Probing the Roles of Calcium-Binding Sites during the Folding of Human Peptidylarginine Deiminase 4

Our recent studies of peptidylarginine deiminase 4 (PAD4) demonstrate that its non-catalytic Ca(2+)-binding sites play a crucial role in the assembly of the correct geometry of the enzyme. Here, we examined the folding mechanism of PAD4 and the role of Ca(2+) ions in the folding pathway. Multiple mutations were introduced into the calcium-binding sites, and these mutants were termed the Ca1_site, Ca2_site, Ca3_site, Ca4_site and Ca5_site mutants. Our data indicate that during the unfolding process, the PAD4 dimer first dissociates into monomers, and the monomers then undergo a three-state denaturation process via an intermediate state formation. In addition, Ca(2+) ions assist in stabilizing the folding intermediate, particularly through binding to the Ca3_site and Ca4_site to ensure the correct and active conformation of PAD4. The binding of calcium ions to the Ca1_site and Ca2_site is directly involved in the catalytic action of the enzyme. Finally, this study proposes a model for the folding of PAD4. The nascent polypeptide chains of PAD4 are first folded into monomeric intermediate states, then continue to fold into monomers, and ultimately assemble into a functional and dimeric PAD4 enzyme, and cellular Ca(2+) ions may be the critical factor governing the interchange.