Rapid characterization of the binding property of HtrA2/Omi PDZ domain by validation screening of PDZ ligand library

Abstract

HtrA2/Omi is a mammalian mitochondrial serine protease, and was found to have dual roles in mammalian cells, not only acting as an apoptosis-inducing protein but also maintaining mitochondrial homeostasis. PDZ domain is one of the most important protein-protein interaction modules and is involved in a variety of important cellular functions, such as signal transduction, degradation of proteins, and formation of cytoskeleton. Recently, it was reported that the PDZ domain of HtrA2/Omi might regulate proteolytic activity through its interactions with ligand proteins. In this study, we rapidly characterized the binding properties of HtrA2/Omi PDZ domain by validation screening of the PDZ ligand library with yeast two-hybrid approach. Then, we predicted its novel ligand proteins in human proteome and reconfirmed them in the yeast two-hybrid system. Finally, we analyzed the smallest networks bordered by the shortest path length between the protein pairs of novel interactions to evaluate the confidence of the identified interactions. The results revealed some novel binding properties of HtrA2/Omi PDZ domain. Besides the reported Class II PDZ motif, it also binds to Class I and Class III motifs, and exhibits restricted variability at P(-3), which means that the P(-3) residue is selected according to the composition of the last three residues. Seven novel ligand proteins of HtrA2/Omi PDZ domain were discovered, providing significant clues for further clarifying the roles of HtrA2/Omi. Moreover, this study proves the high efficiency and practicability of the newly developed validation screening of candidate ligand library method for binding property characterization of peptide-binding domains.

The Serine Protease HtrA1 Specifically Interacts and Degrades the Tuberous Sclerosis Complex 2 Protein

“…These binding experiments indicate that HtrA1 is able to interact with TSC2 but not with the other component of the TSC complex, TSC1, thereby confirming that there is a specific interaction between HtrA1 and TSC2. It is well known that the PDZ domain is the prime site of interactions for HtrA1 (41,(51)(52)(53). Nevertheless, our study shows the first interaction in which HtrA1 binds to another protein through the mac25 domain.…”

Allosteric Regulation of Serine Protease HtrA2 through Novel Non-Canonical Substrate Binding Pocket

“…A 10 mer peptide having the sequence KNNPNNAHQN that does not match the consensus SBP binding peptide pattern was used as a negative control. These combinations were used for searching all possible sequences of known and potential HtrA2 binding partners [38].…”

“…Identification of the putative binding site(s) on HtrA2 was done using SiteMap 2.5 [18] and the selective binding pocket (SBP) for the ligand was chosen based on optimum energy parameters. Peptides at SBP were docked from our peptide library that was generated based on available literature reports [19], [20], [21] and structural complementarities. MDS of the docked structures was done using Desmond 2010 [22] which provided critical information on loop and linker movements in HtrA2.…”

Proteomics Strategy to Identify Substrates of LNX, a PDZ Domain-containing E3 Ubiquitin Ligase

“…Each LNX PDZ domain was used individually as bait to screen this library using the Y2H approach as previously described. 32,33 WebLogo, a sequence logo generator, 34 was used to analyze and graphically display the binding properties of each LNX PDZ domain that recognized the C-terminal binding motifs. Consensus binding sequences were deduced from the sequence alignment of the positive clones and from the comparative analysis of both the positive and negative sequences.…”