Chemistry and Enzymology of Disulfide Cross-Linking in Proteins

Abstract

Cysteine thiols are among the most reactive functional groups in proteins, and their pairing in disulfide linkages is a common post-translational modification in proteins entering the secretory pathway. This modest amino acid alteration, the mere removal of a pair of hydrogen atoms from juxtaposed cysteine residues, contrasts with the substantial changes that characterize most other post-translational reactions. However, the wide variety of proteins that contain disulfides, the profound impact of cross-linking on the behavior of the protein polymer, the numerous and diverse players in intracellular pathways for disulfide formation, and the distinct biological settings in which disulfide bond formation can take place belie the simplicity of the process. Here we lay the groundwork for appreciating the mechanisms and consequences of disulfide bond formation in vivo by reviewing chemical principles underlying cysteine pairing and oxidation. We then show how enzymes tune redox-active cofactors and recruit oxidants to improve the specificity and efficiency of disulfide formation. Finally, we discuss disulfide bond formation in a cellular context and identify important principles that contribute to productive thiol oxidation in complex, crowded, dynamic environments.

Figure 1

Examples of proteins and protein complexes cross-linked by disulfide bonds. Protein backbones are represented as ribbon traces, and the Cβ and sulfur (yellow) atoms of cysteine side chains are shown as spheres. In protein complexes, different polypeptides are shown in distinct colors. PDB codes are as follows: conotoxin, 2LXG; theta-defensin, 5INZ; antifreeze protein, 1EZG; Izumo, 5JK9; ribonuclease A, 3MZR; laminin fragment, 4AQS; insulin, 2BN3; C4b-binding protein oligomerization domain, 4b0f; Kremen1-LRP6-Dickkopf complex, 5FWS.

Figure 2

Examples of diverse disulfide-rich proteins incorporated into biomaterials. The human keratin-associated protein has an ultra-high sulfur content, with cysteine constituting 37% of the residues in the amino acid sequence. In the cnidarian mini-collagens, the six-Cys motifs amino and carboxy terminal to the short collagen triple helical region (green) form disulfide-bridged networks to reinforce the pressurized nematocyst. CREMP proteins, with cysteine contents of ~11%, feature a highly repetitive and long sequence of disulfide-linked modules, eight of which are shown here.

Figure 3

Two proteins demonstrating different ways in which disulfides can decorate a fold. Disulfides link surface loops to the termini of core β-strands in uPAR. Disulfides link core helices to one another and affix a β-hairpin to the helical core in phospholipase A2. PDB codes are UPAR, 2FD6; phospholipase A2, 1C74.

Figure 4

Oxidation of thiols by dehydroascorbate. Generation of a thiohemiacetal intermediate is followed by capture of the adduct by a second thiolate species to yield ascorbate and the disulfide, RS-SR.

Figure 5

Mechanism of disulfide bond formation by 1,4-quinones. Attack of a thiolate on quinone (1) forms the Michael adduct (2) which can be resolved as in (3) to generate the reduced cofactor and the corresponding disulfide, RS-SR.

Figure 6

Mechanism of disulfide formation by flavin cofactors. The isoalloxazine ring system (1) can be attacked by a thiolate nucleophile at the C4a position to yield a thiol-flavin adduct (2). Resolution of this species with a second thiolate (3) leads to the generation of the disulfide. The resulting dihydroflavin (4) can be oxidized in two one-electron steps by molecular oxygen or by other oxidants.

Figure 7

(A) Two proteins with cysteine sulfurs oriented as shown are able to engage in an in-line SN2 reaction, form a mixed disulfide, and complete a thiol-disulfide exchange reaction. (B) The orientation of cysteine sulfurs shown is incompatible with thiol-disulfide exchange.

Figure 8

Representative active site of a PDI family protein. Amino acids commonly observed in PDIfamily active sites are shown in stick representation, with red indicating oxygen atoms and blue nitrogen. The Phe side chain is replaced by Tyr in some cases. Potential interactions of the largely buried S2 sulfur of the CXXC motif are indicated by dashed lines (black—hydrophobic; blue—hydrogen bonding; red— proton transfer). Green arrows indicate sites for hydrogen bonding interactions by the incoming substrate backbone. Red sphere is water. PDB code is 3ED3 (yeast Mpd1p).

Figure 9

Model for substrate interaction with a PDI protein. The P5 protein (gene name PDIA6) is in gray, and the backbone (including a proline side chain) of a peptide from peroxiredoxin Prx4 is in magenta. The cysteine side chain atoms of Prx4 are labeled Cβ and S. Blue dashed lines are hydrogen bonds. Red in stick representations indicates oxygen atoms, blue nitrogens. The S1 sulfur of P5 is labeled. PDB code is 3W8J.

Figure 10

Oxidative protein folding comprises two conceptual steps: oxidation and isomerization of mispaired disulfides. These steps are schematically depicted here for the introduction and rearrangement of a single disulfide bond.

Figure 11

Structure of PDI. The image shows S. cerevisiae PDI. Thioredoxin fold domains are labeled according to conventional nomenclature (a-b-b′-a′). Cysteine side chains are shown as spheres with yellow sulfurs. A set of exposed hydrophobic residues in the b′ domain that may constitute a binding site for folding or misfolded proteins is shown in space-filling representation (carbons in cyan). PDB code is 2B5E.

Figure 12

Structures of the Mia40 oxidoreductase. Sulfurs in the CPC motif are labeled S1 and S2. Gray arrow in the oxidized structure illustrates solvent accessibility of the redox-active disulfide. PDB codes are 2K3J (reduced; H. sapiens Mia40 solution NMR structure) and 2ZXT (oxidized; S. cerevisiae Mia40 crystallized as a fusion with maltose binding protein (not shown)).

Figure 13

Prx4 peroxidase cycle. Isolated segments of two neighboring Prx4 subunits are shown (purple and orange). Spheres are the side chains of cysteines participating in catalytic disulfide formation, with sulfurs colored yellow and Cβ atoms colored according to the parent chain. Based on PDB codes 3TJG and 3TJF.

Figure 14

Active-site regions of quinone-containing disulfide catalysts. Quinone-proximal disulfides are shown as spheres with sulfur atoms in yellow and Cβ atoms in green. The quinone (orange sticks) is buried in the core of the helical bundle in each enzyme, and the amino-terminal cysteine of the quinone-proximal disulfide (S1) is exposed to in-line nucleophilic attack. PDB codes are 2ZUP and 3KP9.

Figure 15

(A) Schematic representation of the DsbA-DsbB system showing the key charge-transfer intermediate observed when reduced DsbA is mixed with oxidized DsbB. Reduction of the quinone is rate-limiting and is coupled to the release of oxidized DsbA. The quinone cofactor is shown as a hexagon, in which blue represents a charge-transfer state. (B) Thermodynamics along the reaction coordinate for the events depicted in panel A. The redox potential for DsbA would appear insufficiently reducing to efficiently transfer electrons to the first disulfide in DsbB. (C) While the net conversion of reactants to products is thermodynamically unfavorable, the mixed disulfide intermediate between them can be highly populated (black line) or destabilized (grey line). Selective stabilization of mixed disulfide intermediates may allow the formation of products if those products are in turn depleted by other favorable reactions, such as electron transfer to quinone or oxygen.

Figure 16

Schematic representation of internal electron transfer events in a bacterial VKOR homolog. The quinone cofactor is shown as a hexagon, in which blue represents a charge-transfer state. It is not known to what extent the electron-transfer steps are concerted in VKOR enzymes, i.e., whether the electron-transfer loop is simultaneously disulfide bonded to both a trx domain active-site cysteine and the partner of the charge-transfer cysteine in the transmembrane domain. For comparison with Figure 14, the trx domain is shown to the left of the transmembrane domain, but it should be noted that the trx domain is fused carboxy terminally to the transmembrane domain.

Figure 17

Gallery of sulfhydryl oxidase flavoenzyme structures. Protein subunits are purple and green, FAD is orange, disulfide bond sulfurs are yellow. Ero1, Saccharomyces cerevisiae Ero1; QSOX1, Rattus norvegicus Quiescin Sulfhydryl Oxidase 1; QSOX (T. brucei), Trypanosoma brucei Quiescin Sulfhydryl Oxidase; ALR, Homo sapiens Augmenter of Liver Regeneration; AcMNPV, Autographa californica multicapsid nucleopolyhedrovirus; ASFV, African swine fever virus; AfGliT, Aspergillus fumigatus Gliotoxin Sulfhydryl Oxidase; APMV, Acanthamoeba polyphaga mimivirus. With the exception of APMV R596, dimer structures are viewed down the two-fold axis. PDB codes are Ero1, 1RP4; QSOX1, 4P2L; QSOX (T. brucei), 3QCP; ALR, 1OQC; AcMNV Ac92, 3QZY; ASFV pB119L, 3GWL; AfGliT, 4NTC; APMV R596, 3GWN.

Figure 18

Active-site regions of flavin-dependent sulfhydryl oxidases. Flavin-proximal disulfides are shown as spheres with sulfur atoms in yellow and Cβ atoms in gray. The sulfur in each enzyme targeted for nucleophilic attack by an incoming thiolate during catalysis (the interchange sulfur) is labeled S1. The relative solvent exposure of the S1 sulfurs and accessibility of the disulfide to in-line attack is evident in ALR, QSOX1, and GliT. Only in Ero1 is the FAD-proximal disulfide buried and the S1 cysteine apparently inaccessible. PDB files are ALR, 1OQC; QSOX1, 3LLI; Ero1, 1RP4; GliT, 4NTC.

Figure 19

Schematic of an Erv/ALR dimeric enzyme with shuttle disulfides delivering electrons, derived from substrate oxidation, to the opposite subunit in the dimer.

Figure 20

Incorporation of the Erv/ALR module into viral sulfhydryl oxidase dimers. Different dimerization modes are used in each case, and the module is embedded in different tertiary structural contexts. ASFV, African swine fever virus; AcMNPV, Autographa californica multicapsid nucleopolyhedrovirus; APMV, Acanthamoeba polyphaga mimivirus. PDB codes are ASFW pB119L, 3GWL; AcMNPV Ac92, 3QZY; APMV R596, 3GWN. For comparison, the structure of the mitochondrial enzyme ALR is also shown.

Figure 21

Schematic representation of conformational flexibility and internal electron transfer events in QSOX. The FAD cofactor is represented by hexagons, with blue representing a charge-transfer state. The rightmost (i.e., most carboxy-terminal) CXXC disulfide does not appear to participate in electron transfer from substrate to the FAD.

Figure 22

QSOX catalytic cycle. This model depicts turnover involving two-electron reduced forms of the enzyme for simplicity. Additional pathways involving four-electron reduced enzyme forms may contribute at high concentration of reducing substrates and/or low oxygen tensions. Curved lines represent motion. Step 1 is reduction of QSOX by substrate protein. Step 2 is the rapid formation of an interdomain disulfide. Step 3 is a rate limiting step involving reduction of the flavin cofactor. Step 4 regenerates the oxidized enzyme and occurs from either the closed or the open (shown here) state.

Figure 23

Structure of rat QSOX with juxtaposed redox-active disulfides. The protein is oriented to correspond roughly to Figure 20. The flexible linker is behind the domains. Disulfides are shown with yellow sulfur atoms. The three CXXC motifs and the FAD cofactor are labeled. Unpaired cysteines are not shown. Disulfides not in CXXC motifs are displayed but not labeled. The rightmost CXXC motif is distant from the redox-active centers. PDB code is 4P2L.

Figure 24

Structure of yeast Ero1 displaying different conformations of the loop containing the shuttle disulfide. Only the flexible loop containing the shuttle disulfide is shown for the “out” conformation (magenta). The FAD is orange. The FAD-proximal cysteine is obscured by the S1 sulfur in this view, such that the thiolate nucleophile would be expected to approach S1 from the direction of the reader. PDB codes are 2RP4 and 1RQ1.

Figure 25

Compounds with disulfide bonds introduced by fungal and bacterial sulfhydryl oxidases related to the thioredoxin reductase enzyme family. Gliotoxin is a epipolythiodioxopiperazine virulence factor in Aspergillus. Holomycin is a broad-spectrum antibiotic from Streptomyces. Romidepsin (FK228) is a depsipeptide anticancer agent from Chromobacterium species used against T-cell lymphomas.

Figure 26

Schematic flow of reducing equivalents in the ER during oxidative protein folding. Electron input: reducing equivalents arrive from the cytosol as cysteine residues in translocating nascent chains and imported glutathione. Output: electrons ultimately reduce molecular oxygen. The major reductive flux in the ER pipeline is believed to involve multiple PDIs in redox communication with one another and with the glutathione redox system, and capable of transferring electrons to sulfhydryl oxidases and peroxidases, which in turn reduce terminal electron acceptors.