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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.

SUBMITTER: Perkins A

PROVIDER: S-EPMC3932808 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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The sensitive balance between the fully folded and locally unfolded conformations of a model peroxiredoxin.

Perkins Arden A Nelson Kimberly J KJ Williams Jared R JR Parsonage Derek D Poole Leslie B LB Karplus P Andrew PA

Biochemistry 20131120 48

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 obt ...[more]

PMID: 24175952

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Project description:Cdt1 is a protein critical for DNA replication licensing and is well-established to be a binding partner of the minichromosome maintenance (MCM) complex. Cdt1 has also been demonstrated to have an emerging, "moonlighting" role at the kinetochore via direct binding to microtubules and to the Ndc80 complex. However, it is not known how the structure and conformations of Cdt1 could allow for these multiple, completely unique sets of protein complexes. And while there exist multiple robust methods to study entirely folded or entirely unfolded proteins, structure-function studies of combined, mixed folded/disordered proteins remain challenging. It this work, we employ multiple orthogonal biophysical and computational techniques to provide a detailed structural characterization of human Cdt1 92-546. DSF and DSCD show both folded winged helix (WH) domains of Cdt1 are relatively unstable. CD and NMR show the N-terminal and the linker regions are intrinsically disordered. Using DLS and SEC-MALS, we show that Cdt1 is polydisperse, monomeric at high concentrations, and without any apparent inter-molecular self-association. SEC-SAXS of the monomer in solution enabled computational modeling of the protein in silico. Using the program SASSIE, we performed rigid body Monte Carlo simulations to generate a conformational ensemble. Using experimental SAXS data, we filtered for conformations which did and did not fit our data. We observe that neither fully extended nor extremely compact Cdt1 conformations are consistent with our SAXS data. The best fit models have the N-terminal and linker regions extended into solution and the two folded domains close to each other in apparent "folded over" conformations. The best fit Cdt1 conformations are consistent with a function as a scaffold protein which may be sterically blocked without the presence of binding partners. Our studies also provide a template for combining experimental and computational biophysical techniques to study mixed-folded proteins.

Project description:BACKGROUND:Mounting evidence shows that ROS regulation by various antioxidants is essential for the expression of enzymes involved in steroidogenesis and maintenance of progesterone production by the corpus luteum (CL). However, the underlying mechanisms of peroxiredoxin 1 (PRDX1), an antioxidant enzyme, in luteal function for progesterone production in mice have not been reported. The aim of this study was to evaluate the functional link between PRDX1 and progesterone production in the CL of Prdx1 knockout (K/O) mice in the functional stage of CL. METHODS:The expression pattern of the unfolded protein response (UPR) signaling pathways, endoplasmic reticulum (ER) stress-induced apoptosis related genes and peroxiredoxins 1 (PRDX1) were investigated by western blotting analysis in CL tissue of 10 weeks mice during functional stage of CL. The protein levels of these genes after ER-stress inducer tunicamycin (Tm), ER-stress inhibitor tauroursodeoxycholic acid (TUDCA) and ROS scavenger, N-acetylcysteine (NAC) stimulation by intraperitoneal (i.p) injection were also investigated in CL tissue of wild type (WT) mice. Finally, we examined progesterone production and UPR signaling related gene expression in CL tissue of Prdx1 K/O mice. RESULTS:We demonstrated that PRDX1 deficiency in the functional stage activates the UPR signaling pathways in response to ER stress-induced apoptosis. Interestingly, CL number, serum progesterone levels, and steroidogenic enzyme expression in Prdx1 K/O mice decreased significantly, compared to those in wild type mice. Levels of UPR signaling pathway markers (GRP78/BIP, P50ATF6, and phosphorylated (p)-eIF2) and ER-stress associated apoptotic factors (CHOP, p-JNK, and cleaved caspase-3) were dramatically increased in the CL tissue of Prdx1 K/O mice. In addition, administration of the NAC, reduced progesterone production and activated ER-stress-induced UPR signaling in the CL tissue obtained from the ovary of Prdx1 K/O mice. Taken together, these results indicated that reduction in serum progesterone levels and activation of ER-stress-induced UPR signaling are restored by NAC injection in the CL of Prdx1 K/O mice. CONCLUSION:These observations provide the first evidence regarding the basic mechanisms connecting PRDX1 and progesterone production in the functional stage of CL.

Dataset Information

The sensitive balance between the fully folded and locally unfolded conformations of a model peroxiredoxin.

Publications

The sensitive balance between the fully folded and locally unfolded conformations of a model peroxiredoxin.

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