title: Molecular Interplay between the Dimer Interface and the Substrate-Binding Site of Human Peptidylarginine Deiminase 4.
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.
authors.affiliation: Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan.|Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan.|Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan.
authors.forename: Chien-Yun
authors.initials: CY
authors.lastname: Lee
authors.affiliation: Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Chu-Cheng
authors.initials: CC
authors.lastname: Lin
authors.affiliation: Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan.|Institute of Biochemistry, Microbiology and Immunology, Chung Shan Medical University, Taichung, Taiwan.
authors.forename: Yi-Liang
authors.initials: YL
authors.lastname: Liu
authors.affiliation: Institute of Biochemistry, Microbiology and Immunology, Chung Shan Medical University, Taichung, Taiwan.|Division of Allergy, Immunology and Rheumatology, Chung Shan Medical University Hospital, Taichung, Taiwan.
authors.forename: Guang-Yaw
authors.initials: GY
authors.lastname: Liu
authors.affiliation: Institute of Genomics and Bioinformatics, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Jyung-Hurng
authors.initials: JH
authors.lastname: Liu
authors.affiliation: Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan.|Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan.|Institute of Genomics and Bioinformatics, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Hui-Chih
authors.initials: HC
authors.lastname: Hung
mesh_terms: D055162:Biocatalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006657:Histones / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D009154:Mutation; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D006657:Histones; D020033:Protein Isoforms; D011994:Recombinant Proteins; C000629235:PADI4 protein, human; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases