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, 24 (10), 1668-1678

Peroxiredoxin Catalysis at Atomic Resolution

Affiliations

Peroxiredoxin Catalysis at Atomic Resolution

Arden Perkins et al. Structure.

Abstract

Peroxiredoxins (Prxs) are ubiquitous cysteine-based peroxidases that guard cells against oxidative damage, are virulence factors for pathogens, and are involved in eukaryotic redox regulatory pathways. We have analyzed catalytically active crystals to capture atomic resolution snapshots of a PrxQ subfamily enzyme (from Xanthomonas campestris) proceeding through thiolate, sulfenate, and sulfinate species. These analyses provide structures of unprecedented accuracy for seeding theoretical studies, and reveal conformational intermediates giving insight into the reaction pathway. Based on a highly non-standard geometry seen for the sulfenate intermediate, we infer that the sulfenate formation itself can strongly promote local unfolding of the active site to enhance productive catalysis. Further, these structures reveal that preventing local unfolding, in this case via crystal contacts, results in facile hyperoxidative inactivation even for Prxs normally resistant to such inactivation. This supports previous proposals that conformation-specific inhibitors may be useful for achieving selective inhibition of Prxs that are drug targets.

Figures

Figure 1
Figure 1
Summary of proposed mechanisms of Prx peroxidation. A. Overlay of active sites of DTT-bound human PrxV (light blue) and peroxide-bound ApTpx (green) with water-bound wild-type XcPrxQ (white), with ligands colored cyan (DTT), lime (H2O2), and dark gray (waters), respectively. Dashed lines indicate active site interactions for XcPrxQ, including a contact between the CδH of Tyr40 with the thiolate that would also contribute weak electrostatic stabilization to the thiolate. This appears to be a conserved interaction in FF Prx structures and involves either a Tyr or His. B–D. Previous studies do not provide a consensus on how Prx catalysis proceeds, as evident by the many different hydrogen bond interactions and residue shifts proposed. Hydrogen bonds seen in the XcPrxQ structures in this study (dark gray arrows pointing from donor to acceptor) for the substrate-bound state, the inferred transition state (based on relative rigidity of active site residues), and the product states, are compared with those proposed by previous studies: brown arrows for Hall et al. (Hall et al., 2010), blue arrows for Portillo et al. (Portillo-Ledesma et al., 2014), green arrows for Zeida et al. (Zeida et al., 2014), and pink arrows for Nagy et al. (Nagy et al., 2011). Shifts in residue positions are noted by thick arrows (Thr shift in blue panels C/D and Pro shift in dark gray in panel D). Forming and breaking covalent bonds are depicted as double dashed lines. Atoms not incorporated into previous QM simulations, presumably because they were not thought to play key roles in the chemistry of catalysis, are highlighted in yellow in panel B. The circled hydrogen and dashed arrow in panel D notes a predicted proton transfer by Portillo et al. (Portillo-Ledesma et al., 2014). See also Fig. S1 for an analysis of available Prx crystal structures.
Figure 2
Figure 2
In solution kinetics of XcPrxQ. A. Bisubstrate kinetics of XcPrxQ (0.2 μM) with hydrogen peroxide and EcTrxA at 5 μM (black), 10 μM (red), 20 μM (green) and 40 μM (blue). The curves are the results of the global fitting of all four data sets. B. Same as A, but for cumene peroxide and EcTrxA at 5 μM (black), 10 μM (red), 20 μM (green) and 50 μM (blue). C–D. Time courses showing XcPrxQ hyperoxidative activity loss during reactions with varying concentrations (as indicated) of hydrogen peroxide and cumene hydroperoxide, respectively. E. The fraction of protein inactivated per turnover (Wood et al., 2003) (finact) is plotted as a function of peroxide concentration for XcPrxQ hyperoxidation by hydrogen peroxide (closed circles) and cumene peroxide (open circles). F. Sensitivity of XcPrxQ to hyperoxidation by hydrogen peroxide (closed circles) is compared to literature data (Nelson et al., 2013) for human PrxI (open squares) and Salmonella typhimurium AhpC (open circles). See also Table S2 and Table S3.
Figure 3
Figure 3
Fully Folded and Locally Unfolded Conformations of XcPrxQ. A. The active FF conformation (left) and the LU disulfide conformation are shown (right) as cartoons colored by local mobility as indicated by B-factor, and with sticks for CP, CR and the conserved active site Pro, Thr and Arg. The largest conformation changes involve the helix 3 region that is highlighted in yellow. This structural rearrangement moves CP away from the active site pocket, disrupting all of its hydrogen bonding interactions, as described by Liao et al. (Liao et al., 2009). B. The active site of the XcPrxQ FF structure with 2FO-FC density (thin blue mesh 1.5 ρrms pink mesh 3.5 ρrms). C. Same as B but for the LU CP-CR disulfide structure.
Figure 4
Figure 4
Atomic resolution snapshots of XcPrxQ catalysis. A–D. Structures FF0, FF3, FF5 and FF8, respectively, are shown with their 2FO-FC electron density (blue contoured at 1.0 ρrms) and difference electron density between the structure of interest and FF0 (green and red contoured at ±3.0 ρrms). In panels B–D, key shifts in active site residues are indicated by brown arrows with the conserved Pro flipping between two positions (noted as P1 and P2) and the Arg transitioning among three positions (R1, R2, R3). E. CP is shown with occupancies of its oxygen adducts indicated by color (from light to dark red). 2FO-FC density is shown (blue contoured at 1.0 ρrms and for the sulfenate oxygen in FF1 olive contoured at 0.3 ρrms). Density near the top of the image in structures FF4–FF7 is from a partially-occupied Arg conformation that occurs as the protein converts from the sulfenate to sulfinate state. F. A single image showing the 2FO-FC electron density peaks for CP oxygens from all structures as single planes (solid colors at 1.0 ρrms, red outline 0.3 ρrms). The overlay shows the progression of the oxidation of CP: from 10% SO (red outline; FF1), 45% SO (orange; FF2), 50% SO (yellow; FF3), 10% SO2 (light green; FF4), 20% SO2 (cyan; FF5), 20% SO2 (blue; FF6), 35% SO2 (dark blue; FF7), 100% SO2, (purple; FF8). All structural images were prepared using Pymol. See also Figure S3 and Figure S4.
Figure 5
Figure 5
Unusual Cys-sulfenate geometry. A. Packing interactions of the Cys-sulfenate are shown for structure FF3, highlighting interactions with the Cys-Sγ (gray dashes and distances in Å), with the sulfenate oxygen (black dashes and distances), and a possibly unfavorable interaction between Thr45-OH and the CP-SO (red dash and distance). 2FO-FC electron density evidence for the sulfenate position is also shown (blue contoured at 1.0 ρrms). B. Interaction of the CP-thiolate with Pro41 (upper image) compared with that of the CP-sulfenate (at 0.5 occupancy and labeled with its unusual bond angle and distance reported; lower image). A brown arrow shows the shift of Pro41-Cγ accommodating the sulfenate formation by relieving a steric clash (red double-headed arrow) to increase the Pro41-Cγ to sulfenate oxygen distance to 3.6 Å. C. Structure of C48S XcPrxQ including bound water and phosphate (pink model) is compared with the XcPrxQ thiolate (FF0) and XcPrxQ sulfenate (FF3) forms (similar models both with white carbons; FF0 waters are gray spheres) and peroxide bound ApTpx (PDB code 3a2v, green carbons). Shift of the Pro is noted with a brown arrow. Notable observations are the C48S water and phosphate being near the FF3 Cys-sulfenate oxygen and the ApTpx peroxide, respectively. D. Gas phase quantum mechanical calculations (Zhao and Truhlar, 2008) of methyl SO, SH2O+, and SH2O species showing their energies as a function of their C-S-O bond angle (upper panel) as well as the optimized geometries and energies for the structures constrained to have a C-S-O angle of 155º. 3D figures were generated using CYLview (Legault, 2009). See Figures S4 and S5 for supporting details.
Figure 6
Figure 6
Non-covalent stabilization of the FF conformation can promote hyperoxidation. A. Protein surface area buried by crystal contacts (gray) has substantial overlap with magnitudes of conformation change (red) on a per residue basis along the chain. The CP (Cys48) and CR (Cys84) positions are denoted (*). Residues 79–81, not well defined in the LU structure, were assigned a shift of 10 Å based on their neighbors. B. One crystal contact region in the FF XcPrxQ crystals (white for main molecule and gray for symmetry mate) overlaid with LU structure (black) shows FF→LU movement (brown arrows) of Phe83 is prevented by a steric clash with Arg103 of the symmetry mate (red) because the two side chains would be 1.3 Å apart. C. A general mechanism for Prx catalysis and hyperoxidation is shown, as has been previously proposed (Sevilla et al., 2015)(Perkins et al., 2015)(Perkins et al., 2014), that highlights the key physiologically-relevant redox states that have been captured at atomic resolution for XcPrxQ. Also emphasized is that inhibiting facile unfolding enhances inactivation. This has been observed to occur for sensitive Prx1-subfamily members by stabilization by the C-terminal tail (Wood et al., 2003). In the case of XcPrxQ, the FF crystal form can be conceptually considered as a large non-covalent FF-conformation-stabilizing inhibitor, trapping the enzyme as FF and promoting inactivation.

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