesis,phage display and yeast two-hybrid,have been successfully employed for characterization of the peptide recognition specificities of individual domain families. However,these methods are all based on large-scale screenings for individual bait domain, which are relatively labor-intensive and time-consuming.For more efficiently characterizing the binding properties of peptide-binding domains,on the basis of the facts that one domain can bind many ligands and one ligand can be recognized by multiple domains,we developed a systematic strategy of high-throughput validation screening of a specialized candidate ligand library using yeast two-hybrid mating array.The library was constructed mainly by collecting the ligand clones isolated from yeast two-hybrid screenings of random peptide libraries with representative members of the selected domain family.By validation screening,consensus sequences were deduced from both positive and negative binding ligands.Novel ligand proteins were identified through database searches with consensus-binding sequences and confirmed in yeast two-hybrid system.The domains that did not interact with enough numbers of ligands from the library were used as bait for de novo screenings of random peptide libraries in the traditional way.This strategy is notably different from other approaches in several features.Firstly,the overall efficiency is dramatically improved by validation screening instead of traditional screening.It can be further improved,as the candidate ligand library expands with the addition of clones from more traditional screenings and clones constructed for confirmations.Secondly,we can achieve high throughput with yeast two-hybrid mating array approach.Multiple bait domains can be tested in parallel and thousands of candidate ligands can be screened on arrays simultaneously.Thirdly,since the library consists of ligands of known sequences,actual positive and negative binding sequences,which are both very important for precisely defining binding properties,can be directly read from arrays without resequencing.●Construction of a high-efficiency strategy for binding property characterization of PDZ domainWe have firstly demonstrated the feasibility of the newly developed strategy by employing it to investigate the PDZ domain interactions.We constructed an initial PDZ ligand library by yeast two-hybrid screenings of random peptide libraries with ZO-1 PDZ1,ZO-1 PDZ3 and Erbin PDZ.Next,we rapidly characterized HtrA2 PDZ and LNX1 PDZ2 by validation screenings of this initial library. MAGI3 PDZ4 and other interested PDZ domains,which could not find enough numbers of ligands from the initial library,were used as bait for de novo screenings of random peptide libraries in the traditional way.The PDZ ligand library was expanded with the addition of new positive clones isolated by new traditional screenings and clones constructed for confirmations.The binding properties of the interested PDZ domains have been successfully defined. Broader binding properties have been identified compared to other methods,including unique novel recognition specificities,such as ZO-1-PDZ3 favors hydrophobic residues at p^-5,ZO-1 PDZ3,LNX1 PDZ2 and MAGI3 PDZ4 select polar or hydrophilic residues at p⁰, five of the six PDZs prefer aromatic residues at p^-1,four of the six PDZs can bind three conventional classes of PDZ ligands,and HtrA2 PDZ and LNX1-PDZ2 exhibit linkage selectivity at the C-termini of ligands.All these findings not only are important for predicting PDZ binding partners but also provide the basis for new definition of PDZ domain and major revision of conventional PDZ classification.In addition,40 novel interactions have been discovered,serving as significant clues for further functional investigation.●Application of the high-efficiency strategy for binding property characterization of PDZ domainLNX is the first described PDZ domain containing member of the RING type E3 ubiquitin ligase.However,the functions of LNX PDZ domains are poorly understood.We systematically characterized the binding properties of human LNX PDZ domains and discovered their potential ligand proteins in human proteome,using our newly developed high-efficiency validation screening method.By validation screenings of the PDZ ligand library and/or yeast two-hybrid screenings of random peptide libraries,the C-terminal binding properties of LNX1 PDZ1,LNX1 PDZ2,LNX1 PDZ3,LNX2 PDZ2 and LNX4 PDZ were characterized.The recognition specificities between these five PDZs showed certain common features and some distinct properties.3 novel PDZ-PDZ interactions in LNX family were identified,which were the interactions between LNX1 PDZ1 and LNX1 PDZ4,LNX1 PDZ1 and LNX2 PDZ4, LNX2 PDZ1 and LNX2 PDZ4.In addition,a few of novel ligand proteins of LNX PDZ domains were discovered.These novel identified interactors are known to be functionally different and expressed extensively.This prompts LNX family proteins may play diverse roles in multiple systems. ●Preliminary investigation on molecular therapy of Liddle syndromeLiddle syndrome,first described by Grant Liddle et al in 1963,is an autosomal dominant form of salt-sensitive hypertension.The pathogenesis of Liddle syndrome has been attributed to missense,deletion or frameshift mutations of PY motifs in cytoplasmic carboxyl termini of beta or gamma subunits of the amiloride-sensitive epithelial sodium channel(ENaC).Disease mutations lead to absence of ENaC interaction with the WW domain of Nedd4,an ubiquitin ligase.The abnormalities result in increased channel activity and excessive Na⁺ absorption in the kidney.We proposed a novel strategy for molecular therapy of Liddle syndrome,which was use of an artificial ubiquitin ligase to target mutational ENaC.The artificial ubiquitin ligase was constructed by replacement of the WW domain of Nedd4 with a peptide that was able to interact with mutational PY motifs,and this peptide could be obtained from screenings of random peptide libraries.In this study,we firstly chose one of Liddle syndrome mutations,Y620H,to screen random peptide library and obtained one positive peptide.We further demonstrated the positive peptide specifically interacted with at least two Liddle syndrome mutations, Y620H and P618L.