The genomics of disulfide bonding and protein stabilization in thermophiles

Thermophilic organisms flourish in varied high-temperature environmental niches that are deadly to other organisms. Recently, genomic evidence has implicated a critical role for disulfide bonds in the structural stabilization of intracellular proteins from certain of these organisms, contrary to the conventional view that structural disulfide bonds are exclusively extracellular. Here both computational and structural data are presented to explore the occurrence of disulfide bonds as a protein-stabilization method across many thermophilic prokaryotes. Based on computational studies, disulfide-bond richness is found to be widespread, with thermophiles containing the highest levels. Interestingly, only a distinct subset of thermophiles exhibit this property. A computational search for proteins matching this target phylogenetic profile singles out a specific protein, known as protein disulfide oxidoreductase, as a potential key player in thermophilic intracellular disulfide-bond formation. Finally, biochemical support in the form of a new crystal structure of a thermophilic protein with three disulfide bonds is presented together with a survey of known structures from the literature. Together, the results provide insight into biochemical specialization and the diversity of methods employed by organisms to stabilize their proteins in exotic environments. The findings also motivate continued efforts to sequence genomes from divergent organisms.     

Engineering disulfide bonds within an antibody

Antibodies have evolved to function in oxidative, extracellular environments. A pair of cysteines in close proximity will oxidatively react to form a disulfide bond that fixes and stabilizes the tertiary structure of a protein. Immunoglobulin G (IgG) includes several disulfide bonds, and the patterns of inter-chain disulfide bonds characterize different IgG sub-classes. Moreover, the Ig-fold domains are characterized by a buried intra-domain disulfide bond, which is important for its structural stability. However, the intra-domain disulfide bond can be replaced without crucial effects on the structure and function, if the domain structure is intrinsically stable or has been stabilized by protein engineering. In previous studies, disulfide bonds were removed by amino-acid substitution indicating that Val and/or Ala (i.e. Ala–Ala, Ala–Val, Val–Ala, and Val–Ala) pairs were preferred for cysteine replacement in the Ig-fold domain. As such, these mutations may be useful for the intracellular use of antibodies. Recently, additional intra-domain disulfide bonds have been shown to stabilize Ig-fold domains and whole IgGs. In heavy chain variable or light chain variable domains, the introduction of additional disulfide bonds into the framework region did not reduce antigen-binding affinity, suggesting that generating disulfide bonds may be a method for stabilizing IgG and antibody fragments, such as the antigen-binding fragment, and single-chain and single-domain antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

Reconsideration of an early dogma,saying “there is no evidence for disulfide bonds in proteins from archaea”

Stability and function of a large number of proteins are crucially dependent on the presence of disulfide bonds. Recent genome analysis has pointed out an important role of disulfide bonds for the structural stabilization of intracellular proteins from hyperthermophilic archaea and bacteria. These findings contradict the conventional view that disulfide bonds are rare in those proteins. A specific protein, known as protein disulfide oxidoreductase (PDO) is recognized as a potential key enzyme in intracellular disulfide-shuffling in hyperthermophiles. The structure of this protein consists of two combined thioredoxin-related units which together, in tandem-like manner, form a closed protein domain. Each of these units contains a distinct CXXC active site motif. Both sites seem to have different redox properties. A relation to eukaryotic protein disulfide isomerase is suggested by the observed structural and functional characteristics of the protein. Enzymological studies have revealed that both, the archaeal and bacterial forms of this protein show oxidative and reductive activity and are able to isomerize protein disulfides. The variety of active site disulfides found in PDO’s from hyperthermophiles is puzzling. It is assumed, that PDO enzymes in hyperthermophilic archaea and bacteria may be part of a complex system involved in the maintenance of protein disulfide bonds.     

Thioredoxin A active-site mutants form mixed disulfide dimers that resemble enzyme-substrate reaction intermediates

Thioredoxin functions in nearly all organisms as the major thiol-disulfide oxidoreductase within the cytosol. Its prime purpose is to maintain cysteine-containing proteins in the reduced state by converting intramolecular disulfide bonds into dithiols in a disulfide exchange reaction. Thioredoxin has been reported to contribute to a wide variety of physiological functions by interacting with specific sets of substrates in different cell types. To investigate the function of the essential thioredoxin A (TrxA) in the low-GC Gram-positive bacterium Bacillus subtilis, we purified wild-type TrxA and three mutant TrxA proteins that lack either one or both of the two cysteine residues in the CxxC active site. The pure proteins were used for substrate-binding studies known as “mixed disulfide fishing” in which covalent disulfide-bonded reaction intermediates can be visualized. An unprecedented finding is that both active-site cysteine residues can form mixed disulfides with substrate proteins when the other active-site cysteine is absent, but only the N-terminal active-site cysteine forms stable interactions. A second novelty is that both single-cysteine mutant TrxA proteins form stable homodimers due to thiol oxidation of the remaining active-site cysteine residue. To investigate whether these dimers resemble mixed enzyme-substrate disulfides, the structure of the most abundant dimer, C32S, was characterized by X-ray crystallography. This yielded a high-resolution (1.5Å) X-ray crystallographic structure of a thioredoxin homodimer from a low-GC Gram-positive bacterium. The C32S TrxA dimer can be regarded as a mixed disulfide reaction intermediate of thioredoxin, which reveals the diversity of thioredoxin/substrate-binding modes.     

Interresidue interactions in protein classes

The free energy difference between folded and unfolded state is about the same for most proteins and it is not more than the energy of a few noncovalent interactions. In addition to the numerous noncovalent interactions, some proteins contain one or more disulfide bonds, which, as covalent crosslinks, significantly stabilize their tertiary structure. Correlation between the presence of disulfide bond(s), and the number noncovalent interresidue interactions of various kinds is analyzed here. The number of interactions per residue is almost the same for all protein. Also the number of long-range interactions per residue is the same in all proteins. Proteins with S(SINGLE BOND)S bond(s) (extracellular proteins) have more medium-range and fewer short-range interactions than those without S(SINGLE BOND)S bonds. However, the difference is independent of the number of these covalent crosslinks. We concluded that the different distributions of the various kinds of noncovalent interaction reflect the needs of proteins in the different environments, the extracellular and the intracellular ones, rather than the presence of the disulfide bond(s). We also pointed out that the observed differences in the distributions of short- and medium-range interactions are in good agreement with different secondary structure compositions of extracellular and intracellular proteins. Proteins 27:360–366, 1997. © 1997 Wiley-Liss, Inc.     

A novel member of the protein disulfide oxidoreductase family from Aeropyrum pernix K1: structure, function and electrostatics

The formation of disulfide bonds between cysteine residues is a rate-limiting step in protein folding. To control this oxidative process, different organisms have developed different systems. In bacteria, disulfide bond formation is assisted by the Dsb protein family; in eukarya, disulfide bond formation and rearrangement are catalyzed by PDI. In thermophilic organisms, a potential key role in disulfide bond formation has recently been ascribed to a new cytosolic Protein Disulphide Oxidoreductase family whose members have a molecular mass of about 26 kDa and are characterized by two thioredoxin folds comprising a CXXC active site motif each. Here we report on the functional and structural characterization of ApPDO, a new member of this family, which was isolated from the archaeon Aeropyrum pernix K1. Functional studies have revealed that ApPDO can catalyze the reduction, oxidation and isomerization of disulfide bridges. Structural studies have shown that this protein has two CXXC active sites with fairly similar geometrical parameters typical of a stable conformation. Finally, a theoretical calculation of the cysteine pK(a) values has suggested that the two active sites have similar functional properties and each of them can impart activity to the enzyme. Our results are evidence of functional similarity between the members of the Protein Disulphide Oxidoreductase family and the eukaryotic enzyme PDI. However, as the different three-dimensional features of these two biological systems strongly suggest significantly different mechanisms of action, further experimental studies will be needed to make clear how different three-dimensional structures can result in systems with similar functional behavior.     

Selective Removal of Individual Disulfide Bonds within a Potato Type II Serine Proteinase Inhibitor from Nicotiana alata Reveals Differential Stabilization of the Reactive-Site Loop

The 53-amino-acid trypsin inhibitor 1 from Nicotiana alata (T1) belongs to the potato type II family also known as the PinII family of proteinase inhibitors, one of the major families of canonical proteinase inhibitors. T1 contains four disulfide bonds, two of which (C4-C41 and C8-C37) stabilize the reactive-site loop. To investigate the influence of these two disulfide bonds on the structure and function of potato II inhibitors, we constructed two variants of T1, C4A/C41A-T1 and C8A/C37A-T1, in which these two disulfide bonds were individually removed and replaced by alanine residues. Trypsin inhibition assays show that wild-type T1 has a K_i of < 5 nM, C4A/C41A-T1 has a weaker K_i of ∼ 350 nM, and the potency of the C8A/C37A variant is further decreased to a K_i of ∼ 1.8 μM. To assess the influence of the disulfide bonds on the structure of T1, we determined the structure and dynamics of both disulfide variants by NMR spectroscopy. The structure of C4A/C41A-T1 and the amplitude of intrinsic flexibility in the reactive-site loop resemble that of the wild-type protein closely, despite the lack of the C4-C41 disulfide bond, whereas the timescale of motions is markedly decreased. The rescue of the structure despite loss of a disulfide bond is due to a previously unrecognized network of interactions, which stabilizes the structure of the reactive-site loop in the region of the missing disulfide bond, while allowing intrinsic motions on a fast (picosecond-nanosecond) timescale. In contrast, no comparable interactions are present around the C8-C37 disulfide bond. Consequently, the reactive-site loop becomes disordered and highly flexible in the structure of C8A/C37A-T1, making it unable to bind to trypsin. Thus, the reactive-site loop of T1 is stabilized differently by the C8-C37 and C4-C41 disulfide bonds. The C8-C37 disulfide bond is essential for the inhibitory activity of T1, whereas the C4-C41 disulfide bond is not as critical for maintaining the three-dimensional structure and function of the molecule but is responsible for maintaining flexibility of the reactive-site loop on a microsecond-nanosecond timescale.     

Disulfide bridges in the mesophilic triosephosphate isomerase from Giardia lamblia are related to oligomerization and activity

Triosephosphate isomerase from the mesophile Giardia lamblia (GlTIM) is the only known TIM with natural disulfide bridges. We previously found that oxidized and reduced thiol states of GlTIM are involved in the interconversion between native dimers and higher oligomeric species, and in the regulation of enzymatic activity. Here, we found that trophozoites and cysts have different oligomeric species of GlTIM and complexes of GlTIM with other proteins. Our data indicate that the internal milieu of G. lamblia is favorable for the formation of disulfide bonds. Enzyme mutants of the three most solvent exposed Cys of GlTIM (C202A, C222A, and C228A) were prepared to ascertain their contribution to oligomerization and activity. The data show that the establishment of a disulfide bridge between two C202 of two dimeric GlTIMs accounts for multimerization. In addition, we found that the establishment of an intramonomeric disulfide bond between C222 and C228 abolishes catalysis. Multimerization and inactivation are both reversed by reducing conditions. The 3D structure of the C202A GlTIM was solved at 2.1 A resolution, showing that the environment of the C202 is prone to hydrophobic interactions. Molecular dynamics of an in silico model of GlTIM when the intramonomeric disulfide bond is formed, showed that S216 is displaced 4.6 A from its original position, causing loss of hydrogen bonds with residues of the active-site loop. This suggests that this change perturb the conformational state that aligns the catalytic center with the substrate, inducing enzyme inactivation.     

Restoration of structural stability and ligand binding after removal of the conserved disulfide bond in tear lipocalin

Disulfide bonds play diverse structural and functional roles in proteins. In tear lipocalin (TL), the conserved sole disulfide bond regulates stability and ligand binding. Probing protein structure often involves thiol selective labeling for which removal of the disulfide bonds may be necessary. Loss of the disulfide bond may destabilize the protein so strategies to retain the native state are needed. Several approaches were tested to regain the native conformational state in the disulfide-less protein. These included the addition of trimethylamine N-oxide (TMAO) and the substitution of the Cys residues of disulfide bond with residues that can either form a potential salt bridge or others that can create a hydrophobic interaction. TMAO stabilized the protein relaxed by removal of the disulfide bond. In the disulfide-less mutants of TL, 1.0 M TMAO increased the free energy change (ΔG⁰) significantly from 2.1 to 3.8 kcal/mol. Moderate recovery was observed for the ligand binding tested with NBD-cholesterol. Because the disulfide bond of TL is solvent exposed, the substitution of the disulfide bond with a potential salt bridge or hydrophobic interaction did not stabilize the protein. This approach should work for buried disulfide bonds. However, for proteins with solvent exposed disulfide bonds, the use of TMAO may be an excellent strategy to restore the native conformational states in disulfide-less analogs of the proteins.     

Characterization of a multifunctional protein disulfide oxidoreductase from Sulfolobus solfataricus

A potential role in disulfide bond formation in the intracellular proteins of thermophilic organisms has recently been ascribed to a new family of protein disulfide oxidoreductases (PDOs). We report on the characterization of SsPDO, isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. SsPDO was cloned and expressed in Escherichia coli. We revealed that SsPDO is the substrate of a thioredoxin reductase in S. solfataricus (K(M) 0.3 microm) and not thioredoxins (TrxA1 and TrxA2). SsPDO/S. solfataricus thioredoxin reductase constitute a new thioredoxin system in aerobic thermophilic archaea. While redox (reductase, oxidative and isomerase) activities of SsPDO point to its central role in the biochemistry of cytoplasmic disulfide bonds, chaperone activities also on an endogenous substrate suggest a potential role in the stabilization of intracellular proteins. Northern and western analysis have been performed in order to analyze the response to the oxidative stress.     

The effect of additional disulfide bonds on the stability and folding of ribonuclease A

The significant contribution of disulfide bonds to the conformational stability of proteins is generally considered to result from an entropic destabilization of the unfolded state causing a faster escape of the molecules to the native state. However, the introduction of extra disulfide bonds into proteins as a general approach to protein stabilization yields rather inconsistent results. By modeling studies, we selected positions to introduce additional disulfide bonds into ribonuclease A at regions that had proven to be crucial for the initiation of the folding or unfolding process, respectively. However, only two out of the six variants proved to be more stable than unmodified ribonuclease A. The comparison of the thermodynamic and kinetic data disclosed a more pronounced effect on the unfolding reaction for all variants regardless of the position of the extra disulfide bond. Native-state proteolysis indicated a perturbation of the native state of the destabilized variants that obviously counterbalances the stability gain by the extra disulfide bond.     

Major kinetic traps for the oxidative folding of leech carboxypeptidase inhibitor

The leech carboxypeptidase inhibitor (LCI) is a 66-amino acid protein, containing four disulfides that stabilize its structure. This polypeptide represents an excellent model for the study and understanding of the diversity of folding pathways in small, cysteine-rich proteins. The pathway of oxidative folding of LCI has been elucidated in this work, using structural and kinetic analysis of the folding intermediates trapped by acid quenching. Reduced and denatured LCI refolds through a rapid, sequential flow of one- and two-disulfide intermediates and reaches a rate-limiting step in which a mixture of three major three-disulfide species and a heterogeneous population of non-native four-disulfide (scrambled) isomers coexist. The three three-disulfide intermediates have been identified as major kinetic traps along the folding pathway of LCI, and their disulfide structures have been elucidated in this work. Two of them contain only native disulfide pairings, and one contains one native and two non-native disulfide bonds. The coexistence of three-disulfide kinetic traps adopting native disulfide bonds together with a significant proportion of fully oxidized scrambled isomers shows that the folding pathway of LCI features properties exhibited by both the bovine pancreatic trypsin inhibitor and hirudin, two diverse models with extreme folding characteristics. The results further demonstrate the large diversity of disulfide folding pathways.     

Structures of scrambled disulfide forms of the potato carboxypeptidase inhibitor predicted by molecular dynamics simulations with constraints

The structures of two species of potato carboxypeptidase inhibitor with nonnative disulfide bonds were determined by molecular dynamics simulations in explicit solvent using disulfide bond constraints that have been shown to work for the native species. Ten structures were determined; five for scrambled A (disulfide bonds between Cys8-Cys27, Cys12-Cys18, and Cys24-Cys34) and five for the scrambled C (disulfide bonds Cys8-Cys24, Cys12-Cys18, and Cys27-Cys34). The two scrambled species were both more solvent exposed than the native structure; the scrambled C species was more solvent exposed and less compact than the scrambled A species. Analysis of the loop regions indicates that certain loops in scrambled C are more nativelike than in scrambled A. These factors, combined with the fact that scrambled C has one native disulfide bond, may contribute to the observed faster conversion to the native structure from scrambled C than from scrambled A. Results from the PROCHECK program using the standard parameter database and a database specially constructed for small, disulfide-rich proteins indicate that the 10 scrambled structures have correct stereochemistry. Further, the results show that a characteristic feature of small, disulfide-rich proteins is that they score poorly using the standard PROCHECK parameter database. Proteins 2000;40:482-493.     

Disulfide bond formation during the folding of influenza virus hemagglutinin

To study the importance of individual sulfhydryl residues during the folding and assembly in vivo of influenza virus hemagglutinin (HA), we have constructed and expressed a series of mutant HA proteins in which cysteines involved in three disulfide bonds have been substituted by serine residues. Investigations of the structure and intracellular transport of the mutant proteins indicate that (a) cysteine residues in the ectodomain are essential both for efficient folding of HA and for stabilization of the folded molecule; (b) cysteine residues in the globular portion of the ectodomain are likely to form native disulfide bonds rapidly and directly, without involvement of intermediate, nonnative linkages; and (c) cysteine residues in the stalk portion of the ectodomain also appear not to form intermediate disulfide bonds, even though they have the opportunity to do so, being separated from their correct partners by hundreds of amino acids including two or more other sulfhydryl residues. We propose a role for the cellular protein BiP in shielding the cysteine residues of the stalk domain during the folding process, thus preventing them from forming intermediate, nonnative disulfide bonds.     

Reexamination of the cysteine residues in glucocerebrosidase

Glucocerebrosidase, the deficient enzyme in Gaucher disease, catalyzes the cleavage of the beta-glycosidic linkage of glucosylceramide. A previous study on the enzyme identified three disulfide bridges and a single sulfhydryl [Lee, Y., Kinoshita, H., Radke, G., Weiler, S., Barranger, J.A. and Tomich, J.M. (1995) Position of the sulfhydryl group and the disulfide bonds of human glucocerebrosidase. J. Protein Chem. 14(3), 127-137] but recent publication of the X-ray structure identifies only two disulfide bridges with three free sulfhydryls [Dvir, H., Harel, M., McCarthy, A.A., Toker, L., Silman, I., Futerman, A.H. and Sussman, J.L. (2003) X-ray structure of human acid-beta-glucosidase, the defective enzyme in Gaucher disease. EMBO. 4(7), 704-709]. Using chemical modifications, acid cleavage and enzymatic digestion methods, we report that three free sulfhydryls exist and that the remaining four cysteines form two disulfide bonds located within the first 25 amino-terminal residues, supporting the X-ray structure.     

The crystal structure of adenylosuccinate lyase from Pyrobaculum aerophilum reveals an intracellular protein with three disulfide bonds

Adenylosuccinate lyase catalyzes two separate reactions in the de novo purine biosynthetic pathway. Through its dual action in this pathway, adenylosuccinate lyase plays an integral part in cellular replication and metabolism. Mutations in the human enzyme can result in severe neurological disorders, including mental retardation with autistic features. The crystal structure of adenylosuccinate lyase from the hyperthermophilic archaebacterium Pyrobaculum aerophilum has been determined to 2.1 A resolution. Although both the fold of the monomer and the architecture of the tetrameric assembly are similar to adenylosuccinate lyase from the thermophilic eubacterium Thermotoga maritima, the archaebacterial lyase contains unique features. Surprisingly, the structure of adenylosuccinate lyase from P. aerophilum reveals that this intracellular protein contains three disulfide bonds that contribute significantly to its stability against thermal and chemical denaturation. The observation of multiple disulfide bonds in the recombinant form of the enzyme suggests the need for further investigations into whether the intracellular environment of P. aerophilum, and possibly other hyperthermophiles, may be compatible with protein disulfide bond formation. In addition, the protein is shorter in P. aerophilum than it is in other organisms. This abbreviation results from an internal excision of a cluster of helices that may be involved in protein-protein interactions in other organisms and may relate to the observed clinical effects of human mutations in that region.     

Solution structure and dynamics of epidermal growth factor and transforming growth factor alpha.

Circular dichroism (CD) and Fourier transform infrared spectroscopic studies have shown that the secondary structure of transforming growth factor alpha (TGF-alpha) is very similar to that of epidermal growth factor (EGF). The infrared spectra revealed a minor difference between the two proteins, in particular in the beta-sheet structure. A large difference was observed with CD between the two proteins in the apparent conformation each adopts when the disulfide bonds are reduced. Reduced TGF-alpha showed a distinct alpha-helical conformation only at a high trifluoroethanol concentration, whereas reduced EGF assumed an alpha-helical conformation in the absence of trifluoroethanol. This indicates that these two proteins adopt different secondary structures in the absence of disulfide bonds, although they assume similar folding structures in their presence. These data suggest that the disulfide bonds to a large degree dictate the conformation of these two proteins. Additionally, differences in the dynamic behavior between EGF and TGF-alpha were also observed. Infrared experiments showed that the hydrogen-deuterium exchange rate is much higher for TGF-alpha than for EGF, indicating that TGF-alpha is a more flexible molecule. The rate of reduction of the disulfide bonds by dithiothreitol was also faster for TGF-alpha. Therefore, it can be concluded that although EGF and TGF-alpha have a similar overall conformation, TGF-alpha is a more flexible molecule than EGF.     

An Unconventional Role for Cytoplasmic Disulfide Bonds in Vaccinia Virus Proteins

Previous data have shown that reducing agents disrupt the structure of vaccinia virus (vv). Here, we have analyzed the disulfide bonding of vv proteins in detail. In vv-infected cells cytoplasmically synthesized vv core proteins became disulfide bonded in the newly assembled intracellular mature viruses (IMVs). vv membrane proteins also assembled disulfide bonds, but independent of IMV formation and to a large extent on their cytoplasmic domains. If disulfide bonding was prevented, virus assembly was only partially impaired as shown by electron microscopy as well as a biochemical assay of IMV formation. Under these conditions, however, the membranes around the isolated particles appeared less stable and detached from the underlying core. During the viral infection process the membrane proteins remained disulfide bonded, whereas the core proteins were reduced, concomitant with delivery of the cores into the cytoplasm. Our data show that vv has evolved an unique system for the assembly of cytoplasmic disulfide bonds that are localized both on the exterior and interior parts of the IMV.     

Structural and Biochemical Characterization of Yeast Monothiol Glutaredoxin Grx6

Glutaredoxins (Grxs) are a ubiquitous family of proteins that reduce disulfide bonds in substrate proteins using electrons from reduced glutathione (GSH). The yeast Saccharomyces cerevisiae Grx6 is a monothiol Grx that is localized in the endoplasmic reticulum and Golgi compartments. Grx6 consists of three segments, a putative signal peptide (M1-I36), an N-terminal domain (K37-T110), and a C-terminal Grx domain (K111-N231, designated Grx6C). Compared to the classic dithiol glutaredoxin Grx1, Grx6 has a lower glutathione disulfide reductase activity but a higher glutathione S-transferase activity. In addition, similar to human Grx2, Grx6 binds GSH via an iron-sulfur cluster in vitro. The N-terminal domain is essential for noncovalent dimerization, but not required for either of the above activities. The crystal structure of Grx6C at 1.5 Å resolution revealed a novel two-strand antiparallel β-sheet opposite the GSH binding groove. This extra β-sheet might also exist in yeast Grx7 and in a group of putative Grxs in lower organisms, suggesting that Grx6 might represent the first member of a novel Grx subfamily.