Abstract
Our previous studies suggest that the fully active form of Peptidylarginine deiminase 4 (PAD4) should be a dimer and not a monomer. This paper provides a plausible mechanism for the control of PAD4 catalysis by molecular interplay between its dimer-interface loop (I-loop) and its substrate-binding loop (S-loop). Mutagenesis studies revealed that two hydrophobic residues, W347 and V469, are critical for substrate binding at the active site; mutating these two residues led to a severe reduction in the catalytic activity. We also identified several hydrophobic amino acid residues (L6, L279 and V283) at the dimer interface. Ultracentrifugation analysis revealed that interruption of the hydrophobicity of this region decreases dimer formation and, consequently, enzyme activity. Molecular dynamic simulations and mutagenesis studies suggested that the dimer interface and the substrate-binding site of PAD4, which consist of the I-loop and the S-loop, respectively, are responsible for substrate binding and dimer stabilization. We identified five residues with crucial roles in PAD4 catalysis and dimerization: Y435 and R441 in the I-loop, D465 and V469 in the S-loop, and W548, which stabilizes the I-loop via van der Waals interactions with C434 and Y435. The molecular interplay between the S-loop and the I-loop is crucial for PAD4 catalysis.
Highlights
Peptidylarginine deiminases (PADs) are a family of enzymes that catalyze the conversion of protein-arginine to protein-citrulline, referred to as protein deimination or citrullination
In 2003, an rheumatoid arthritis (RA)-associated Peptidylarginine deiminase 4 (PAD4) haplotype consisting of 4 exonic single nucleotide polymorphisms (SNPs) was identified[24], and the increase in PAD4 protein levels in the synovium of RA patients compared to healthy individuals is due to the increased stability of the PAD4 mRNA containing these SNPs
As part of the elucidation of the relationship between structure and function in PAD4, we previously demonstrated that fully functional PAD4 should be in the dimeric form and that the non-catalytic calcium ions in the N-terminal domain are indispensable for the overall conformational stability of PAD4, which in turn is essential for full activation of the PAD4 enzyme[34,35]
Summary
Kinetic and Biophysical Properties of Human PAD4 Substrate-Binding Site Mutants. The crystal structures of PAD4 complexed with an artificial substrate, benzoyl-L-arginine amide (BA), and its histone peptide substrates[33,38] indicate that three arginyl residues, R372, R374 and R639, form hydrogen bonds with the amide oxygen and nitrogen of the substrate. The kcat value of R639A was 5.0 s−1, approximately 40% of the activity of the WT enzyme (11.7 s−1); its Km,BAEE was 0.7 mM, similar to WT, and the kcat/Km of R639A was 30% that of the WT enzyme (Table 1). These data indicate that R374 and R639 are important but not critical for PAD4 catalysis. The kinetic data for the two hydrophobic amino acid residues W347 and V469 revealed that mutations at these sites severely impaired the catalytic activity of PAD4 (Table 1). There are several hydrophobic amino acid residues in the dimer interface, including L6, L279, V283, V284 and F285 in the A
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