Role of disulfide bonds in conformational stability and folding of 5′-deoxy-5′-methylthioadenosine phosphorylase II from the hyperthermophilic archaeon Sulfolobus solfataricus

Sulfolobus solfataricus 5′-deoxy-5′-melthylthioadenosine phosphorylase II (SsMTAPII), is a hyperthermophilic hexameric protein with two intrasubunit disulfide bonds (C138–C205 and C200–C262) and a CXC motif (C259–C261). To get information on the role played by these covalent links in stability and folding, the conformational stability of SsMTAPII and C262S and C259S/C261S mutants was studied by thermal and guanidinium chloride (GdmCl)-induced unfolding and analyzed by fluorescence spectroscopy, circular dichroism, and SDS-PAGE. No thermal unfolding transition of SsMTAPII can be obtained under nonreducing conditions, while in the presence of the reducing agent Tris-(2-carboxyethyl) phosphine (TCEP), a Tm of 100 °C can be measured demonstrating the involvement of disulfide bridges in enzyme thermostability. Different from the wild-type, C262S and C259S/C261S show complete thermal denaturation curves with sigmoidal transitions centered at 102 °C and 99 °C respectively. Under reducing conditions these values decrease by 4 °C and 8 °C respectively, highlighting the important role exerted by the CXC disulfide on enzyme thermostability. The contribution of disulfide bonds to the conformational stability of SsMTAPII was further assessed by GdmCl-induced unfolding experiments carried out under reducing and nonreducing conditions. Thermal unfolding was found to be reversible if the protein was heated in the presence of TCEP up to 90 °C but irreversible above this temperature because of aggregation. In analogy, only chemical unfolding carried out in the presence of reducing agents resulted in a reversible process suggesting that disulfide bonds play a role in enzyme denaturation. Thermal and chemical unfolding of SsMTAPII occur with dissociation of the native hexameric state into denatured monomers, as indicated by SDS-PAGE.     

A novel hyperthermostable 5'-deoxy-5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus

We report herein the first molecular characterization of 5'-deoxy-5'-methylthio-adenosine phosphorylase II from Sulfolobus solfataricus (SsMTAPII). The isolated gene of SsMTAPII was overexpressed in Escherichia coli BL21. Purified recombinant SsMTAPII is a homohexamer of 180 kDa with an extremely low Km (0.7 microm) for 5'-deoxy-5'-methylthioadenosine. The enzyme is highly thermophilic with an optimum temperature of 120 degrees C and extremely thermostable with an apparent Tm of 112 degrees C that increases in the presence of substrates. The enzyme is characterized by high kinetic stability and remarkable SDS resistance and is also resistant to guanidinium chloride-induced unfolding with a transition midpoint of 3.3 m after 22-h incubation. Limited proteolysis experiments indicated that the only one proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is necessary for the integrity of the active site. Moreover, the binding of 5'-deoxy-5'-methylthioadenosine induces a conformational transition that protected the enzyme against protease inactivation. By site-directed mutagenesis we demonstrated that Cys259, Cys261 and Cys262 play an important role in the enzyme stability since the mutants C259S/C261S and C262S show thermophilicity and thermostability features significantly lower than those of the wild-type enzyme. In order to get insight into the physiological role of SsMTAPII a comparative kinetic analysis with the homologous 5'-deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) was carried out. Finally, the alignment of the protein sequence of SsMTAPII with those of SsMTAP and human 5'-deoxy-5'-methylthioadenosine phosphorylase (hMTAP) shows several key residue changes that may account why SsMTAPII, unlike hMTAP, is able to recognize adenosine as substrate.     

Thermal unfolding of nucleoside hydrolases from the hyperthermophilic archaeon Sulfolobus solfataricus: role of disulfide bonds

Nucleoside hydrolases are metalloproteins that hydrolyze the N-glycosidic bond of β-ribonucleosides, forming the free purine/pyrimidine base and ribose. We report the stability of the two hyperthermophilic enzymes Sulfolobus solfataricus pyrimidine-specific nucleoside hydrolase (SsCU-NH) and Sulfolobus solfataricus purine-specific inosineadenosine- guanosine nucleoside hydrolase (SsIAG-NH) against the denaturing action of temperature and guanidine hydrochloride by means of circular dichroism and fluorescence spectroscopy. The guanidine hydrochloride-induced unfolding is reversible for both enzymes as demonstrated by the analysis of the refolding process by activity assays and fluorescence measurements. The evidence that the denaturation of SsIAG-NH carried out in the presence of reducing agents proved to be reversible indicates that the presence of disulfide bonds interferes with the refolding process of this enzyme. Both enzymes are highly thermostable and no thermal unfolding transition can be obtained up to 108°C. SsIAG-NH is thermally denatured under reducing conditions (T(m)=93°C) demonstrating the contribution of disulfide bridges to enzyme thermostability.     

The crystal structure of 5'-deoxy-5'-methylthioadenosine phosphorylase II from Sulfolobus solfataricus, a thermophilic enzyme stabilized by intramolecular disulfide bonds

The crystal structure of Sulfolobus solfataricus 5'-deoxy-5'-methylthioadenosine phosphorylase II (SsMTAPII) in complex with 5'-deoxy-5'-methylthioadenosine (MTA) and sulfate was determined to 1.45A resolution. The hexameric structure of SsMTAPII is a dimer-of-trimers with one active site per monomer. The oligomeric assembly of the trimer and the monomer topology of SsMTAPII are almost identical with trimeric human 5'-deoxy-5'-methylthioadenosine phosphorylase (hMTAP). SsMTAPII is the first reported hexameric member in the trimeric class of purine nucleoside phosphorylase (PNP) from Archaea. Unlike hMTAP, which is highly specific for MTA, SsMTAPII also accepts adenosine as a substrate. The residues at the active sites of SsMTAPII and hMTAP are almost identical. The broad substrate specificity of SsMTAPII may be due to the flexibility of the C-terminal loop. SsMTAPII is extremely thermoactive and thermostable. The three-dimensional structure of SsMTAPII suggests that the unique dimer-of-trimers quaternary structure, a CXC motif at the C terminus, and two pairs of intrasubunit disulfide bridges may play an important role in its thermal stability.     

Biochemical analysis and kinetic modeling of the thermal inactivation of MBP-fused heparinase I: implications for a comprehensive thermostabilization strategy

Enzymatic degradation of heparin by heparin lyases has not only largely facilitated heparin structural analysis and contamination detection, but also showed great potential to be a green and cost-effective way to produce low molecular weight heparin (LMWH). However, the commercial use of heparinase I (HepI), one of the most studied heparin lyases, has been largely hampered by its low productivity and extremely poor thermostability. Here we report the thermal inactivation mechanism and strategic thermal stabilization of maltose-binding protein (MBP)-HepI, a fusion HepI produced in E. coli with high yield, solubility and activity. Biochemical studies demonstrated that the thermal inactivation of MBP-HepI involves an unfolding step that is temperature-dependently reversible, followed by an irreversible dimerization step induced by intermolecular disulfide bonds. A good consistency between the kinetic modeling and experimental data of the inactivation was obtained within a wide range of temperature and enzyme concentration, confirming the adequacy of the proposed inactivation model. Based on the inactivation mechanism, a comprehensive strategy was proposed for the thermal stabilization of MBP-HepI, in which Ca(2+) and Tween 80 were used to inhibit unfolding while site mutation at Cys297 and DTT were employed to suppress dimerization. The engineered enzyme exhibits remarkably improved storage and operational thermostability, for example, 16-fold increase in half-life at its optimum temperature of 30 °C and 8-fold increase in remaining activity of 95% after 1-week storage at 4 °C, and therefore shows great potential as a commercial biocatalyst for heparin degradation in the pharmaceutical industry.     

Interchain disulfide bonds promote protein cross-linking during protein folding

We provide evidence that in vitro protein cross-linking can be accomplished in three concerted steps: (i) a change in protein conformation; (ii) formation of interchain disulfide bonds; and (iii) formation of interchain isopeptide cross-links. Oxidative refolding and thermal unfolding of ribonuclease A, lysozyme, and protein disulfide isomerase led to the formation of cross-linked dimers/oligomers as revealed by SDS-polyacrylamide gel electrophoresis. Chemical modification of free amino groups in these proteins or unfolding at pH < 7.0 resulted in a loss of interchain isopeptide cross-linking without affecting interchain disulfide bond cross-linking. Furthermore, preformed interchain disulfide bonds were pivotal for promoting subsequent interchain isopeptide cross-links; no dimers/oligomers were detected when the refolding and unfolding solution contained the reducing agent dithiothreitol. Similarly, the Cys326Ser point mutation in protein disulfide isomerase abrogated its ability to cross-link into homodimers. Heterogeneous proteins become cross-linked following the formation of heteromolecular interchain disulfide bonds during thermal unfolding of a mixture of of ribonuclease A and lysozyme. The absence of glutathione and glutathione disulfide during the unfolding process attenuated both the interchain disulfide bond cross-links and interchain isopeptide cross-links. No dimers/oligomers were detected when the thermal unfolding temperature was lower than the midpoint of thermal denaturation temperature.     

Thermostable hexameric form of Eis (Rv2416c) protein of M. tuberculosis plays an important role for enhanced intracellular survival within macrophages

Eis protein is reported to enhance the intracellular survival of Mycobacterium tuberculosis in human macrophages. Eis protein is not only known to skew away the immunity by disturbing the protective T(H)1 response, but aminoglycoside acetyltransferase activity of Eis is reported to regulate autophagy, inflammation and cell death. Here we have gained insight into the structure-function properties of Eis. Eis protein is a hexameric αβ protein. Although urea and guanidinium hydrochloride (GdmCl) was found to induce one-step unfolding of Eis but size exclusion chromatography showed that GdmCl treated Eis maintained its hexameric form. SDS-PAGE assay confirmed that hexameric form of Eis is partially stable to SDS and converts into trimers and monomers. Out of these three forms, aminoglycoside acetyltransferase activity is found to be associated only with hexamers. The Tm of Eis was found to be ～75°C. Aminoglycoside acetyltransferase Eis demonstrated remarkable heat stability retaining >80% of their activity at 70°C which falls down to ～50% at 75°C and is completely inactive at 80°C. Further, intracellular survival assay with heated samples of M. smegmatis harboring eis gene of M. tuberculosis H37Rv demonstrated a possible role for the thermostability associated with Eis protein in the enhanced intracellular survival within macrophages. In sum, these data reveal that only hexameric form of Eis has a thermostable aminoglycoside acetyltransferase activity. This is the first report showing the thermostability associated with aminoglycoside acetyltransferase activity of Eis protein being one of the essential features for the execution of its biological role.     

The location of an engineered inter-subunit disulfide bond in factor for inversion stimulation (FIS) affects the denaturation pathway and cooperativity

Two crossed-linked variants of the homodimeric DNA binding protein factor for inversion stimulation (FIS) were created via engineering of single intermolecular disulfide bonds. The conservative S30C and the nonconservative V58C FIS independent mutations resulted in FIS crossed-linked at the A helix (C30-C30) and at the middle of the B helix (C58-C58). This study sought to investigate how the location of an intermolecular disulfide bond may determine the effect on stability and its propagation through the structure to preserve or alter the denaturation cooperativity of FIS. The oxidized and reduced S30C and V58C FIS exhibited a far-UV CD spectrum and DNA binding affinities that were similar to WT FIS, indicating no significant changes in secondary and tertiary structure. However, the reduced and oxidized forms of the mutants revealed significant differences in the stability and equilibrium denaturation mechanism between the two mutants. In the reduced state, S30C FIS had very little effect on FIS stability, whereas V58C FIS was 2-3 kcal/mol less stable than WT FIS. Interestingly, while both disulfide bonds significantly increased the resistance to urea- and guanidine hydrochloride (GuHCl)-induced denaturation, oxidized V58C FIS exhibited a three-state GuHCl-induced transition. In contrast, oxidized S30C FIS displayed a highly cooperative WT-like transition with both denaturants. The three-state denaturation mechanism of oxidized V58C FIS induced by the GuHCl salt was reproduced by urea denaturation at pH 4, suggesting that disruption of a C-terminus salt-bridge network is responsible for the loss of denaturation cooperativity of V58C FIS in GuHCl or urea, pH 4. A second mutation on V58C FIS created to place a single tryptophan probe (Y95W) at the C-terminus further implies that the denaturation intermediate observed in disulfide crossed-linked V58C FIS results from a decoupling of the stabilities of the C-terminus and the rest of the protein. These results show that, unlike the C30-C30 intermolecular disulfide bond, the C58-C58 disulfide bond did not evenly stabilize the FIS structure, thereby highlighting the importance of the location of an engineered disulfide bond on the propagation of stability and the denaturation cooperativity of a protein.     

Intramolecular Disulfide Bonds for Biogenesis of CALHM1 Ion Channel Are Dispensable for Voltage-Dependent Activation

Calcium homeostasis modulator 1 (CALHM1) is a membrane protein with four transmembrane helices that form an octameric ion channel with voltage-dependent activation. There are four conserved cysteine (Cys) residues in the extracellular domain that form two intramolecular disulfide bonds. We investigated the roles of C42-C127 and C44-C161 in human CALHM1 channel biogenesis and the ionic current (I _CALHM1). Replacing Cys with Ser or Ala abolished the membrane trafficking as well as I _CALHM1. Immunoblotting analysis revealed dithiothreitol-sensitive multimeric CALHM1, which was markedly reduced in C44S and C161S, but preserved in C42S and C127S. The mixed expression of C42S and wild-type did not show a dominant-negative effect. While the heteromeric assembly of CALHM1 and CALHM3 formed active ion channels, the co-expression of C42S and CALHM3 did not produce functional channels. Despite the critical structural role of the extracellular cysteine residues, a treatment with the membrane-impermeable reducing agent tris(2-carboxyethyl) phosphine (TCEP, 2 mM) did not affect I _CALHM1 for up to 30 min. Interestingly, incubation with TCEP (2 mM) for 2-6 h reduced both I _CALHM1 and the surface expression of CALHM1 in a time-dependent manner. We propose that the intramolecular disulfide bonds are essential for folding, oligomerization, trafficking and maintenance of CALHM1 in the plasma membrane, but dispensable for the voltage-dependent activation once expressed on the plasma membrane.     

Heterologous expression of 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus: characterization of the recombinant protein and involvement of disulfide bonds in thermophilicity and thermostability.

The gene for the extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus was expressed at a high level in Escherichia coli thus providing a basis for detailed structural and functional studies of the enzyme. The recombinant enzyme was purified to homogeneity by means of a heat treatment (10 min at 100 degrees C) and by a single affinity chromatography step. The appropriate expression vector and host strain were selected and the culture conditions were determined that would ensure a consistent yield of 6 mg of pure enzyme per liter of culture. The heterologously expressed enzyme is identical to the original S. solfataricus 5'-methylthioadenosine phosphorylase regarding molecular weight, substrate specificity, and the presence of intersubunit disulfide bonds. On the other hand, the recombinant 5'-methylthioadenosine phosphorylase is less thermophilic and thermostable than the S. solfataricus enzyme, since an incorrect positioning of disulfide bonds within the molecule generates structures less stable to thermal unfolding.     

Impact of disulfide bonds on the folding and refolding capability of a novel thermostable GH45 cellulase

A new cellulase (TaCel45) of glycoside hydrolase family 45 was identified in the thermophilic fungus Thielavia arenaria XZ7 and was successfully expressed in Pichia pastoris. The specific activities of TaCel45 towards lichenin, sodium carboxymethylcellulose (CMC-Na), and barley β-glucan were 769, 498, and 486 U/mg protein, respectively, which are higher than the values for all other reported GH45 cellulases. TaCel45 had maximum activity at pH 5.0–6.0 and 60–65 °C with barley β-glucan and CMC-Na as substrates and had a melting temperature (T_m) of 68.4 °C. However, TaCel45 exhibited extraordinary thermostability at 90 and 100 °C, retaining more than 70 and 45% of its activity after a 1-h incubation, respectively. Seven mutants (C11S, C12S, C16S, C31S, C171S, C193S, and C203S) were then constructed to investigate the effects of each disulfide bond on the structure, activity, and stability of TaCel45. As a result, six disulfide bonds (C11-C136, C16-C87, C31-C57, C88-C203, C90-C193, and C160-Cy171) were found to be indispensable for the folding, secretion, and activity of TaCel45, while C12-C48 was critical for thermal adaptation and refolding. The mutant C12S showed decreased optimal temperature and T_m values of 50 and 60.2 °C, respectively, and retained less than 50% of the thermal refolding ability of the wild type. Overall, this study demonstrated that disulfide bonds play a vital role in the folding and refolding capability and thermostability of this GH45 cellulase.

     

Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus. Mechanism of the reaction and assignment of disulfide bonds.

The extremely heat-stable 5'-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, expressed to high levels in Escherichia coli, and purified to homogeneity by heat precipitation and affinity chromatography. The recombinant enzyme was subjected to a kinetic analysis including initial velocity and product inhibition studies. The reaction follows an ordered Bi-Bi mechanism and phosphate binding precedes nucleoside binding in the phosphorolytic direction. 5'-Methylthioadenosine phosphorylase from Pyrococcus furiosus is a hexameric protein with five cysteine residues per subunit. Analysis of the fragments obtained after digestion of the protein alkylated without previous reduction identified two intrasubunit disulfide bridges. The enzyme is very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 3.0 M after 22 h incubation. This value decreases to 2.0 M in the presence of 30 mM dithiothreitol, furnishing evidence that disulfide bonds are needed for protein stability. The guanidinium chloride-induced unfolding is completely reversible as demonstrated by the analysis of the refolding process by activity assays, fluorescence measurements and SDS/PAGE. The finding of multiple disulfide bridges in 5'-methylthioadenosine phosphorylase from Pyrococcus furiosus argues strongly that disulfide bond formation may be a significant molecular strategy for stabilizing intracellular hyperthermophilic proteins.     

Disulfide bonds of phospholipase A2 from bee venom yield discrete contributions to its conformational stability

Disulfide bonds are known to be crucial for protein stability. To probe the contribution of each of the five disulfide bonds (C9-C31, C30-C70, C37-C63, C61-C95, and C105-C113) in bee venom phospholipase A₂ to stability, variants with deleted disulfide bonds were produced by substituting two serine residues for each pair of cysteine residues. The mutations started from the pseudo-wild-type variant (pWT) with the mutation I1A (Markert et al., Biotechnol. Bioeng. 98 (2007) 48-59). All variants were expressed in Escherichia coli, refolded from inclusion bodies and purified as pWT. The activity of the variants ranged from 12 to 82% of pWT. From the transition curves of guanidine hydrochloride-induced unfolding, the contributions of the individual disulfide bonds to conformational stability were estimated. They increased in the sequence C9-C31 < C105-C113 < C30-C70 ≈ C37-C63 < C61-C95. For two disulfide bonds (C9-C31, C105-C113) the effects were confirmed on additionally produced variants with the substitution of cysteine by alanine. Despite distinct differences in stability, all variants showed similar cooperativity in unfolding. Selected variants were also probed for proteolytic stability toward thermolysin. The removal of disulfide bonds increased the proteolytic susceptibility of the native proteins in the same way as the stability decreased. From the comparison of the results with literature data on phospholipase A₂ from bovine pancreas possessing seven disulfide bonds, it was concluded that conserved disulfide bonds in homologous proteins fulfill related functions in conformational stability.     

Contribution of individual disulfide bonds to the oxidative folding of ribonuclease A

The eight cysteine residues of ribonuclease A form four disulfide bonds in the native protein. We have analyzed the folding of three double RNase A mutants (C65A/C72A, C58A/C110A, and C26A/C84A, lacking the C65-C72, C58-C110, and C26-C84 disulfide bonds, respectively) and two single mutants (C110A and C26A), in which a single cysteine is replaced with an alanine and the paired cysteine is present in the reduced form. The folding of these mutants was carried out in the presence of oxidized and reduced glutathione, which constitute the main redox agents present within the ER. The use of mass spectrometry in the analysis of the folding processes allowed us (i) to follow the formation of intermediates and thus the pathway of folding of the RNase A mutants, (ii) to quantitate the intermediates that formed, and (iii) to compare the rates of formation of intermediates. By comparison of the folding kinetics of the mutants with that of wild-type RNase A, the contribution of each disulfide bond to the folding process has been evaluated. In particular, we have found that the folding of the C65A/C72A mutant occurs on the same time scale as that of the wild-type protein, thus suggesting that the removal of the C65-C72 disulfide bond has no effect on the kinetics of RNase A folding. Conversely, the C58A/C110A and C26A/C84A mutants fold much more slowly than the wild-type protein. The removal of the C58-C110 and C26-C84 disulfide bonds has a dramatic effect on the kinetics of RNase A folding. Results described in this paper provide specific information about conformational folding events in the regions involving the mutated cysteine residues, thus contributing to a better understanding of the complex mechanism of oxidative folding.     

Roles of disulfide bridges in scorpion toxin BmK M1 analyzed by mutagenesis.

The unique fold of scorpion toxins is a natural scaffold for protein engineering, in which multiple disulfide bonds are crucial structural elements. To understand the respective roles of these disulfide bridges, a mutagenesis analysis for the four disulfide bonds, 12-63, 16-36, 22-46 and 26-48, of a representative toxin BmK M1 from the scorpion Buthus martensii Karsch was carried out. All cysteines were replaced by serine with double mutations. The recombinant mutants were expressed in the Saccharomyces cerevisiae S-78 system. Toxic activities of the expressed mutants were tested on ICR mice in vivo and on neuronal Na+ channels (rNav1.2) by electrophysiological analysis. Recombinant variants M1 (C22S,C46S) and M1 (C26S,C48S) were not expressed at all; M1 (C16S,C36S) could be expressed at trace levels but was extremely unstable. Variant M1 (C12S,C63S) could be expressed in an amount comparable with that of unmodified rBmK M1, but had no detectable bioactivities. The results indicated that among the four disulfide bonds for long-chain scorpion toxins, loss of either bridge C22-C46 or C26-C48 is fatal for the general folding of the molecule. Bridge C16-C36 mainly contributes to the global stability of the folded scaffold, and bridge C12-C63 plays an essential role in the functional performance of scorpion toxins.     

A C-terminal interdomain disulfide bond significantly stabilizes the Fc fragment of IgG

We describe the stabilization of human IgG1 Fc by an engineered interdomain disulfide bond at the C-terminal end of the molecule. Covalently interconnecting the C-termini of the CH(3) domains led to an increase of the melting temperatures by 5.6 and 9.1°C respectively as compared to CH(3) domains in the context of the wild-type Fc. Combined with a recently described additional intradomain disulfide bond, both novel disulfide bonds led to an increase of the Tm by about 18.1°C to 100.7°C. The interdomain disulfide bond had no impact on the thermal stability of the CH(2) domain. Far- and near-UV CD spectroscopy showed very similar overall CD profiles, indicating that secondary and tertiary structure of the Fc was not negatively affected. When introduced into an Fc fragment that had been engineered to bind to Her2/neu via a novel antigen binding site located at the C-terminus of the CH(3) domain, the novel inter- and intra-domain bonds also brought about a significant increase in thermostability. Using them in combination, the Tm of the CH(3) domain was raised by 18°C and thus restored to the Tm of the wild-type CH(3) domain. Importantly, antigen binding of the modified Fc was not affected by the engineered disulfide bonds.     

Methionine adenosyltransferase alpha-helix structure unfolds at lower temperatures than beta-sheet: a 2D-IR study

Two-dimensional infrared spectroscopy has been used to characterize rat liver methionine adenosyltransferase and the events taking place during its thermal unfolding. Secondary structure data have been obtained for the native recombinant enzyme by fitting the amide I band of infrared spectra. Thermal denaturation studies allow the identification of events associated with individual secondary-structure elements during temperature-induced unfolding. They are correlated to the changes observed in enzyme activity and intrinsic fluorescence. In all cases, thermal denaturation proved to be an irreversible process, with a T(m) of 47-51 degrees C. Thermal profiles and two-dimensional infrared spectroscopy show that unfolding starts with alpha-helical segments and turns, located in the outer part of the protein, whereas extended structure, associated with subunit contacts, unfolds at higher temperatures. The data indicate a good correlation between the denaturation profiles obtained from activity measurements, fluorescence spectroscopy, and the behavior of the infrared bands. A study of the sequence of events that takes place is discussed in light of the previous knowledge on methionine adenosyltransferase structure and oligomerization pathway.     

Characterization of disulfide bonds in human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3): implications for NTPDase structural modeling

Cell-surface nucleotidases (NTPDases) contain 10 invariant cysteine residues in their extracellular regions. To investigate disulfide structure in human NTPDase3, we made single and double mutants of these 10 cysteines, and analyzed their enzymatic activity, glycosylation pattern, trafficking to the cell membrane, and sensitivity to reduction. The mutants constituted five distinct phenotypes, thus, strongly suggesting disulfide bonds between C92-C116 (first bond), C261-C308 (second bond), C289-C334 (third bond), C347-C353 (fourth bond), and C399-C422 (fifth bond). Due to conservation of the 10 cysteines, the identified five disulfide bonds are likely to exist in all cell-surface NTPDases. The third and fifth bonds are also present in the soluble NTPDases and are critical for processing, trafficking, and enzymatic activity. The fourth bond has minimal effect on processing and function, while the first and second bonds are of intermediate importance. Most of the N-linked glycosylation sites in the wild-type enzyme are processed to complex oligosaccharides, but at least one site is high-mannose or hybrid in structure. Interestingly, disruption of the first disulfide bond resulted in some enzyme that lost sensitivity to endoglycosidase H, suggesting that the first disulfide bond in the wild-type enzyme shields some high-mannose glycans from terminal glycosylation. Comparative modeling by threading and homology modeling of the NTPDase3 sequence revealed a high degree of structural fold similarity with a bacterial exopolyphosphatase (PDB ). The resultant theoretical 3-D model of the extracellular portion of NTPDase3, based on homology with this exopolyphosphatase, is consistent with the assignment of the disulfide bonds occurring in regions of good fold similarity between NTPDase3 and the exopolyphosphatase. The 3-D model obtained for NTPDase3 also suggests the structural basis for the importance of several apyrase conserved regions for the nucleotidase activities of the NTPDases.     

Conformational landscape and pathway of disulfide bond reduction of human alpha defensin

Human alpha defensins are a class of antimicrobial peptides with additional antiviral activity. Such antimicrobial peptides constitute a major part of mammalian innate immunity. Alpha defensins contain six cysteines, which form three well defined disulfide bridges under oxidizing conditions. Residues C3-C31, C5-C20, and C10-C30 form disulfide pairs in the native structure of the peptide. The major tissue in which HD5 is expressed is the crypt of the small intestine, an anaerobic niche that should allow for substantial pools of both oxidized and (partly) reduced HD5. We used ion mobility coupled to mass spectrometry to track the structural changes in HD5 upon disulfide bond reduction. We found evidence of stepwise unfolding of HD5 with sequential reduction of the three disulfide bonds. Alkylation of free cysteines followed by tandem mass spectrometry of the corresponding partially reduced states revealed a dominant pathway of reductive unfolding. The majority of HD5 unfolds by initial reduction of C5-C20, followed by C10-C30 and C3-C31. We find additional evidence for a minor pathway that starts with reduction of C3-C31, followed by C5-C20 and C10-C30. Our results provide insight into the pathway and conformational landscape of disulfide bond reduction in HD5.     

Stabilization of Escherichia coli ribonuclease H by introduction of an artificial disulfide bond.

To examine the effect of the introduction of a disulfide bond on the stability of Escherichia coli ribonuclease H, a disulfide bond was engineered between Cys13, which is present in the wild-type enzyme, and Cys44, which is substituted for Asn44 by site-directed mutagenesis. The disulfide bond was only formed between these residues upon oxidation in vitro with redox buffer. The conformational and thermal stabilities were estimated from the guanidine hydrochloride and thermal denaturation curves, respectively. The oxidized (cross-linked) mutant enzyme showed a Tm of 62.3 degrees C, which was 11.8 degrees C higher than that observed for the wild-type enzyme. The free energy change of unfolding in the absence of denaturant, delta G[H2O], and the mid-point of the denaturation curve, [D]1/2, of the oxidized mutant enzyme were also increased by 2.1-2.8 kcal/mol and 0.36-0.48 M, respectively. Introduction of a disulfide bond thus greatly enhanced both the thermal and conformational stabilities of the enzyme. In addition, kinetic analyses for the enzymatic activities of mutant enzymes suggest that Thr43 and Asn44 are involved in the substrate-binding site of the enzyme.