Binding Property Characterization Of PDZ And SH2 Domains

Erbin (ErbB2 interacting protein) plays a critical role in restricting ErbB2 to thelateral membrane of human intestinal epithelial cells in PDZ domain dependent manner.Because of the potential therapeutic value of disrupting ErbB2 basolateral targeting, weinitially set out to further characterize the binding ligands of Erbin PDZ domain. Yeasttwo-hybrid screening of random peptide library was employed here. The experimentsshowed that besides the reported ClassⅠ-[E/D]-[T/S]-W-V motif. Erbin PDZ can alsorecognize the ClassⅡ-E-[V/A]-W-V and ClassⅢ-D-E-W-V motifs. At the -1 position,acidic residues were observed although aromatic residues W and F were the predominantselections. Interestingly, both high affinity ligands carrying the C-terminal ETWV and lowaffinity ones with ESDV were identified by once screening. Alignment ofscreening-derived ligands established clear consensus sequences at the C termini. We usedthe consensus sequences to search the Swiss-Prot Database to retrieve the potential nativeligands. 4 out of 13 most promising candidate ligands were confirmed in one-to-one yeasttwo-hybrid experiments. The novel interactions identified here may reveal significant cluesfor further functional investigation. The results suggested that yeast two-hybird screeningof random peptide library was not only sufficient for binding property characterization ofPDZ domains, but also suitable for predicting physiologically relevant ligands for PDZdomains. A large proportion of protein-protein interaction is mediated by families ofpeptide-binding domains. Comprehensive characterization of each of them is critical forunderstanding the mechanisms and networks of protein interaction at the domain level.However. existing methods are all based on large-scale screenings for each domain, whichare inefficient to deal with hundreds of members in major domain families. To improveefficiency of current methods, we developed a systematic strategy for efficient bindingproperty characterization of peptide-binding domains based on high-throughput validationscreening of a specialized candidate ligand library. The library was constructed bycollecting the ligand clones isolated from Y2H screenings of random peptide libraries(RPY2H) with representative members of a domain family. By validation screening,consensus sequences were deduced from both positive and negative sequences. Novelinteractors were identified through database searches and confirmed in Y2H system. Thedomains that did not bind enough numbers of ligands from the ligand library wereconsidered as new representative ones and used as baits for de novo RPY2H. The ligandlibrary is gradually expanded with the addition of clones from more traditional screeningsand clones constructed for confirmations. The distinct feature of the strategy is that theefficiency is dramatically improved and will go higher as the system cycles, making itmore probable to characterize peptide-binding domain family in proteomic scale.PDZ domain family was first used to assess the strategy. We constructed an initial PDZligand library by RPY2H with Erbin PDZ, ZO-1 PDZ1,ZO-1 PDZ3 and GIPC PDZ. Next,we rapidly investigated Densin-180 PDZ,HtrA2 PDZ,LNX1 PDZ1 and LNX1 PDZ2 byvalidation screening of this initial ligand library. The library was further expanded with theaddition of new positive clones isolated by RGS3 PDZ and clones constructed forconfirmations. Broader binding properties have been identified compared to other methods,including novel recognition specificities that provided the basis for major revision ofconventional PDZ classification. Several novel interactions have been discovered, serving as significant clues for further functional investigation. This strategy can be extended to avariety of peptide-binding domains, as a powerful tool for comprehensive analysis ofdomain binding property in proteomic scale. SH2 domains recognize between 3-6 residues C-terminal to the phosphorylatedtyrosine in a fashion that differs from one SH2 domain to another. Binding propertycharacterization of SH2 domain is critical for the description of phosphotyrosine relatedsignal transduction. To analysis the phosphorylation-dependent and sequence specificrecognition of SH2 domains, a modified yeast two-hybrid method, which we calledyeast-three hybrid system, was employed here. In this system, SH2 domain was cloned intothe multiple cloning sitesⅠ(MCSI) of pBridge, Abl tyrosine kinase domain was cloned intothe MCSⅡ, in which expression of the kinase was controlled by a conditional methioninepromoter (P_MET25). In the absence of methionine, yeast was engineered to express thetyrosine kinase domain of Abl as an effector, in addition to two fusion proteins with GAL4DNA-binding and GAL4 activation domains as bait and prey proteins respectively.Using this system with Abl SH2 domain as bait, we obtained only one positive clone,which expressed the potential recognition sequence of YEIF. For Grb14 SH2 domain,another bait studied here, two consensus sequences, Y-x-x-(basic/ST) and Y-φ-x-φ(whereφis hydrophobic amino acid) were deduced from the positive clones that present tyrosineresidues. However, some positive clones, which lack Tyr but present Ser or Thr were alsoidentified. Mutational studies suggested that the interactions were not S/T-dependent. Wefailed to determine whether the interactions are specific. The results indicated that it maybe necessary to test several different kinases in order to ascertain the best forphosphorylation of tyrosine residues.