title: A redox 2-Cys mechanism regulates the catalytic activity of divergent cyclophilins.
abstract: The citrus (Citrus sinensis) cyclophilin CsCyp is a target of the Xanthomonas citri transcription activator-like effector PthA, required to elicit cankers on citrus. CsCyp binds the citrus thioredoxin CsTdx and the carboxyl-terminal domain of RNA polymerase II and is a divergent cyclophilin that carries the additional loop KSGKPLH, invariable cysteine (Cys) residues Cys-40 and Cys-168, and the conserved glutamate (Glu) Glu-83. Despite the suggested roles in ATP and metal binding, the functions of these unique structural elements remain unknown. Here, we show that the conserved Cys residues form a disulfide bond that inactivates the enzyme, whereas Glu-83, which belongs to the catalytic loop and is also critical for enzyme activity, is anchored to the divergent loop to maintain the active site open. In addition, we demonstrate that Cys-40 and Cys-168 are required for the interaction with CsTdx and that CsCyp binds the citrus carboxyl-terminal domain of RNA polymerase II YSPSAP repeat. Our data support a model where formation of the Cys-40-Cys-168 disulfide bond induces a conformational change that disrupts the interaction of the divergent and catalytic loops, via Glu-83, causing the active site to close. This suggests a new type of allosteric regulation in divergent cyclophilins, involving disulfide bond formation and a loop-displacement mechanism.
authors.affiliation: Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP CP6192, Brazil.
authors.forename: Bruna Medéia
authors.initials: BM
authors.lastname: Campos
authors.forename: Mauricio Luis
authors.initials: ML
authors.lastname: Sforça
authors.forename: Andre Luis Berteli
authors.initials: AL
authors.lastname: Ambrosio
authors.forename: Mariane Noronha
authors.initials: MN
authors.lastname: Domingues
authors.forename: Tatiana de Arruda Campos
authors.initials: Tde A
authors.lastname: Brasil de Souza
authors.forename: João Alexandre Ribeiro Gonçalvez
authors.initials: JA
authors.lastname: Barbosa
authors.forename: Adriana Franco
authors.initials: AF
authors.lastname: Paes Leme
authors.forename: Carlos Alberto
authors.initials: CA
authors.lastname: Perez
authors.forename: Sara Britt-Marie
authors.initials: SB
authors.lastname: Whittaker
authors.forename: Mario Tyago
authors.initials: MT
authors.lastname: Murakami
authors.forename: Ana Carolina de Matos
authors.initials: AC
authors.lastname: Zeri
authors.forename: Celso Eduardo
authors.initials: CE
authors.lastname: Benedetti
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D032084:Citrus sinensis / Q000378:metabolism*; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D021983:Cyclophilins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016572:Cyclosporine / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011487:Protein Conformation; D012319:RNA Polymerase II / Q000378:metabolism*; D013879:Thioredoxins / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
chemical_list: D004220:Disulfides; D018698:Glutamic Acid; D013879:Thioredoxins; D016572:Cyclosporine; D012319:RNA Polymerase II; D021983:Cyclophilins; D015032:Zinc; D003545:Cysteine