Consequence of the Removal of Evolutionary Conserved Disulfide Bridges on the Structure and Function of Charybdotoxin and Evidence That Particular Cysteine Spacings Govern Specific Disulfide Bond Formation

Abstract

Scorpion toxins are miniglobular proteins containing a common structural motif formed by an alpha-helix on one face, an antiparallel beta-sheet on the opposite face, and three disulfide bonds making up most of its internal volume. We have investigated the role of these evolutionary conserved bonds by replacing each couple of bridged cysteine residues of the scorpion charybdotoxin by a pair of nonbridging L-alpha-aminobutyric acid (Aba) residues. Three analogues were obtained by solid-phase synthesis, Chab I, Chab II, and Chab III, containing the Aba residues in positions 7 and 28, 13 and 33, 17 and 35, respectively. Circular dichroism analysis showed that the purified Chab II acquired a conformation similar to that of charybdotoxin, while the Chab I and Chab III possess decreased nativelike characteristics. All analogues block single high-conductance Ca(2+)-activated K+ channels from rat skeletal muscle inserted into planar lipid bilayers, but with different potencies. Chab II is the most active analogue (KD = 8.0 x 10(-8) M), with a 9-fold lower affinity as compared to native charybdotoxin. Chab I and Chab III have, respectively, 180- and 580-fold lower affinity. Therefore, the removal of evolutionary conserved disulfide bridges does not prevent the toxin to adopt a functional and presumably nativelike structure. However, removal of one disulfide bond affects the yields of formation of correct pairing between the remaining cysteine residues, and only Chab I preserves the ability to form the native disulfide pairings with high efficiency. This is the only analogue to preserve particular spacings of three and one residue between the cysteines, which have been described to thermodynamically disfavor disulfide bond formation between the cysteines [Zhang R., and Snyder, G. H. (1989) J. Biol. Chem. 264, 18472-18479]. Therefore, we conclude that the position of the cysteine residues in the sequence of charybdotoxin, by disfavoring specific pairings and favoring others, may govern selective formation of specific disulfide bonds, thus, explaining the efficient folding properties of Chab I and of native charybdotoxin. The structural properties of the Chab analogues and the discovered role of the cysteine spacings have interesting implications in protein design and engineering.

Acid-sensing ion channels interact with and inhibit BK K + channels

“…The Cys in the R⅐F/-⅐G⅐K⅐C sequence forms a disulfide bridge that stabilizes scorpion toxin structure, thereby positioning Arg and Lys to interact with BK. Mutating that Cys dramatically reduced CTx affinity for BK (19). We found that mASIC1a-C194A still inhibited BK current and pH 6 solution relieved inhibition (Fig.…”

“…1A) (17). In ␣-KTx toxins, the Lys side chain plugs the channel pore, the Arg interacts with residues in the outer vestibule of K ϩ channels, and the Cys forms a disulfide bond that stabilizes toxin structure (17)(18)(19). Although it seemed clear that the ASIC extracellular domain did not resemble an ␣-KTx toxin, the conserved sequence led us to hypothesize that ASICs might interact with and inhibit K ϩ channels.…”

Structure–function study of a chlorotoxin-chimer and its activity on Kv1.3 channels

“…The analogs were fully active in vitro on ion channels and in vivo when tested for neurotoxicity in mice [20]. Similarly, charybdotoxin (ChTx) analogs, lacking the first disulfide bridge, preserve the ability to form native disulfide pairings, in contrast to analogs lacking the second or third bridge [21]. On the other hand, studies on mono-loop analogues of iberiotoxin (IbTx) indicated that no single loop derivative displayed biological activity [22].…”

Structural determinants of protein folding

“…Deletion of this critical disulfide bridge significantly disrupted the ordered secondary structure and increased the proinsulin's susceptibility to proteolysis [119]. At the other extreme of the spectrum, multiple disulfide bridges have occasionally been noted to be redundant, with folding and structural stability maintained with a reduced number of native disulfide bridges [121,122].…”

“…In the case of GI a-conotoxin, Zhang and Snyder [128] demonstrated that the propensity of the peptide toxin to fold into the native globular conformation is largely dependent on the loop sizes of the two intercysteine loops. Similarly, the disulfide connectivity of charybdotoxin from the venom of the scorpion, Leiurus quinquestriatus, was influenced by the position of the cysteine residues and intercysteine loop spaces [122]. In an engineered barnase structure, the inclusion of disulfide bridges reduced the rate of protein unfolding [129].…”