title: Protein arginine deiminase 2 binds calcium in an ordered fashion: implications for inhibitor design.
abstract: Protein arginine deiminases (PADs) are calcium-dependent histone-modifying enzymes whose activity is dysregulated in inflammatory diseases and cancer. PAD2 functions as an Estrogen Receptor (ER) coactivator in breast cancer cells via the citrullination of histone tail arginine residues at ER binding sites. Although an attractive therapeutic target, the mechanisms that regulate PAD2 activity are largely unknown, especially the detailed role of how calcium facilitates enzyme activation. To gain insights into these regulatory processes, we determined the first structures of PAD2 (27 in total), and through calcium-titrations by X-ray crystallography, determined the order of binding and affinity for the six calcium ions that bind and activate this enzyme. These structures also identified several PAD2 regulatory elements, including a calcium switch that controls proper positioning of the catalytic cysteine residue, and a novel active site shielding mechanism. Additional biochemical and mass-spectrometry-based hydrogen/deuterium exchange studies support these structural findings. The identification of multiple intermediate calcium-bound structures along the PAD2 activation pathway provides critical insights that will aid the development of allosteric inhibitors targeting the PADs.
authors.affiliation: †Department of Chemistry, The Scripps Research Institute, Jupiter, Florida 33458, United States.|¶Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, United States.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Slade
authors.affiliation: ‡Department of Cancer Biology, The Scripps Research Institute, Jupiter, Florida 33458, United States.
authors.forename: Pengfei
authors.initials: P
authors.lastname: Fang
authors.affiliation: †Department of Chemistry, The Scripps Research Institute, Jupiter, Florida 33458, United States.|∇Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, United States.
authors.forename: Christina J
authors.initials: CJ
authors.lastname: Dreyton
authors.affiliation: §Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri 63130, United States.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: †Department of Chemistry, The Scripps Research Institute, Jupiter, Florida 33458, United States.
authors.forename: Jakob
authors.initials: J
authors.lastname: Fuhrmann
authors.affiliation: §Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri 63130, United States.
authors.forename: Don
authors.initials: D
authors.lastname: Rempel
authors.affiliation: ∥Molecular Discovery Research, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts., SG1 2NY, United Kingdom.
authors.forename: Benjamin D
authors.initials: BD
authors.lastname: Bax
authors.affiliation: ⊥Department of Biomedical Sciences, Baker Institute for Animal Health, Cornell University, Ithaca, New York 14853, United States.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Coonrod
authors.affiliation: #EpiNova DPU, Immuno-Inflammation Therapy Area, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts., SG1 2NY, United Kingdom.
authors.forename: Huw D
authors.initials: HD
authors.lastname: Lewis
authors.affiliation: ‡Department of Cancer Biology, The Scripps Research Institute, Jupiter, Florida 33458, United States.
authors.forename: Min
authors.initials: M
authors.lastname: Guo
authors.affiliation: §Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri 63130, United States.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Gross
authors.affiliation: †Department of Chemistry, The Scripps Research Institute, Jupiter, Florida 33458, United States.|∇Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, United States.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Thompson
mesh_terms: D000595:Amino Acid Sequence; D001562:Benzimidazoles / Q000737:chemistry / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D006801:Humans; D006867:Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D011487:Protein Conformation; D000080064:Protein-Arginine Deiminase Type 2; D000076342:Protein-Arginine Deiminases
chemical_list: D001562:Benzimidazoles; D004791:Enzyme Inhibitors; C000607177:GSK199; D006867:Hydrolases; C000620352:PADI2 protein, human; D000080064:Protein-Arginine Deiminase Type 2; D000076342:Protein-Arginine Deiminases; D003545:Cysteine; D002118:Calcium