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, 44 (24), 8634-42

Structural Basis for the Retroreduction of Inactivated Peroxiredoxins by Human Sulfiredoxin

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Structural Basis for the Retroreduction of Inactivated Peroxiredoxins by Human Sulfiredoxin

Thomas J Jönsson et al. Biochemistry.

Abstract

Sufiredoxins (Srx) repair the inactivated forms of typical two-Cys peroxiredoxins (Prx) implicated in hydrogen peroxide-mediated cell signaling. The reduction of the cysteine sulfinic acid moiety within the active site of the Prx by Srx involves novel sulfur chemistry and the use of ATP and Mg(2+). The 1.65 A crystal structure of human Srx (hSrx) exhibits a new protein fold and a unique nucleotide binding motif containing the Gly98-Cys99-His100-Arg101 sequence at the N-terminus of an alpha-helix. HPLC analysis of the reaction products has confirmed that the site of ATP cleavage is between the beta- and gamma-phosphate groups. Cys99 and the gamma-phosphate of ATP, modeled within the active site of the 2.0 A ADP product complex structure, are adjacent to large surface depressions containing additional conserved residues. These features and the necessity for significant remodeling of the Prx structure suggest that the interactions between hSrx and typical two-Cys Prxs are specific. Moreover, the concave shape of the hSrx active site surface appears to be ideally suited to interacting with the convex surface of the toroidal Prx decamer.

Figures

Figure 1
Figure 1
Interrelationships between the catalytic and inactivation pathways of Prxs and the proposed mechanism of Srx. In the top part of the figure, the Cys-SPH and Cys-SRH residues of the Prx dimer (each from one subunit of the dimer; indicated by the divided rectangle) reduce peroxides, e.g., H2O2, through the formation of a sulfenic acid intermediate (Cys-SPOH) and an intermolecular disulfide bond which is ultimately reduced by a disulfide reductase (1, 2). Reaction of the Cys-SPOH moiety with another molecule of substrate results in the formation of Cys sulfinic acid (Cys-SPO2) and consequently the inactivation of the Prx molecule. The proposed mechanism of Srx (dashed box) begins with the activation of the sulfinic acid group by reaction with ATP (9). Additional steps include the thiol-dependent (R-SH) resolution of a sulfinic phosphoryl ester (Cys-SPO2PO32–) and thiosulfinate (Prx-Cys-SPO-SCys-Srx) intermediates. The second subunit of the Prx dimer and the Cys-SRH residue are not shown in the Srx mechanism for clarity. See the text for further details.
Figure 2
Figure 2
Structure of human sulfiredoxin. (A) Overall fold of hSrx in complex with phosphate. The α-helices (pink) and β-strands (blue) are numbered consecutively on the basis of the primary sequence. The N-terminal extension (residues 28–36) that protrudes from the protein surface to establish an unusual crystal contact (see Figure 1 of the Supporting Information for details) and the 310-helicies are not shown for clarity. Arg51, Cys99, His100, Arg101, and a phosphate molecule (PO4) are shown as sticks to illustrate the relationship of the active site structures to the novel protein fold. Atom colors are as follows: green for carbon, blue for nitrogen, red for oxygen, orange for sulfur, and magenta for phosphate. (B) Electron density (simulated annealing, FoFc omit map contoured at 3.0σ) within the active site of the refined model of wild-type ET-hSrx. Three water molecules (W1–W3) that interact with the phosphate ion are colored red. (C) Molecular interactions among the conserved GCHR motif of hSrx, phosphate, and solvent. Putative hydrogen bonding interactions are shown as dashed yellow lines. The phosphate molecule is bound by interactions with His100 and Arg101. The sulfur atom of the side chain of Cys99 is oriented outside the R2 helical axis and is 3.5 Å from the NH2 atom of Arg51 (as shown in panels A and B). This figure and subsequent molecular images were generated with PYMOL (54).
Figure 3
Figure 3
Structure of ADP bound to hSrx and the site of ATP cleavage. (A) Electron density and molecular interactions among the enzyme, solvent, and ADP. Simulated annealing, FoFc omit maps are contoured at 2σ and 4σ for ADP (cyan) and the oxidized, sulfinic acid form of Cys99 (Csd99, blue), respectively. Putative hydrogen bonding interactions are shown as dashed yellow lines. The adenine ring of ADP interacts with Ser64 and Thr68. One of the oxygen atoms of the α-phosphate group interacts with Lys61. The oxygen atoms of the β-phosphate group interact with His100, Arg101, W29, and W38. The backbone nitrogen atom of Arg101 also interacts with the oxygen atom (O1B) of the β-phosphate (not shown). Additional hydrogen bonding interactions extend from water molecules W29 and W38 to include Gly98, W52, W54, and Val56. Csd99 forms interactions with the conserved Arg51 and W18. (B) HPLC analysis of the site of ATP cleavage during the Srx reaction. Nucleotide standards (black line) were eluted from the Mono Q anion exchange column (see Materials and Methods) with monitoring at 254 nm. The Srx reaction was initiated by mixing wild-type, overoxidized hPrx2, wild-type ET-hSrx, ATP, and MgCl2 (red line). After incubation at 37 °C for 1 h, the samples were analyzed. Reactions were also carried out in which Cys99 of ET-hSrx was either prederivatized with iodoacetamide (green line) or mutated to Ser (cyan line).
Figure 4
Figure 4
hSrx surface features, hPrx2 active site, and model for the Srx-Prx interaction. (A) Solvent contact surface representation (1.7 Å probe) of hSrx with ATP modeled within the active site. The atom colors are the same as in Figure 1. Residues highlighted with black lines are strictly conserved. Gly98, Cys99, His100, Arg101, and Lys61 mediate ATP binding. These residues along with Pro52, Asp58, Asp80, and Phe96 may also play a key role in establishing the Srx-Prx macromolecular interface. Other residues that may also contribute to the latter role are highlighted in red: Pro59, Ser64, Thr68, Lys116, Asp124, Val127, and Tyr128. (B) Close-up of the hPrx2 α2 dimer (purple and yellow chains) on the perimeter of the sulfinic acid form of the hPrx2 decamer (PDB entry 1QMV) (40). The hydrogen bonds between Csd51 (Cys51-SPO2) and Arg127 stabilize the active site. Nearby within the same chain is the Gly-Gly-Leu-Gly (GGLG) motif (residues 93-96). The Tyr-Phe (YF) motif within the C-terminal helix (residues 187-197) and the resolving Cys residue, Cys172, are contributed from the adjacent subunit of the dimer. (C) Model for the Srx-Prx interaction. The top panel is an edge-on view of four subunits of the Prx decamer before the binding of Srx. The α2 dimer (blue) is shown in the center with the active sites and the noncrystallographic, 2-fold axis colored yellow and red, respectively. Adjacent Prx subunits within the decamer are represented by the nonfilled, black circles. The bottom panel shows Srx molecules (green) bound in an antiparallel manner to the circumference of the Prx ringlike decamer. Only the Srx molecules bound to the central Prx dimer are shown. The plus sign indicates directionality. The Srx molecules may interact with each other and adjacent Prx subunits.
Figure 5
Figure 5
Proposed mode of binding of ATP and the first step of the hSrx reaction. Putative hydrogen bonding interactions between hSrx and ATP (nucleotide and sugar structures denoted by A) are indicated by dashed lines. The Cys sulfinic acid moiety of the Prx molecule to be repaired is shown in bold. See the text for details.

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