| Summary: | 碩士 === 國立中興大學 === 生命科學系所 === 97 === The peptidylarginine deiminase (PAD) is a Ca2+-dependent enzyme that can carry out protein citrullination (deimination) which is the conversion of protein-bound arginine to citrulline (Arg -> Cit). PAD4 is the only one isotype with a nuclear localization signal (NLS) region and the gene of PAD4 has a susceptible locus for rheumatoid arthritis (RA). The structures of PAD4 combined without or with calcium ions and the substrate have been resolved by X-ray crystallography. PAD4 is a dimer consisted of two monomer head-to-tail contact. However, it is still unclear the relationship between enzyme regulation and subunit-subunit interaction of PAD4. To gain insight into the quaternary structure stability of PAD4, we used LIGPLOT and DIMPLOT to find the important residues at dimer interface of PAD4 with hydrogen-bonded interaction. The Arg8 of one monomer is hydrogen-bonded with Asp547 of the other monomer and these two amino acid residues are charged residues containing the ionic interaction. On the other hand, the Tyr435 of one PAD4 monomer also has the hydrogen-bonded interaction with Tyr237 and Glu281 on the other monomer. Then we analyzed a series of PAD4 mutations at residues Arg8, Tyr237, Glu281, Tyr435, and Asp547 on the dimer interface by site-directed mutagenesis to disrupt the dimer organization combined with the analytical ultracentrifugation data and kinetic analysis. The mutations of PAD4 dimer interface caused quaternary structural change accompanied by the decreased activities and Hill coefficient (h) compared to WT. Here, our data indicated that the role of the dimeric state of PAD4 is not only important to maintain the stability of the overall structure but also to regulate its catalysis. Moreover, the ionic interaction between Arg8 and Asp547 slightly affect the dimer interface and the hydrophobic interaction of Tyr435 is the most important factor for the structure stability and activity of PAD4.
|
|---|
