Structural and functional consequences of methionine oxidation in thrombomodulin

Thrombomodulin (TM) is an endothelial cell surface glycoprotein that is responsible for switching the catalytic activity of thrombin away from fibrinogen cleavage (pro-coagulant) and towards protein C cleavage (anticoagulant). Although TM is a large protein, only the fourth and fifth epidermal growth factor-like (EGF-like) domains are required for anticoagulant function. These two domains must work together, and the linker between the two domains contains a single methionine residue, Met 388. Oxidation of Met 388 is deleterious for TM activity. Structural studies, both X-ray and NMR, of wild type and variants at position 388 show that Met 388 provides a key linkage between the two domains. Oxidation of the methionine has consequences for the structure of the fifth domain, which binds to thrombin. Oxidation also appears to disrupt the interdomain contacts resulting in structural and dynamic changes. The functional consequences of oxidation of Met 388 include decreased anticoagulant activity. Oxidative stress from several causes is reflected in lower serum levels of activated protein C and a higher thrombotic tendency, and this is thought to be linked to the oxidation of Met 388 in TM. Thus, TM structure and function are altered in a subtle but functionally critical way upon oxidation of Met 388.     

Increasing the storage and oxidation stabilities of N-acyl-d-amino acid amidohydrolase by site-directed mutagenesis of critical methionine residues

The recombinant N-acyl-d-amino acid amidohydrolase (N-d-AAase) of Variovorax paradoxus Iso1 was unstable during protein purification and storage at 4 °C. Since the methionine oxidation might be the artificial factor leading to the inactivation of N-d-AAase, eight potential oxidation sensitive methionine residues of the enzyme were individually substituted with leucine utilizing site-directed mutagenesis. Among them, five mutants, M39L, M56L, M221L, M254L, and M352L remained at least 70% of wild-type specific activity. The enzyme kinetic parameters of M221L revealed a 44% decrease in K_m, and finally reflected a 2.4-fold increase in k_cat/K_m. Moreover, its half-life at 4 °C increased up to 6-fold longer than that of the wild-type. Structural analysis of each methionine substitution was carried out based on the crystal structure of N-d-AAase from Alcaligenes faecalis DA1. Met²²¹ spatial closeness to the zinc-assistant catalytic center is highly potential as the primary site for oxidative inactivation. We conclude that the replacement of methionine M221 with leucine in N-d-AAase successfully enhances the oxidative resistance, half-life, and enzyme activity. This finding provides a promising basis for the engineering the stability and activity of N-d-AAase.     

Structural and functional changes associated with modification of the ubiquitin methionine

The effects of oxidation and cleavage of Met-1 of ubiquitin on conformation and biological activity were individually investigated. Proton NMR studies demonstrated that oxidation to the sulfone led to restricted structural perturbations at neutral pH, particularly in the vicinity of Ile-61. Below pH 3, in the presence of acetic acid, oxidation to the sulfone facilitated a conformational expansion demonstrable by retardation on gel electrophoresis and CD changes below 210 nm. The predominant phase of the low-pH transition did not involve significant changes in alpha-helix content, indicating the capacity of ubiquitin for limited structural transitions. Cleavage of Met-1 by CNBr, on the other hand, was associated with a global unfolding transition below pH 4 that involved a major loss of alpha-helix. Differences in the behavior of the native and des-Met proteins at low pH indicate that Met-1 contributes a minimum of 3.4 kcal/mol to the stability of the native conformation. Two Met-1 sulfoxide isomers, of markedly different conformational stability, were formed by treatment of ubiquitin with H2O2. One isomer was similar in stability to the sulfone, while the other was intermediate in stability between the sulfone and des-Met proteins, the differences potentially interpretable in terms of the geometry of the Met-1-Lys-63 hydrogen bond. The overall activities of the oxidized and des-Met derivatives in ATP-dependent proteolysis differed subtly from that of native ubiquitin. The unresolved sulfoxides exhibited an approximately 50% increase in activity, while the sulfone and des-Met proteins exhibited a 50% decrease in activity at low concentrations and normal activity at higher concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calmodulin oxidation and methionine to glutamine substitutions reveal methionine residues critical for functional interaction with ryanodine receptor-1

Calmodulin (CaM) binds to the skeletal muscle ryanodine receptor Ca(2+) release channel (RyR1) with high affinity, and it may act as a Ca(2+)-sensing subunit of the channel. Apo-CaM increases RyR1 channel activity, but Ca(2+)-CaM is inhibitory. Here we examine the functional effects of CaM oxidation on RyR1 regulation by both apo-CaM and Ca(2+)-CaM, as assessed via determinations of [(3)H]ryanodine and [(35)S]CaM binding to skeletal muscle sarcoplasmic reticulum vesicles. Oxidation of all nine CaM Met residues abolished functional interactions of CaM with RyR1. Incomplete CaM oxidation, affecting 5-8 Met residues, increased the CaM concentration required to modulate RyR1, having a greater effect on the apo-CaM species. Mutating individual CaM Met residues to Gln demonstrated that Met-109 was required for apo-CaM activation of RyR1 but not for Ca(2+)-CaM inhibition of the channel. Furthermore, substitution of Gln for Met-124 increased the apo- and Ca(2+)-CaM concentrations required to regulate RyR1. These results thus identify Met residues critical for the productive association of CaM with RyR1 channels and suggest that oxidation of CaM may contribute to altered regulation of sarcoplasmic reticulum Ca(2+) release during oxidative stress.     

Myeloperoxidase-mediated oxidation of methionine

The myeloperoxidase-mediated oxidation of methionine was studied using a purified canine myeloperoxidase preparation. The system required the simultaneous presence of myeloperoxidase, H₂O₂, and a halide anion. When 0.1 mM H₂O₂ was used, the system has a pH optimum of approximately pH 5–5.5. Bromide and iodide were much more effective than chloride in the myeloperoxidase-mediated oxidation of methionine. Horseradish peroxidase was unable to oxidize methionine whether chloride or iodide was used. In contrast, lactoperoxidase oxidized methionine in the presence of iodide but not chloride. Based on studies of (1) the effect of various inhibitors and singlet oxygen quenchers, as well as (2) the effect of D₂O on the oxidation of methionine, by the myeloperoxidase system, OCl^?, or methylene blue, it was shown that the oxidation of methionine by the myeloperoxidase system was not mediated by OCl^? or ¹O₂. The mechanism of the myeloperoxidase-mediated oxidation of methionine remains unclear. However, it may be one mechanism by which the myeloperoxidase system damage microorganisms.     

Structural and functional aspects of the myosin essential light chain in cardiac muscle contraction

The myosin essential light chain (ELC) is a structural component of the actomyosin cross-bridge, but its function is poorly understood, especially the role of the cardiac specific N-terminal extension in modulating actomyosin interaction. Here, we generated transgenic (Tg) mice expressing the A57G (alanine to glycine) mutation in the cardiac ELC known to cause familial hypertrophic cardiomyopathy (FHC). The function of the ELC N-terminal extension was investigated with the Tg-Δ43 mouse model, whose myocardium expresses a truncated ELC. Low-angle X-ray diffraction studies on papillary muscle fibers in rigor revealed a decreased interfilament spacing (≈ 1.5 nm) and no alterations in cross-bridge mass distribution in Tg-A57G mice compared to Tg-WT, expressing the full-length nonmutated ELC. The truncation mutation showed a 1.3-fold increase in I(1,1)/I(1,0), indicating a shift of cross-bridge mass from the thick filament backbone toward the thin filaments. Mechanical studies demonstrated increased stiffness in Tg-A57G muscle fibers compared to Tg-WT or Tg-Δ43. The equilibrium constant for the cross-bridge force generation step was smallest in Tg-Δ43. These results support an important role for the N-terminal ELC extension in prepositioning the cross-bridge for optimal force production. Subtle changes in the ELC sequence were sufficient to alter cross-bridge properties and lead to pathological phenotypes.     

Periodate oxidation of methionine in proteins

Treatment of free methionine with an equimolar amount of periodate gave nearly quantitative formation of the sulfoxide; treatment of free methionine sulfoxide with equimolar periodate gave nearly equal amounts of the original sulfoxide and the sulfone. Treatments of 0.5-1.0% solutions of the following proteins with relatively low concentration of periodate (5 mm) gave the following approximate values for conversion of methionine sulfoxide from total methionine: bovine pancreatic ribonuclease A (2 of 4), chicken ovalbumin (14 or 17), chicken ovotransferrin (5 of 11), human serum transferrin (2 of 8), bovine α-chymotrypsin (1 of 2). It is recommended that when proteins are treated with sodium periodate (and probably with oxidizing agents in general), especially when changes in properties are observed, determinations of methionine sulfoxide should be done.     

Structural and functional changes of myosin during development: comparison with adult fast, slow and cardiac myosin.

ATPase (Ca²⁺ and K⁺ activated) activity of myosin prepared from muscles of 3–4 week rabbit embryos (EM) is slighly lower than that of adult fast muscle myosin (FM), but in contrast to the less active adult slow muscle myosin (SM) is stable on exposure to pH 9.2. Studies of the time course, by means of Na dodecyl-SO₄ polyacrylamide gel electrophoresis, of changes in the pattern of polypeptides released by tryptic digestion show that in this regard EM is closest to SM. The light chain complement of EM appears identical with that of FM rather than of SM or cardiac myosin (CM) by the criteria of coelectrophoresis and removal by 5,5′-dithio-2-dinitrobenzoate treatment of LC₂ except that the relative amount of LC₃ is less in EM than in FM. The staining pattern of light meromyosin (EMM) paracrystals prepared from EM is distinct from either the FM, SM or CM LMM staining pattern. These studies suggest that different genes are involved in the coding for embryonic and adult heavy chains.     

Electron microscopic mappings of myosin head with site-directed antibodies

Site-directed antibodies were raised against three synthetic peptides whose sequences correspond to a region around the reactive lysine residue and two protease-sensitive regions of subfragment 1 (S1) of skeletal muscle myosin (one at the junction of the 23,000 Mr and 50,000 Mr segments, the J1 junction; and the other at the junction of the 50,000 Mr and 20,000 Mr segments of the heavy chain, the J2 junction). The antisera cross-reacted with intact myosin with titres of 5 x 10(4) (anti-J1 antiserum) and 10(4) (anti-J2 and anti-reactive lysine residue antisera). Site-specific antibodies purified by S1-Sepharose readily bound to myosin. Electron microscopic examinations of antibody-myosin complexes revealed that the J1 and J2 junctions are located 15 nm and 16 nm from the head-rod junction, respectively, while the reactive lysine residue region is 13 nm from the junction.     

Structural and functional differences between carbonic anhydrase isoenzymes I and II as studied by site-directed mutagenesis

Site-specific mutagenesis has been used to replace amino acid residues in the active site of human carbonic anhydrase II with residues characterizing carbonic anhydrases I. Previous studies of [Thr200----His]isoenzyme II [Behravan, G., Jonsson, B.-H. & Lindskog, S. (1990) Eur. J. Biochem. 190, 351-357] showed that His200 is important for the specific catalytic properties of isoenzymes I. In this paper some properties of two single mutants, Asn62----Val and Asn67----His, as well as a double mutant, Asn67----His/Thr200----His, are described. The results show that neither Val62 nor His67 give rise to isoenzyme-I-like properties, while the double mutant behaves like the single mutant with His200. At pH 8.9, the variant with Val62 has a higher value of kcat/Km for CO2 hydration than unmodified isoenzyme II, whereas the variant with His67 has an enhanced kcat value. The replacement of Asn62 with Val results in a 20% increase of the 4-nitrophenyl acetate hydrolase activity. For the double mutant, the esterase activity is quite close to that calculated on the assumption that the effects of the two single mutations on the free energy of activation are additive.     

The quantitative oxidation of methionine to methionine sulfoxide by peroxynitrite

Both peroxynitrous acid and peroxynitrite react with methionine, k(acid) = (1.7 +/- 0.1) x 10(3) M(-1) s(-1) and k(anion) = 8.6 +/- 0.2 M(-1) s(-1), respectively, and with N-acetylmethionine k(acid) = (2.8 +/- 0.1) x 10(3) M(-1) s(-1) and k(anion) = 10.0 +/- 0.1 M(-1) s(-1), respectively, to form sulfoxides. In contrast to the results of Pryor et al. (1994, Proc. Natl. Acad. Sci. USA 91, 11173-11177), a linear correlation between k(obs) and [met] was obtained. Surprisingly, for every two sulfoxides and nitrites formed, one peroxynitrite is converted to nitrate. Thus, methionine also catalyzes the isomerization of peroxynitrite to nitrate. Neither the pH nor the concentration of methionine affected the distribution of the yields of nitrite, nitrate, and methionine sulfoxide, which were the only products detected. No products other than nitrite, nitrate, and methioninesulfoxide could be detected. The reactions of methionine and N-acetylmethionine with peroxynitrous acid and peroxynitrite are simple bimolecular reactions that do not involve an activated form of peroxynitrous acid or of peroxynitrite. Nitrite, produced together with methionine sulfoxide, or present as a contamination in the peroxynitrite preparation, is not innocuous, but oxidizes methionine by one electron, which leads to the formation of methional and ethylene.     

Structural and functional characterization of human microsomal prostaglandin E synthase-1 by computational modeling and site-directed mutagenesis

Microsomal prostaglandin (PG) E synthase-1 (mPGES-1) has recently been recognized as a novel, promising drug target for inflammation-related diseases. Functional and pathological studies on this enzyme further stimulate to understand its structure and the structure-function relationships. Using an approach of the combined structure prediction, molecular docking, site-directed mutagenesis, and enzymatic activity assay, we have developed the first three-dimensional (3D) model of the substrate-binding domain (SBD) of mPGES-1 and its binding with substrates prostaglandin H2 (PGH2) and glutathione (GSH). In light of the 3D model, key amino acid residues have been identified for the substrate binding and the obtained experimental activity data have confirmed the computationally determined substrate-enzyme binding mode. Both the computational and experimental results show that Y130 plays a vital role in the binding with PGH2 and, probably, in the catalytic reaction process. R110 and T114 interact intensively with the carboxyl tail of PGH2, whereas Q36 and Q134 only enhance the PGH2-binding affinity. The modeled binding structure indicates that substrate PGH2 interacts with GSH through hydrogen binding between the peroxy group of PGH2 and the -SH group of GSH. The -SH group of GSH is expected to attack the peroxy group of PGH2, initializing the catalytic reaction transforming PGH2 to prostaglandin E2 (PGE2). The overall agreement between the calculated and experimental results demonstrates that the predicted 3D model could be valuable in future rational design of potent inhibitors of mPGES-1 as the next-generation inflammation-related therapeutic.     

Redox proteomics of protein-bound methionine oxidation

We here present a new method to measure the degree of protein-bound methionine sulfoxide formation at a proteome-wide scale. In human Jurkat cells that were stressed with hydrogen peroxide, over 2000 oxidation-sensitive methionines in more than 1600 different proteins were mapped and their extent of oxidation was quantified. Meta-analysis of the sequences surrounding the oxidized methionine residues revealed a high preference for neighboring polar residues. Using synthetic methionine sulfoxide containing peptides designed according to the observed sequence preferences in the oxidized Jurkat proteome, we discovered that the substrate specificity of the cellular methionine sulfoxide reductases is a major determinant for the steady-state of methionine oxidation. This was supported by a structural modeling of the MsrA catalytic center. Finally, we applied our method onto a serum proteome from a mouse sepsis model and identified 35 in vivo methionine oxidation events in 27 different proteins.     

Structural basis for the functional differences between type I and type II human methionine aminopeptidases

Determination of the crystal structure of human MetAP1 makes it possible, for the first time, to compare the structures of a Type I and a Type II methionine aminopeptidase (MetAP) from the same organism. Comparison of the Type I enzyme with the previously reported complex of ovalicin with Type II MetAP shows that the active site of the former is reduced in size and would incur steric clashes with the bound inhibitor. This explains why ovalicin and related anti-angiogenesis inhibitors target Type II human MetAP but not Type I. The differences in both size and shape of the active sites between MetAP1 and MetAP2 also help to explain their different substrate specificity. In the presence of excess Co(2+), a third cobalt ion binds in the active site region, explaining why metal ions in excess can be inhibitory. Also, the N-terminal region of the protein contains three distinct Pro-x-x-Pro motifs, supporting the prior suggestion that this region of the protein may participate in binding to the ribosome.     

Mediating molecular recognition by methionine oxidation: conformational switching by oxidation of methionine in the carboxyl-terminal domain of calmodulin

The C-terminus of calmodulin (CaM) functions as a sensor of oxidative stress, with oxidation of methionine 144 and 145 inducing a nonproductive association of the oxidized CaM with the plasma membrane Ca(2+)-ATPase (PMCA) and other target proteins to downregulate cellular metabolism. To better understand the structural underpinnings and mechanism of this switch, we have engineered a CaM mutant (CaM-L7) that permits the site-specific oxidation of M144 and M145, and we have used NMR spectroscopy to identify structural changes in CaM and CaM-L7 and changes in the interactions between CaM-L7 and the CaM-binding sequence of the PMCA (C28W) due to methionine oxidation. In CaM and CaM-L7, methionine oxidation results in nominal secondary structural changes, but chemical shift changes and line broadening in NMR spectra indicate significant tertiary structural changes. For CaM-L7 bound to C28W, main chain and side chain chemical shift perturbations indicate that oxidation of M144 and M145 leads to large tertiary structural changes in the C-terminal hydrophobic pocket involving residues that comprise the interface with C28W. Smaller changes in the N-terminal domain also involving residues that interact with C28W are observed, as are changes in the central linker region. At the C-terminal helix, (1)H(alpha), (13)C(alpha), and (13)CO chemical shift changes indicate decreased helical character, with a complete loss of helicity for M144 and M145. Using (13)C-filtered, (13)C-edited NMR experiments, dramatic changes in intermolecular contacts between residues in the C-terminal domain of CaM-L7 and C28W accompany oxidation of M144 and M145, with an essentially complete loss of contacts between C28W and M144 and M145. We propose that the inability of CaM to fully activate the PMCA after methionine oxidation originates in a reduced helical propensity for M144 and M145, and results primarily from a global rearrangement of the tertiary structure of the C-terminal globular domain that substantially alters the interaction of this domain with the PMCA.     

Selective oxidation of methionine residues in proteins.

Methionine residues in peptides and proteins were oxidized to methionine sulfoxides by mild oxidizing reagents such as chloramine-T and N-chlorosuccinimide at neutral and slightly alkaline pH. With chloramine-T cysteine was also oxidized to cystine but no other amino acid was modified; with N-chlorosuccinimide tryptophans were oxidized as well. In peptides and denaturated proteins all methionine residues were quantitatively oxidized, while in native proteins only exposed methionine residues could be modified. Extent of oxidation of methionine residues was determined by quantitative modification of the unoxidized methionine residues with cyanogen bromide (while methionine sulfoxide residues remained intact), followed by acid hydrolysis and amino acid analysis. Methionine was determined as homoserine and methionine sulfoxide was reduced back to methionine. Sites of oxidation were identified in a similar way by cleaving the unoxidized methionyl peptide bonds with cyanogen bromide, followed by quantitative end-group analysis of the new amino-terminal amino acids (by an automatic sequencer).     

Interference with myosin subfragment-1 binding by site-directed mutagenesis of actin

Three N-terminal double mutants of beta-actin expressed in the yeast Saccharomyces cerevisiae have been characterized with respect to DNase-I interaction, N-terminal post-translational modification, polymerizability and myosin subfragment-1 binding. The results strongly support earlier suggestions that the acidic residues at the N-terminus of actin are part of the myosin-binding site, while they seem to be of no importance for the other aspects of actin biochemistry tested. The suitability of this expression system for production of recombinant actin in general is discussed.     

The physiological role of reversible methionine oxidation

Sulfur-containing amino acids such as cysteine and methionine are particularly vulnerable to oxidation. Oxidation of cysteine and methionine in their free amino acid form renders them unavailable for metabolic processes while their oxidation in the protein-bound state is a common post-translational modification in all organisms and usually alters the function of the protein. In the majority of cases, oxidation causes inactivation of proteins. Yet, an increasing number of examples have been described where reversible cysteine oxidation is part of a sophisticated mechanism to control protein function based on the redox state of the protein. While for methionine the dogma is still that its oxidation inhibits protein function, reversible methionine oxidation is now being recognized as a powerful means of triggering protein activity. This mode of regulation involves oxidation of methionine to methionine sulfoxide leading to activated protein function, and inactivation is accomplished by reduction of methionine sulfoxide back to methionine catalyzed by methionine sulfoxide reductases. Given the similarity to thiol-based redox-regulation of protein function, methionine oxidation is now established as a novel mode of redox-regulation of protein function. This article is part of a Special Issue entitled: Thiol-Based Redox Processes.