PDZ Domain Binding Characteristics Intermediate Sequence Binding Studies

●In Part I, we studied the non-canonical binding properties of PDZ domains to internal sequences.There are enormous protein-protein interaction that mainly mediated by protein domains in multiple biological processes. Large-scale protein interactions can be investigated by studying the binding properties of protein domain. Protein domains (such as PDZ, SH3, SH2, WW, etc.) can canonically recognize a conservative short peptide sequence through one or more binding "pocket". However, there are probably more ways of protein interaction than we currently realize, some non-canonical protein interactions were still needed to be investigated. Like PDZ domains, they are important protein interaction modules that are considered mainly recognize C-terminal4-5amino acids, they can also non-canonically bind internal sequences, polymerize with themselves or other PDZ domains, or can bind to some lipids. Protein interactions become much more diverse and complicate by discovering those non-canonical interaction modes which expand the research field and provide more powerful evidence.First, to systematically and high-throughput study the binding characteristics of PDZ domains to internal sequences, a nearly random octapeptide library with no stop codon designed in the insert was constructed in the yeast two-hybrid vector. To ensure the PDZ domains really interact with internal sequences, all the C-termini of sequences of the whole library were the same. Then, a total of twenty-four PDZ domains were used as baits to screen the library, including some reported internal sequence-binding PDZ domains, some constructed PDZ domain clones of our lab and some PDZ domains which can bind with membrane lipids. Among the twenty-four PDZ domains, fourteen were found to bind internal sequences, including six that were not previously reported to bind internal sequences. PDZ domains showed strong amino acids preferences; only the consensuse of ZO1-PDZ1was similar to its C-terminal ligands, but the other consensuses were more diverse than those of common C-terminal ligands. In addition, HtrA2-PDZ with no strong consensus recognized extended hydrophobic peptides. The results suggested that more PDZ domains can bind internal sequences. Moreover, using the binding properties of internal sequences can help to find the potential binding site of reported interactions and to search the database to find potential natural binding partners. ●In Part Ⅱ, we presented the molecular characteristics of Schistosoma japonicum PDZ domain-containing protein, SjGIPC3and investigated the ligand binding properties of its PDZ domain.Schistosomiasis remains a serious global health problem, which afflicts more than230million people in77countries. The long-term mass treatments with the only available drug, praziquantel, have caused the concern of the drug resistance. PDZ domain containing proteins are the potential target for next-generation of drug development. In this second part, we presented the molecular characteristics of the Schistosoma japonicum PDZ domain-containing protein, SjGIPC3, and explored its ligand binding specificity by yeast two-hybrid (Y2H) using random peptide libraries.SjGIPC3is a single PDZ domain-containing protein. Multiple sequences alignment revealed that GH1and PDZ domain of SjGIPC3was relatively conserved with its orthologs, however, the carboxylate-binding loop within the PDZ domain was not, and the classic GLGF motif is substituted by SFGL. The phylogenetic analysis revealed that SjGIPC3and other trematode orthologs clustered into a well-defined cluster but was distinguishable from those of other phyla. Transcriptional analysis by qRT-PCR showed that SjGIPC3gene was particularly expressed in the stage within the host, especially in male adult worms.In the system of Y2H, we investigated the C-termini binding properties of SjGIPC3-PDZ. We used SjGIPC3full-length protein or only its PDZ domain as bait, to screen an arbitrary peptide library. We found that the SjGIPC3PDZ domain can both bind class Ⅰ and Ⅱ ligands, but more preferred to bind class I ligand. The binding sequences of SjGIPC3full-length protein have more proterties than those of SjGIPC3-PDZ, indicating that the N-terminal and C-terminal regions flanking the PDZ domain may affect the binding proterties of the PDZ domain. Further, native ligands were predicted based on the C-termini binding properties with Tailfit software, four potential ligands were confirmed by Y2H system, one of them was NMDA receptor.