title: The sensitive balance between the fully folded and locally unfolded conformations of a model peroxiredoxin.
abstract: To reduce peroxides, peroxiredoxins (Prxs) require a key "peroxidatic" Cys that, in a substrate-ready fully folded (FF) conformation, is oxidized to sulfenic acid and then, after a local unfolding (LU) of the active site, forms a disulfide bond with a second "resolving" Cys. For Salmonella typhimurium alkyl hydroperoxide reductase C (StAhpC) and some other Prxs, the FF structure is only known for a peroxidatic Cys→Ser variant, which may not accurately represent the wild-type enzyme. Here, we obtain the structure of authentic reduced wild-type StAhpC by dithiothreitol treatment of disulfide form crystals that fortuitously accommodate both the LU and FF conformations. The unique environment of one molecule in the crystal reveals a thermodynamic linkage between the folding of the active site loop and C-terminal regions, and comparisons with the Ser variant show structural and mobility differences from which we infer that the Cys→Ser mutation stabilizes the FF active site. A structure for the C165A variant (a resolving Cys to Ala mutant) in the same crystal form reveals that this mutation destabilizes the folding of the C-terminal region. These structures prove that subtle modifications to Prx structures can substantially influence enzymatic properties. We also present a simple thermodynamic framework for understanding the various mixtures of FF and LU conformations seen in these structures. On the basis of this framework, we rationalize how physiologically relevant regulatory post-translational modifications may modulate activity, and we propose a nonconventional strategy for designing selective Prx inhibitors.
authors.affiliation: Department of Biochemistry and Biophysics, Oregon State University , Corvallis, Oregon 97331, United States.
authors.forename: Arden
authors.initials: A
authors.lastname: Perkins
authors.forename: Kimberly J
authors.initials: KJ
authors.lastname: Nelson
authors.forename: Jared R
authors.initials: JR
authors.lastname: Williams
authors.forename: Derek
authors.initials: D
authors.lastname: Parsonage
authors.forename: Leslie B
authors.initials: LB
authors.lastname: Poole
authors.forename: P Andrew
authors.initials: PA
authors.lastname: Karplus
mesh_terms: D019943:Amino Acid Substitution; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D054464:Peroxiredoxins / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000502:physiology; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics; D012694:Serine / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D012694:Serine; D054464:Peroxiredoxins; D003545:Cysteine