Conflicting three-dimensional structures of charybdotoxin (Chtx), a blocker of K+ channels, have been previously reported. A high-resolution model depicting the tertiary structure of Chtx has been obtained by DIANA and X-PLOR calculations from new proton nuclear magnetic resonance (NMR) data. The protein possesses a small triple-stranded antiparallel beta sheet linked to a short helix by two disulfides and to an extended fragment by one disulfide, respectively. This motif also exists in all known structures of scorpion toxins, irrespective of their size, sequence, and function. Strikingly, antibacterial insect defensins also adopt this folding pattern.

The spatial organization of side chains on a refined model of charybdotoxin is presented. First, the structural role of two groups of well-defined, low-accessible side chains (Thr3, Val5, Val16, Leu20, Cys33 and Leu20, His21, Thr23, Cys17, Cys35) is discussed. These side chains are conserved in three out of the five known scorpion toxins acting on K+ channels. Interestingly, they are not conserved in scyllatoxin which presents a slightly different secondary structure organization. Second, the spatial organization of all positively charged residues is analyzed. Comparison with the results presented by Park and Miller [(1992) Biochemistry (preceding paper in this issue)] shows that all functionally important positive residues are located on the beta-sheet side of the toxin. These results are different from those obtained by Auguste et al. [(1992) Biochemistry 31, 648-654] on scyllatoxin, which blocks a different type of K+ channel. This study shows, in fact, that functionally important positive residues are located on the helix side of the toxin. Thus, charybdotoxin and scyllatoxin, which present the same global fold, interact with two different classes of K+ channels by two different parts of the motif.

A compact, well-organized, and natural motif, stabilized by three disulfide bonds, is proposed as a basic scaffold for protein engineering. This motif contains 37 amino acids only and is formed by a short helix on one face and an antiparallel triple-stranded 1l-sheet on the opposite face. It has been adopted by scorpions as a unique scaffold to express a wide variety of powerful toxic ligands with tuned specificity for different ion channels. We further tested the potential of this fold by engineering a metal binding site on it, taking the carbonic anhydrase site as a model. By chemical synthesis we introduced nine residues, including three histidines, as compared to the original amino acid sequence of the natural charybdotoxin and found that the new protein maintains the original fold, as revealed by CD and IH NMR analysis. Cu2+ ions are bound with Kd = 4.2 x 10-8 M and other metals are bound with affinities in an order mirroring that observed in carbonic anhydrase. The a/fl scorpion motif, small in size, easily amenable to chemical synthesis, highly stable, and tolerant for sequence mutations represents, therefore, an appropriate scaffold onto which polypeptide sequences may be introduced in a predetermined conformation, providing an additional 'means for design and engineering of small proteins.By engineering several disulfide bridges in short sequences (10-70 amino acids) nature has produced small proteins, toxins and protease inhibitors, that are able to adopt stable and biologically active structures. These small proteins bind to their biological targets with high affinity and specificity by virtue of their particular structure: an exposed loop fixed in a characteristic "canonical" conformation, which fits into a protease active site (e.g., in protease inhibitors; see refs. 1 and 2) or a more variegated molecular surface able to interact with a specific receptor or channel (e.g., in toxins; see refs. 3-5). In all these cases, the protein structure functions as a scaffold able to present a specific sequence and a determined fixed conformation to the biological target.These natural miniproteins constitute a priori interesting candidates as core structures for protein design, since the disulfide bonds provide most of their stabilization energy, leaving a large part of the protein structure available for mutations. In this respect, the natural motif of charybdotoxin (6-8) appears particularly well-suited; it is short (37 amino acids), it is composed of an antiparallel triple-stranded X3-sheet on one face and a short a-helix on the opposite face, and it is stabilized by three disulfide bonds in the interior core (8-10). The same fold is also adopted by (i) all known scorpion toxins (6-8), irrespective of their size, amino acid sequence, and function (blockage of K+ channels, Na+ channels, Cl-channels, etc.) (11-13); (ii) insect defensins (14); and (iii) plant y-thionins (15). The only residues commonly shared by all these proteins are the six cysteines involved in disulfide bonds (6, 7), indicating a hi...

The DNA‐binding domain of the oncoprotein Myb comprises three imperfect repeats, R1, R2 and R3. Only R2 and R3 are required for sequence‐specific DNA‐binding. Both are assumed to contain helix‐turn‐helix (HTH)‐related motifs, but multidimensional heteronuclear NMR spectroscopy revealed a disordered structure in R2 where the second HTH helix was predicted [Jamin et al. (1993) Eur. J. Biochem., 216, 147‐154]. We propose that the disordered region folds into a ‘recognition’ helix and generates a full HTH‐related motif upon binding to DNA. This would move Cys43 into the hydrophobic core of R2. We observed that Cys43 was accessible to N‐ethylmaleimide alkylation in the free protein, but inaccessible in the DNA complex. Mutant proteins with charged (C43D) or polar (C43S) side chains in position 43 bound DNA with reduced affinity, while hydrophobic replacements (C43A, C43V and C43I) gave unaltered or improved DNA‐binding. Specific DNA‐binding enhanced protease resistance dramatically. Fluorescence emission spectra and quenching experiments supported a DNA‐induced conformational change. Moreover, reversible oxidation of Cys43 had an effect similar to the inactivating C43D mutation. The highly oxidizable Cys43 could function as a molecular sensor for a redox regulatory mechanism turning specific DNA‐binding on or off by controlling the DNA‐induced conformational change in R2.

A 600-MHz proton NMR study of natural charybdotoxin, a toxin acting on K + channels, is reported. The unambiguous sequential assignment of all the protons of the toxin was achieved. The analysis of NOES and of backbone coupling constants showed the existence of an a-helix (residues 10-19) and of an antiparallel P-sheet in the 26-35 part. Three-dimensional structures were generated by distance geometry, using a set of 114 interresidual calibrated constraints (63 sequential, 47 medium and long range, 4 hydrogen bonds) and 29 @ angles. These structures show that charybdotoxin is composed of a ,&sheet linked to an a-helix by two disulphide bridges and to an extended fragment by the third disulphide bridge. Comparison with the other known structures of long and short scorpion toxins shows that this structural motif is common to all these proteins.Charybdotoxin is a small protein (37 residues, three disulphide bridges) isolated a few years ago from the venom of the scorpion Leiurus yuinquestriutus hebraeus, in which it is present as a minor protein component ( z 0.2%) [l]. Charybdotoxin was originally described as the first specific inhibitor of Ca2+-activated K C channels [2]. Further work established that charybdotoxin binds to a variety of K + channels from vertebrate tissues [3].Successively, other toxic proteins acting like charybdotoxin as blockers of K + channels have been isolated from venom of different scorpion species [4]. All of them are singlechain polypeptides of 31 -40 residues with three disulphide bridges, presenting variable sequence similarities and systematically the same relative position of the six half-cystines.The elucidation of the three-dimensional organisation of this new class of toxins is of considerable interest for future pharmacological studies of K C channel blocking agents. The possible folding of charybdotoxin was originally predicted on the basis of circular dichroic data and sequence comparison with snake neurotoxins of known three-dimensional structure [l]. The model of charybdotoxin presented in that work consisted of a single P-pleated sheet mimicking the second loop of a snake neurotoxin with no helicity. In a very recent NMR paper, the structure of charybdotoxin has been described as consisting of three antiparallel @-sheets with no helical content 151.Data reported independently and almost simultaneously, however, hint at a different folding of charybdotoxin. Thus, Ahhreuiutions. Glp, 5-oxoproline (pyroglutamic acid); NOESY, nuclear Overhauser enhancement spectroscopy; COSY, correlated spectroscopy; DQFCOSY, double quantum-filtered correlated spectroscopy; TOCSY, total correlated spectroscopy.in a preliminary short report we presented results on the natural toxin showing that its structure comprises an a-helix and an antiparallel @-sheet [6]. Another preliminary report appearing subsequently mentioned a similar finding on synthetic charybdotoxin [7]. Moreover, another member of the same familly of toxins, leiurotoxin I, present as charybdotoxin in the venom of the scorpion Le...