Protein Engineering vol. 16 no. 9 pp. 637-639, 2003
© 2003 Oxford University Press
Vicinal disulfide turns
a 
ema
ar11International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, 2Department of General Chemistry, University of Pavia, Viale Taramelli 12, 27100 Pavia, Italy and 3Department of Organic Chemistry, Eötvös L. University, 1117, Pázmány P. s. 1/a Budapest, Hungary
4 To whom correspondence should be addressed. e-mail: pongor{at}icgeb.trieste.it
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The formation of a disulfide bond between adjacent cysteine residues is accompanied by the formation of a tight turn of the protein backbone. In nearly 90% of the structures analyzed a type VIII turn was found. The peptide bond between the two cysteines is in a distorted trans conformation, the omega torsion angle ranges from 159 to –133°, with an average value of 171°. The constrained nature of the vicinal disulfide turn and the pronounced difference observed between the oxidized and reduced states, suggests that vicinal disulfides may be employed as a ‘redox-activated’ conformational switch.
Keywords: ß-turns/disulfide bridge/protein structure/secondary structure
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The disulfide bond formed between the side chains of adjacent cysteines is a rare structural element. In the last few years, vicinal disulfides have been structurally characterized in a variety of proteins including enzymes, receptors and toxins (see Table I). Even though there are no arguments in favor of a common biological function (Blake et al., 1994
; Czajkowski and Karlin, 1995; Engst and Miller, 1999; Wang et al., 2000), it seems plausible that vicinal disulfide bond formation can have both a structural role (e.g. by stabilizing the local backbone conformation) and a redox role (e.g. via facilitating electron exchanges). It has been shown that the acethylcholine cannot bind its receptor when the vicinal disulfide bond of the latter is reduced (Czajkowski and Karlin, 1995). Similarly, the reduction of a vicinal disulfide leads to the inactivation of the methanol dehydrogenase (Blake et al., 1994). A Janus-faced atracotoxin keeps its three-dimensional structure upon the reduction of its vicinal disulfide bond, although its neurotoxic activity is completely lost (Wang et al., 2000). The disulfide ring is also believed to be necessary in binding and reducing the cation in mercuric ion reductase (Engst and Miller, 1999). A vicinal disulfide bond is supposed to be a significant feature in the function of hepcidin, a polypeptide involved in iron uptake in the human intestine and iron release in macrophages (Hunter et al., 2002). Recently, non-native vicinal disulfide bonds were found in transient intermediates observed during the oxidative folding of a small cysteine knot protein, the Amaranth 
-amylase inhibitor (Cemazar et al., 2003). This points to the fact that this structure may be more frequent than originally thought.
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In Figure 1 the structure of the vicinal disulfides is compared with a set of structures in which there are vicinal cysteines in the reduced form. The polypeptide segments containing the oxidized Cys–Cys pair are clearly bent and the two cysteine side chains protrude on the same side of the backbone (Figure 1A). On the contrary, protein segments that contain adjacent cysteines in the reduced form, possess a more-or-less extended backbone and the sulfhydryl side chains tend to be on opposite sides (Figure 1B). The oxidized octapeptide segments also seem to be more constrained than the reduced structures. This is shown by the root-mean-square distance (r.m.s.d.) that is on average smaller for the oxidized octa-peptides (2.51 ± 0.14 Å) than for the reduced octa-peptides (3.31 ± 0.03 Å). These comparisons were calculated only between non-homologous proteins. Figure 1C and D show representative structures of an oxidized and a reduced Cys–Cys motif, respectively.
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The backbone conformation of the turn associated with the vicinal disulfide bridge has two characteristic features. (i) The peptide unit formed by the two cysteines frequently deviates from planarity. The omega torsion angle ranges from 159 to –133°, with an average value of 171°. The peptide unit is thus in a distorted trans conformation, which is reminiscent of the cis–trans isomerization observed in solution of small model peptides (Kim et al., 1999
). Actually, in the solution structure of hepcidin, the polypeptide moiety around the two cysteines involved in the vicinal disulfide bond is clearly conformationally disordered. (ii) The predominant conformation (Table I) found in 17 out of 20 structures resembles the type VIII ß-turn (Wilmot and Thornton, 1990). A different conformation (type II ß-turn) is found in the carboxylic esterase [Protein Data Bank (PDB) code 1qlw] and in the complex between a nicotinic acethylcholine receptor fragment with -bungarotoxin (1idh and 1idg). In this conformation, the 
and 
torsions of the first cysteine and the torsion of the second cysteine have values of –59(7), 161(4) and 58(5)°, respectively. Both conformations compare well with the conformations observed in the solution structure of the model compound Ac–ox–[Cys–Cys]–NH2 (Creighton et al., 2001).
Even though the resolution of several of the structures in Table I may not allow the accurate determination of side chain stereochemistry, the overall stereochemical features of the eight-membered heterocycle containing the disulfide bond appear to be close to those observed in Ac–ox–[Cys–Cys]–NH2 (Creighton et al., 2001). 
1 and 2 are 
77 and 69° for the first cysteine and 58 and –44° for the second cysteine, and 3 is –100°.
The conserved nature of the vicinal disulfide turn and the pronounced difference that can be expected between the oxidized and reduced states suggests that the vicinal disulfides may be used in protein engineering to construct ‘redox activated’ conformational switches. While this hypothesis can be tested with peptides end-labeled with suitable spectroscopic probes, it is worth mentioning that the activity of ribonuclease A has been reversibly modulated via the reduction and oxidation of two vicinal cysteines ‘engineered’ into the molecule (Park and Raines, 2001).
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Received December 16, 2002; revised June 30, 2003; accepted July 30, 2003.
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