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• W3134409844 abstract "Peroxiredoxin 2 (Prdx2) is a thiol peroxidase with an active site Cys (C52) that reacts rapidly with H2O2 and other peroxides. The sulfenic acid product condenses with the resolving Cys (C172) to form a disulfide which is recycled by thioredoxin or GSH via mixed disulfide intermediates or undergoes hyperoxidation to the sulfinic acid. C172 lies near the C terminus, outside the active site. It is not established whether structural changes in this region, such as mixed disulfide formation, affect H2O2 reactivity. To investigate, we designed mutants to cause minimal (C172S) or substantial (C172D and C172W) structural disruption. Stopped flow kinetics and mass spectrometry showed that mutation to Ser had minimal effect on rates of oxidation and hyperoxidation, whereas Asp and Trp decreased both by ∼100-fold. To relate to structural changes, we solved the crystal structures of reduced WT and C172S Prdx2. The WT structure is highly similar to that of the published hyperoxidized form. C172S is closely related but more flexible and as demonstrated by size exclusion chromatography and analytical ultracentrifugation, a weaker decamer. Size exclusion chromatography and analytical ultracentrifugation showed that the C172D and C172W mutants are also weaker decamers than WT, and small-angle X-ray scattering analysis indicated greater flexibility with partially unstructured regions consistent with C-terminal unfolding. We propose that these structural changes around C172 negatively impact the active site geometry to decrease reactivity with H2O2. This is relevant for Prdx turnover as intermediate mixed disulfides with C172 would also be disruptive and could potentially react with peroxides before resolution is complete. Peroxiredoxin 2 (Prdx2) is a thiol peroxidase with an active site Cys (C52) that reacts rapidly with H2O2 and other peroxides. The sulfenic acid product condenses with the resolving Cys (C172) to form a disulfide which is recycled by thioredoxin or GSH via mixed disulfide intermediates or undergoes hyperoxidation to the sulfinic acid. C172 lies near the C terminus, outside the active site. It is not established whether structural changes in this region, such as mixed disulfide formation, affect H2O2 reactivity. To investigate, we designed mutants to cause minimal (C172S) or substantial (C172D and C172W) structural disruption. Stopped flow kinetics and mass spectrometry showed that mutation to Ser had minimal effect on rates of oxidation and hyperoxidation, whereas Asp and Trp decreased both by ∼100-fold. To relate to structural changes, we solved the crystal structures of reduced WT and C172S Prdx2. The WT structure is highly similar to that of the published hyperoxidized form. C172S is closely related but more flexible and as demonstrated by size exclusion chromatography and analytical ultracentrifugation, a weaker decamer. Size exclusion chromatography and analytical ultracentrifugation showed that the C172D and C172W mutants are also weaker decamers than WT, and small-angle X-ray scattering analysis indicated greater flexibility with partially unstructured regions consistent with C-terminal unfolding. We propose that these structural changes around C172 negatively impact the active site geometry to decrease reactivity with H2O2. This is relevant for Prdx turnover as intermediate mixed disulfides with C172 would also be disruptive and could potentially react with peroxides before resolution is complete. Human peroxiredoxin 2 (Prdx2) is a cytoplasmic thiol peroxidase that is a major contributor to cellular antioxidant defense and redox regulation. It is a typical 2-Cys peroxiredoxin with an active site (peroxidatic) Cys residue (CP) that reacts extremely rapidly with H2O2 (1Rhee S.G. Kil I.S. Multiple functions and regulation of mammalian peroxiredoxins.Annu. Rev. Biochem. 2017; 86: 749-775Crossref PubMed Scopus (158) Google Scholar, 2Perkins A. Nelson K.J. Parsonage D. Poole L.B. Karplus P.A. Peroxiredoxins: Guardians against oxidative stress and modulators of peroxide signaling.Trends Biochem. Sci. 2015; 40: 435-445Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). The initial product is a CP-sulfenic acid that undergoes a structural rearrangement to condense with the resolving Cys (CR) located in the C-terminal region of the opposing chain of the functional homodimer (as shown in Fig. 1). At higher H2O2 concentrations, this occurs in competition with further oxidation (hyperoxidation) to the sulfinic acid. During turnover, the CP-CR disulfide is recycled by reaction with thioredoxin/thioredoxin reductase (Trx/TrxR). The sulfenic acid and the CP-CR disulfide also react with GSH to form a mixed disulfide which is reduced by glutaredoxin (3Peskin A.V. Pace P.E. Behring J.B. Paton L.N. Soethoudt M. Bachschmid M.M. Winterbourn C.C. Glutathionylation of the active site cysteines of peroxiredoxin 2 and recycling by glutaredoxin.J. Biol. Chem. 2016; 291: 3053-3062Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The active site geometry dictates the exquisite specificity and high reactivity of CP with H2O2 and other peroxides (4Perkins A. Parsonage D. Nelson K.J. Ogba O.M. Cheong P.H. Poole L.B. Karplus P.A. Peroxiredoxin catalysis at atomic resolution.Structure. 2016; 24: 1668-1678Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 5Hall A. Nelson K. Poole L.B. Karplus P.A. Structure-based insights into the catalytic power and conformational dexterity of peroxiredoxins.Antioxid. Redox Signal. 2011; 15: 795-815Crossref PubMed Scopus (246) Google Scholar), and it is often assumed that mutations and/or modifications of CR would have little impact on the CP environment and reactivity. On this basis, a number of studies, especially those investigating interacting partners or redox relays involving 2-Cys peroxiredoxins, have been performed with the resolving Cys mutated to Ser or Ala (e.g., (6Kwak M.S. Kim H.S. Lkhamsuren K. Kim Y.H. Han M.G. Shin J.M. Park I.H. Rhee W.J. Lee S.K. Rhee S.G. Shin J.S. Peroxiredoxin-mediated disulfide bond formation is required for nucleocytoplasmic translocation and secretion of HMGB1 in response to inflammatory stimuli.Redox Biol. 2019; 24: 101203Crossref PubMed Scopus (30) Google Scholar, 7Anschau V. Ferrer-Sueta G. Aleixo-Silva R.L. Bannitz Fernandes R. Tairum C.A. Tonoli C.C.C. Murakami M.T. de Oliveira M.A. Netto L.E.S. Reduction of sulfenic acids by ascorbate in proteins, connecting thiol-dependent to alternative redox pathways.Free Radic. Biol. Med. 2020; 156: 207-216Crossref PubMed Scopus (12) Google Scholar)). However, this assumption is not necessarily justified as, for example, the C172S variant of bacterial AhpC has a higher CP pKa and only 0.5% of the H2O2 reactivity of the WT enzyme (8Nelson K.J. Perkins A. Van Swearingen A.E.D. Hartman S. Brereton A.E. Parsonage D. Salsbury Jr., F.R. Karplus P.A. Poole L.B. Experimentally dissecting the origins of peroxiredoxin catalysis.Antioxid. Redox Signal. 2018; 28: 521-536Crossref PubMed Scopus (23) Google Scholar). Also, the reaction of the C173S/C83S mutant of Prdx1 with urate hydroperoxide is 10-times slower than for the WT (9Carvalho L.A.C. Truzzi D.R. Fallani T.S. Alves S.V. Toledo Jr., J.C. Augusto O. Netto L.E.S. Meotti F.C. Urate hydroperoxide oxidizes human peroxiredoxin 1 and peroxiredoxin 2.J. Biol. Chem. 2017; 292: 8705-8715Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Structural studies of both the C172S (10Nelson K.J. Parsonage D. Hall A. Karplus P.A. Poole L.B. Cysteine pK(a) values for the bacterial peroxiredoxin AhpC.Biochemistry. 2008; 47: 12860-12868Crossref PubMed Scopus (91) Google Scholar) and C172A (11Perkins A. Nelson K.J. Williams J.R. Parsonage D. Poole L.B. Karplus P.A. The sensitive balance between the fully folded and locally unfolded conformations of a model peroxiredoxin.Biochemistry. 2013; 52: 8708-8721Crossref PubMed Scopus (47) Google Scholar) mutants of AhpC showed that the mutations destabilized the folding of the C-terminal region of the protein and that because of their proximity (see Fig. 1B), this indirectly destabilized and perhaps even shifted the folding of the peroxidatic active site. If structural disruption around CR alters active site geometry, this could impact on peroxide reactivity during catalytic turnover. As demonstrated for Prdx2, turnover by GSH involves initial formation and reduction of a GS-Prdx disulfide (3Peskin A.V. Pace P.E. Behring J.B. Paton L.N. Soethoudt M. Bachschmid M.M. Winterbourn C.C. Glutathionylation of the active site cysteines of peroxiredoxin 2 and recycling by glutaredoxin.J. Biol. Chem. 2016; 291: 3053-3062Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and resolution of the CP-CR disulfide by Trx proceeds via an intermediate mixed disulfide inferred to be between Trx and CR. Such mixed-disulfide derivatives of CR would be expected to be more structurally disruptive than a Cys to Ser or Ala mutation. Moreover, mixed disulfide formation with CR would release reduced CP–SH, which could potentially react with H2O2 before resolution of the conjugate. If this were the case, the mechanism of catalytic turnover of Prdx2 would involve steps involving mixed disulfides. To investigate the physiological relevance of these intermediates and how they affect structure and CP reactivity, we generated CR mutants designed to mimic the disruptive effect of mixed disulfide formation. We have performed a combination of functional analysis and structural characterizations, and also report the first high-resolution crystal structures of WT Prdx2 as well as the CR-to-serine mutation (C172S) in the reduced state. Until now the WT structure has been inferred from the original Prdx2 crystallographic study performed on the hyperoxidized form (12Schroder E. Littlechild J.A. Lebedev A.A. Errington N. Vagin A.A. Isupov M.N. Crystal structure of decameric 2-Cys peroxiredoxin from human erythrocytes at 1.7 A resolution.Structure. 2000; 8: 605-615Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). The structure of the locally unfolded (LU) disulfide form has also been reported (13Bolduc J.A. Nelson K.J. Haynes A.C. Lee J. Reisz J.A. Graff A.H. Clodfelter J.E. Parsonage D. Poole L.B. Furdui C.M. Lowther W.T. Novel hyperoxidation resistance motifs in 2-Cys peroxiredoxins.J. Biol. Chem. 2018; 293: 11901-11912Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). For our study, we selected the commonly used C172S mutation, which is predicted to cause minimal disruption to the overall structure, and two mutants (C172D and C172W) that either through charge or bulk should be more disruptive and are therefore potential models for the effects of conjugation of CR to Trx or GSH during Prdx recycling. We find that the modification of CR-to-Ser yields nearly identical rate constants to WT for oxidation and hyperoxidation by H2O2, whereas CR-to-Asp and CR-to-Trp have highly decreased rates. We demonstrate that both latter mutants are largely unstructured at the C terminus and propose that this is how they influence CP reactivity. Our study highlights the important regulatory role of CR and the C-terminal region of Prdx2 in its redox activities. Stopped flow analysis of changes in Trp fluorescence was used to determine rate constants for the reaction of the reduced Prdx mutants with H2O2. As described previously (9Carvalho L.A.C. Truzzi D.R. Fallani T.S. Alves S.V. Toledo Jr., J.C. Augusto O. Netto L.E.S. Meotti F.C. Urate hydroperoxide oxidizes human peroxiredoxin 1 and peroxiredoxin 2.J. Biol. Chem. 2017; 292: 8705-8715Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 14Trujillo M. Clippe A. Manta B. Ferrer-Sueta G. Smeets A. Declercq J.P. Knoops B. Radi R. Pre-steady state kinetic characterization of human peroxiredoxin 5: Taking advantage of Trp84 fluorescence increase upon oxidation.Arch. Biochem. Biophys. 2007; 467: 95-106Crossref PubMed Scopus (137) Google Scholar, 15Parsonage D. Nelson K.J. Ferrer-Sueta G. Alley S. Karplus P.A. Furdui C.M. Poole L.B. Dissecting peroxiredoxin catalysis: Separating binding, peroxidation, and resolution for a bacterial AhpC.Biochemistry. 2015; 54: 1567-1575Crossref PubMed Scopus (48) Google Scholar, 16Dalla Rizza J. Randall L.M. Santos J. Ferrer-Sueta G. Denicola A. Differential parameters between cytosolic 2-Cys peroxiredoxins, PRDX1 and PRDX2.Protein Sci. 2019; 28: 191-201Crossref PubMed Scopus (29) Google Scholar), WT Prdx2 gave a rapid fluorescence loss attributable to oxidation of the CP thiol group, followed by slower recovery (Fig. 2A). This recovery is associated with disulfide formation, plus at higher H2O2 concentrations, hyperoxidation (16Dalla Rizza J. Randall L.M. Santos J. Ferrer-Sueta G. Denicola A. Differential parameters between cytosolic 2-Cys peroxiredoxins, PRDX1 and PRDX2.Protein Sci. 2019; 28: 191-201Crossref PubMed Scopus (29) Google Scholar, 17Peskin A.V. Meotti F.C. De Souza L.F. Anderson R.F. Winterbourn C.C. Intra-dimer cooperativity between the active site cysteines during the oxidation of peroxiredoxin 2 Free.Radic. Biol. Med. 2020; 158: 115-125Crossref PubMed Scopus (6) Google Scholar). The CR mutants cannot form disulfides, and the recovery phase is not expected unless there is hyperoxidation. The mutants all showed an initial drop in fluorescence (Fig. 2, B–D), but the returning fluorescence was detected only with C172S. With a 2-fold excess of H2O2 recovery was slower for C172S than for the WT (Fig. 2B), but as expected for hyperoxidation and described in detail in (17Peskin A.V. Meotti F.C. De Souza L.F. Anderson R.F. Winterbourn C.C. Intra-dimer cooperativity between the active site cysteines during the oxidation of peroxiredoxin 2 Free.Radic. Biol. Med. 2020; 158: 115-125Crossref PubMed Scopus (6) Google Scholar), the rate increased with increasing H2O2 concentration. In contrast, the C172D (Fig. 2C) and C172W (Fig. 2D) mutants did not present this returning fluorescence even at higher H2O2 concentrations but showed a slow, H2O2 concentration-independent, continuous drop of fluorescence similar to that observed with no H2O2 added, which is most likely attributable to photobleaching. Analysis of the fast phase over a range of H2O2 concentrations was performed for WT Prdx2 and for each mutant (as shown in inserts, Fig. 3). Second-order rate constants, determined from plots of the pseudo first-order rate constants versus H2O2 (Fig. 3), are shown in Table 1. The value for WT Prdx2 is similar to other reported values using this method (16Dalla Rizza J. Randall L.M. Santos J. Ferrer-Sueta G. Denicola A. Differential parameters between cytosolic 2-Cys peroxiredoxins, PRDX1 and PRDX2.Protein Sci. 2019; 28: 191-201Crossref PubMed Scopus (29) Google Scholar). The C172S mutant was equally reactive. However, substituting C172 with Trp or Asp decreased reactivity with H2O2 by at least two orders of magnitude.Table 1Rate constants determined for oxidation and hyperoxidation of WT and mutants of Prdx2Prdx2kox (M−1s−1)khyp (M−1s−1)WT(1.3 ± 0.3) × 1084200 (6000)C172S(1.1 ± 0.4) × 1083400 (6000)C172D(2.3 ± 1.2) ×10624 ± 3C172W(1.35 ± 0.05) × 10633Prdx2, peroxiredoxin 2.Values of kox were determined from the stopped flow data in Figure 3. Analyses were performed in duplicate, and means and ranges are shown. Values of khyp for WT and C172S were determined by stopped flow and are taken from (17Peskin A.V. Meotti F.C. De Souza L.F. Anderson R.F. Winterbourn C.C. Intra-dimer cooperativity between the active site cysteines during the oxidation of peroxiredoxin 2 Free.Radic. Biol. Med. 2020; 158: 115-125Crossref PubMed Scopus (6) Google Scholar). Those in parenthesis were determined by catalase competition (3Peskin A.V. Pace P.E. Behring J.B. Paton L.N. Soethoudt M. Bachschmid M.M. Winterbourn C.C. Glutathionylation of the active site cysteines of peroxiredoxin 2 and recycling by glutaredoxin.J. Biol. Chem. 2016; 291: 3053-3062Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 18Peskin A.V. Dickerhof N. Poynton R.A. Paton L.N. Pace P.E. Hampton M.B. Winterbourn C.C. Hyperoxidation of Peroxiredoxins 2 and 3: Rate constants for the reactions of the sulfenic acid of the peroxidative cysteine.J. Biol. Chem. 2013; 288: 14170-14177Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The value for C172D is from Figure 4B (slope +SE) and for C172W from the single analysis in Figure 4. Open table in a new tab Prdx2, peroxiredoxin 2. Values of kox were determined from the stopped flow data in Figure 3. Analyses were performed in duplicate, and means and ranges are shown. Values of khyp for WT and C172S were determined by stopped flow and are taken from (17Peskin A.V. Meotti F.C. De Souza L.F. Anderson R.F. Winterbourn C.C. Intra-dimer cooperativity between the active site cysteines during the oxidation of peroxiredoxin 2 Free.Radic. Biol. Med. 2020; 158: 115-125Crossref PubMed Scopus (6) Google Scholar). Those in parenthesis were determined by catalase competition (3Peskin A.V. Pace P.E. Behring J.B. Paton L.N. Soethoudt M. Bachschmid M.M. Winterbourn C.C. Glutathionylation of the active site cysteines of peroxiredoxin 2 and recycling by glutaredoxin.J. Biol. Chem. 2016; 291: 3053-3062Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 18Peskin A.V. Dickerhof N. Poynton R.A. Paton L.N. Pace P.E. Hampton M.B. Winterbourn C.C. Hyperoxidation of Peroxiredoxins 2 and 3: Rate constants for the reactions of the sulfenic acid of the peroxidative cysteine.J. Biol. Chem. 2013; 288: 14170-14177Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The value for C172D is from Figure 4B (slope +SE) and for C172W from the single analysis in Figure 4. We previously analyzed Prdx2 hyperoxidation by monitoring the second phase, slow increase in Trp fluorescence using stopped flow (as in Fig. 2, A and B) (17Peskin A.V. Meotti F.C. De Souza L.F. Anderson R.F. Winterbourn C.C. Intra-dimer cooperativity between the active site cysteines during the oxidation of peroxiredoxin 2 Free.Radic. Biol. Med. 2020; 158: 115-125Crossref PubMed Scopus (6) Google Scholar) and by product analysis using LC/MS (3Peskin A.V. Pace P.E. Behring J.B. Paton L.N. Soethoudt M. Bachschmid M.M. Winterbourn C.C. Glutathionylation of the active site cysteines of peroxiredoxin 2 and recycling by glutaredoxin.J. Biol. Chem. 2016; 291: 3053-3062Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 18Peskin A.V. Dickerhof N. Poynton R.A. Paton L.N. Pace P.E. Hampton M.B. Winterbourn C.C. Hyperoxidation of Peroxiredoxins 2 and 3: Rate constants for the reactions of the sulfenic acid of the peroxidative cysteine.J. Biol. Chem. 2013; 288: 14170-14177Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). WT Prdx2 gave an [H2O2]-independent increase in fluorescence because of disulfide formation plus an [H2O2]-dependent component because of hyperoxidation of the sulfenic acid (Fig. 2A). Analysis of these data, described in (17Peskin A.V. Meotti F.C. De Souza L.F. Anderson R.F. Winterbourn C.C. Intra-dimer cooperativity between the active site cysteines during the oxidation of peroxiredoxin 2 Free.Radic. Biol. Med. 2020; 158: 115-125Crossref PubMed Scopus (6) Google Scholar), gave a second-order rate constant (khyp) of 4200 M−1s−1 (Table 1). With the C172S mutant, only the [H2O2]-dependent recovery was seen, and analysis of this reaction (17Peskin A.V. Meotti F.C. De Souza L.F. Anderson R.F. Winterbourn C.C. Intra-dimer cooperativity between the active site cysteines during the oxidation of peroxiredoxin 2 Free.Radic. Biol. Med. 2020; 158: 115-125Crossref PubMed Scopus (6) Google Scholar) gave a similar khyp of 3400 M−1s−1. Using LC/MS to monitor sulfinic acid formation and catalase competition to determine a khyp, we previously obtained values of 6000 M−1s−1 for both WT Prdx2 and the C172S mutant (3Peskin A.V. Pace P.E. Behring J.B. Paton L.N. Soethoudt M. Bachschmid M.M. Winterbourn C.C. Glutathionylation of the active site cysteines of peroxiredoxin 2 and recycling by glutaredoxin.J. Biol. Chem. 2016; 291: 3053-3062Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 18Peskin A.V. Dickerhof N. Poynton R.A. Paton L.N. Pace P.E. Hampton M.B. Winterbourn C.C. Hyperoxidation of Peroxiredoxins 2 and 3: Rate constants for the reactions of the sulfenic acid of the peroxidative cysteine.J. Biol. Chem. 2013; 288: 14170-14177Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). These are somewhat higher but in general agreement with those obtained by stopped flow. For the C172D and C172W mutants, no [H2O2]-dependent recovery phase was seen over the same time scale (Fig. 2, C and D), indicating that mutation of C172 to Asp or Trp dramatically decreases the rate of Prdx2 hyperoxidation. To measure the rate directly, we carried out LC/MS analysis to follow accumulation of the sulfinic acid over time. As shown in Figure 4, this occurred over several minutes. Under these conditions, oxidation of the reduced Prdx to the sulfenic acid is relatively rapid, the rate determining step is the subsequent slower oxidation to the sulfinic acid, and the rate constant measured is khyp. As shown for the C172D mutant, the rate of oxidation increased with H2O2 concentration (Fig. 4A), and a plot of pseudo first-order rate constant versus H2O2 concentration (Fig. 4B) gave a second-order rate constant, khyp of 24 M−1s−1. The reaction of C172W with H2O2 followed a similar time course to that of the C172D mutant (Fig. 4C) and gave a similar second-order rate constant of 33 M−1s−1 (Table 1). It was not possible to use the same approach to obtain a rate constant for the C172S mutant, as even with 10 μM H2O2, LC/MS showed that all the protein was hyperoxidized within 20 s. This is consistent with the khyp values obtained from stopped flow and catalase competition. Thus, our results establish that the mutation of CR to Ser has little effect, but replacement with Trp or Asp decreases the hyperoxidation rate constant by more than 100-fold. To demonstrate that the reaction of the sulfenic acid with H2O2 is the rate determining step, we also followed the reaction with the C172D and C172W mutants by trapping the sulfenic acid with N-ethylmaleimide (NEM) (instead of reducing before analysis as in Fig. 4). With this method, MS analysis of the untreated proteins showed major peaks corresponding to the addition of 1, 2, and 3 NEM (Fig. 5, A and D). At 25 s after addition of H2O2 (B,D), these were replaced with peaks with an additional 16 Da. This is consistent with partial alkylation of the two Cys residues present in the mutants plus an additional non-Cys site, with the 16 Da (one oxygen) corresponding to conversion of one Cys residue to a sulfenic acid and derivatization by NEM. At 150 s (C,F), these peaks had declined and were replaced by peaks with one less NEM and an additional 32 Da, as expected for conversion of the sulfenic to the sulfinic acid. These results support a mechanism in which the sulfenic acid is formed and decays slowly on reaction with H2O2. Recombinant Prdx2 (Prdx2SH) and the C172S variant (Prdx2C172S) crystallized under the same conditions, and their structures were solved at 1.7 Å and 2.15 Å resolution, respectively (Table 2). As the Prdx2SH crystals were catalytically active, we also produced the hyperoxidized sulfinate form in crystallo using a H2O2 soak (see Experimental procedures; Fig. S2) and solved the structure (Prdx2SO2) at 2.3 Å resolution (Table 2). Extensive efforts to crystallize the C172D and C172W variants were not successful. The three solved structures all include a decamer (Chains A through J) in the asymmetric unit of the crystal, and all residues (Ala2-Asn198) were modeled for each chain. All subunits adopt the fully folded (FF) conformation, shown as a ribbon diagram for Prdx2SH in Figure 1B and have unambiguous electron density for the CP and CR thiols in Prdx2SH, the CP sulfinate in Prdx2SO2, and the Ser172 side chain in Prdx2C172S (Fig. 6). Also, all of the structures are highly similar to each other (rmsd <0.3 Å) and to that originally published (12Schroder E. Littlechild J.A. Lebedev A.A. Errington N. Vagin A.A. Isupov M.N. Crystal structure of decameric 2-Cys peroxiredoxin from human erythrocytes at 1.7 A resolution.Structure. 2000; 8: 605-615Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar) for the sulfinate form of the natural enzyme (Fig. 6E). The close alignment of all these structures confirms the long held assumption that for this enzyme, the CP sulfinate form mimics the functional FF active site (12Schroder E. Littlechild J.A. Lebedev A.A. Errington N. Vagin A.A. Isupov M.N. Crystal structure of decameric 2-Cys peroxiredoxin from human erythrocytes at 1.7 A resolution.Structure. 2000; 8: 605-615Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar).Table 2Data collection and refinement statisticsPrdx2SHPrdx2SO2aData merged from three crystals.Prdx2C172SData statisticsbHighest resolution shell in parentheses. Space groupC2C2C2 Wavelength (Å)1.00.9761.0 Unit cell: a, b, c (Å); β (°)229.8, 88.1, 127.1; 100.0231.0, 88.0, 125.6; 100.2233.2, 88.3, 125.2; 99.2 Resolution (Å)49.7–1.70 (1.74–1.70)49.6–2.29 (2.35–2.29)35.2–2.15 (2.21–2.15) Unique reflections271,919 (19,847)109,799 (8119)136,298 (10,028) Multiplicity6.7 (6.4)22.5 (21.6)5.0 (4.7) Average I/σ8.9 (0.6)10.6 (0.9)7.5 (0.7) Rmeas (%)11.6 (303)46.1 (533)cIn the lowest resolution bin, the Rmeas value for this dataset is just 4.7%; the high overall Rmeas value results from the many weak high resolution reflections in the data set that thanks to the ∼23-fold multiplicity have an improved signal-to-noise that is not reflected in their Rmeas values (38).25.2 (283) Completeness (%)99.3 (98.5)97.9 (98.8)99.8 (99.9) CC1/299.8 (19)99.5 (32)99.2 (20)Refinement statistics Amino acid residues197019701970 Solvent atoms168917381753 Non-H atoms17,58717,66917,662 RMS bonds (Å)0.0110.0020.002 RMS angles (˚)1.00.60.5 φ, ψ favored (%) dBased on Molprobity (19).979797 φ, ψ outliers (%) dBased on Molprobity (19).0.20.40.3 <Bprotein> (Å2)425050 <Bsolvent> (Å2)545455 Rwork (%)18.318.219.5 Rfree (%)22.123.323.7 PDB code7KIZ7KJ07KJ1Prdx2, peroxiredoxin 2.a Data merged from three crystals.b Highest resolution shell in parentheses.c In the lowest resolution bin, the Rmeas value for this dataset is just 4.7%; the high overall Rmeas value results from the many weak high resolution reflections in the data set that thanks to the ∼23-fold multiplicity have an improved signal-to-noise that is not reflected in their Rmeas values (38Karplus P.A. Diederichs K. Linking crystallographic model and data quality.Science. 2012; 336: 1030-1033Crossref PubMed Scopus (1303) Google Scholar).d Based on Molprobity (19Williams C.J. Headd J.J. Moriarty N.W. Prisant M.G. Videau L.L. Deis L.N. Verma V. Keedy D.A. Hintze B.J. Chen V.B. Jain S. Lewis S.M. Arendall 3rd, W.B. Snoeyink J. Adams P.D. et al.MolProbity: More and better reference data for improved all-atom structure validation.Protein Sci. 2018; 27: 293-315Crossref PubMed Scopus (1464) Google Scholar). Open table in a new tab Prdx2, peroxiredoxin 2. These structures provided a basis for assessing the impact of the C172S mutation on protein structure and dynamics. While a Ser side chain is nominally isosteric with a Cys side chain, the hydroxyl is both smaller and much more polar than the thiol, meaning it can fit in smaller spaces and requires a polar, hydrogen-bonding environment for stability. As seen in the Prdx2C172S structure, the Ser172 side chain adopts a different χ1 side chain rotamer so it does not sit in the fully hydrophobic pocket in which the Cys172 thiol resides (Figs. 6D and S3). Instead, it is inserted into a tightly packed position in which it can form one hydrogen bond with a buried water molecule and make additional weak polar interactions with nearby backbone amides involved in β-sheet hydrogen-bonding polar interactions. The ability of the Ser side chain to adopt a conformation that allows it to donate a hydrogen bond and have a weakly polar environment mitigates what would have been a major destabilizing factor if it was oriented into the pocket filled by the Cys172 side chain and had no hydrogen bonding partners at all. According to a MolProbity analysis (19Williams C.J. Headd J.J. Moriarty N.W. Prisant M.G. Videau L.L. Deis L.N. Verma V. Keedy D.A. Hintze B.J. Chen V.B. Jain S. Lewis S.M. Arendall 3rd, W.B. Snoeyink J. Adams P.D. et al.MolProbity: More and better reference data for improved all-atom structure validation.Protein Sci. 2018; 27: 293-315Crossref PubMed Scopus (1464) Google Scholar), the Ser172 hydroxyl does not clash with neighboring atoms (not shown). The loss of hydrophobic driving force related to the burial of Cys172-SG and the small cavity left at its position would be expected to be destabilizing (e.g., (20Eriksson A.E. Baase W.A. Zhang X.J. Heinz D.W. Blaber M. Baldwin E.P. Matthews B.W. Response of a protein structure to cavity-creating mutations and its relation to the hydrophobic effect.Science. 1992; 255: 178-183Crossref PubMed Scopus (891) Google Scholar, 21Xu J. Baase W.A. Baldwin E. Matthews B.W. The response of T4 lysozyme to large-to-small substitutions within the core and its relation to the hydrophobic effect.Protein Sci. 1998; 7: 158-177Crossref PubMed Scopus (213) Google Scholar)), but fulfilling the full hydrogen-bonding potential of a buried water would provide some stabilization. Because all of the chains of the Prdx2C172S structure remain in the FF form, we sought evidence of more subtle impacts on stability by looking at the mobility of the chains as seen in crystallographic B-factors. For" @default.
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• W3134409844 title "Modifying the resolving cysteine affects the structure and hydrogen peroxide reactivity of peroxiredoxin 2" @default.
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