Comparison of the (30-51, 14-38) two-disulphide folding intermediates of the homologous proteins dendrotoxin K and bovine pancreatic trypsin inhibitor by two-dimensional 1H nuclear magnetic resonance.

The disulphide folding pathway of bovine pancreatic trypsin inhibitor (BPTI) revealed that the native conformation is still stable in each intermediate state with two native disulphide linkages, in the absence of each of the corresponding third disulphide bonds. This is thought to be a consequence of the extreme stability of the native BPTI conformation. The current study addresses the question of whether the native-like conformation would be populated significantly at the two-disulphide stage in disulphide refolding if the final structure is less stable than in the case of BPTI. Dendrotoxin K from black mamba venom provides a good model to test this, since it contains the BPTI fold and was shown to fold predominantly via the same pathway, but its native conformation is stable than that of BPTI. The conformation of a chemically trapped two-disulphide intermediate in the disulphide refolding of dendrotoxin K, with blocking groups on Cys5 and Cys55 and disulphide bonds between Cys30 and Cys51, and Cys14 and Cys38, respectively, has been determined by 1H NMR spectroscopy and compared to those of the native protein and of the corresponding intermediate in BPTI. The analysis reveals that the dendrotoxin K intermediate adopts a partly-folded conformation, in contrast to the quasi-native conformation of the corresponding BPTI intermediate. It is similar to the partly-folded conformation of the BPTI intermediate with just the Cys30-Cys-51 disulphide bond, but with a more fixed conformation in the region of the Cys14-Cys38 disulphide bond. The destabilisation of the fully native conformation of the dendrotoxin K intermediate, relative to BPTI, appears to reduce the cooperativeity of the folding process.

NMR and restrained molecular dynamics study of the three-dimensional solution structure of toxin FS2, a specific blocker of the L-type calcium channel, isolated from black mamba venom.

The three-dimensional solution structure of toxin FS2, a 60-residue polypeptide isolated from the venom of black mamba snake (Dendroaspis polylepis polylepis), has been determined by nuclear magnetic resonance spectroscopy. Using 600 NOE constraints and 55 dihedral angle constraints, a set of 20 structures obtained from distance-geometry calculations was further refined by molecular dynamics calculations using a combined simulated annealing-restrained MD protocol. The resulting 20 conformers, taken to represent the solution structure, give an average rmsd of 1.2 A for their backbone atoms, relative to the average structure. The overall resulting three-fingered structure is similar to those already observed in several postsynaptic neurotoxins, cardiotoxins, and fasciculins, which all share with toxin FS2 the same network of four disulfide bridges. The overall concavity of the molecule, considered as a flat bottomed dish, is oriented toward the C-terminal loop of the molecule. This orientation is similar to that of fasciculins and cardiotoxins but opposite to that of neurotoxins. On the basis of the local rms displacements between the 20 conformers, the structure of the first loop appears to be less well defined in FS2 than in the previously reported neurotoxin structures, but fasciculin 1 shows a similar trend with particularly high temperature factors for this part of the X-ray structure. The concave side which presents most of the positively charged residues is quite similar in FS2 and fasciculin 1. The main difference is shown by the convex side of the third loop, mostly hydrophobic in FS2, in contrast to the pair of negatively charged aspartates in fasciculin 1. This difference could be one of the factors leading to the distinct pharmacological properties-L-type calcium channel blocker for FS2 and cholinesterase inhibitor for fasciculin--observed for these two subgroups of the "angusticeps-type" toxins.

Proteinase inhibitor homologues as potassium channel blockers.

We report here the NMR structure of dendrotoxin I, a powerful potassium channel blocker from the venom of the African Elapidae snake Dendroaspis polylepis polylepis (black mamba), calculated from an experimentally-derived set of 719 geometric restraints. The backbone of the toxin superimposes on bovine pancreatic trypsin inhibitor (BPTI) with a root-mean-square deviation of < 1.7 A. The surface electrostatic potential calculated for dendrotoxin I and BPTI, reveal an important difference which might account for the differences in function of the two proteins. These proteins may provide examples of adaptation for specific and diverse biological functions while at the same time maintaining the overall three-dimensional structure of a common ancestor.

Structural features important for the biological activity of the potassium channel blocking dendrotoxins.

1. Dendrotoxins from mamba snake venoms are small proteins that block neuronal K+ channels. In order to investigate structural features associated with their biological activity, partially folded versions of dendrotoxins I and K from black mamba (Dendroaspis polylepis) were prepared by selectively reducing one or more of their three S-S bonds. 2. The modified toxins were tested for ability to compete with 125I-labelled native toxin I to high affinity binding sites on rat brain synaptosomal membranes and for the ability to increase acetylcholine release in a neuromuscular preparation. 3. Binding affinity increased progressively as the toxins folded to the native conformation and the most biologically active of the modified species were those in which only the disulphide bond between residues 14 and 38 was not formed. These intermediates had native-like conformations as determined by circular dichroism but still had about 5-10 times lower affinity than native toxins. 4. Addition of negatively charged groups to block the free sulthydryls at positions 14 and 38 caused a further, marked loss of activity. 5. The results are consistent with the existence of two important regions in the dendrotoxin molecules. The region containing two of the disulphide bonds (around Cys5-Cys55 and Cys30-Cys51) and much of the secondary structure is essential for the binding affinity of the toxins, while the region around Cys14 and Cys38, equivalent to part of the antiprotease site of the homologous protease inhibitor from bovine pancreas (BPTI), plays an important role in the potency of dendrotoxins.

Sequence-specific 1H-NMR assignment and secondary structure of black mamba dendrotoxin I, a highly selective blocker of voltage-gated potassium channels.

The secondary structure of dendrotoxin I, an important constituent of the venom of the African black mamba snake Dendroaspis polylepis polylepis, was determined in aqueous solution by two-dimensional methods. Complete sequence-specific 1H-NMR assignment was obtained with the exception of the backbone amide proton of Gly39 and Cys40. Dendrotoxin I is based on a central antiparallel beta-sheet and two small helices located at the N- and the C-terminal extremities. These secondary-structural units occur at exactly the same places in the amino acid sequence as those of bovine pancreatic trypsin inhibitor (BPTI), with which dendrotoxin I shares 33% sequence similarity. According to the disulfide-bridge positions and the long-range NOE observed these secondary-structural elements fold in a similar manner to BPTI. This similarity allows an hypothesis according to which dendrotoxin I could derive from an ancestral Künitz-type proteinase inhibitor. This ancestor would have been heavily mutated at amino acid positions not critical for gross structure. The spatial locations of the solvent-exposed amino acids concerned could therefore serve as a guideline for interpretation of the structure/activity relationship of dendrotoxin I for the blockage of voltage-sensitive potassium channels of which dendrotoxin I is a strong inhibitor. The possible connections with other polypeptide toxins that block related ion currents is discussed.

Conformational forces affecting the folding pathways of dendrotoxins I and K from black mamba venom.

The conformations of the major intermediates trapped during the folding of dendrotoxins I and K from venom of black mamba snakes, have been investigated by circular-dichroism spectroscopy. Local alterations to the native, folded conformations are observed in toxins I and K and in a protein of similar sequence, bovine pancreatic trypsin inhibitor. The inability of intermediates (30-51, 14-38) to complete refolding by forming directly the 5-55 disulphide bond is explained. The following observations on the role of secondary structure in the folding of the three proteins are of interest. 1. It is not necessary for the three proteins to acquire elements of secondary structure at the same stage of folding in order to attain similar three-dimensional conformations. 2. The stability of the final folded state is not directly correlated to an early appearance of secondary structure. 3. The degree of secondary structure already present in intermediates (30-51) seems to determine the pathway of refolding preferred by the corresponding protein.

Single amino acid mutations block a late step in the folding of beta-lactamase from Staphylococcus aureus.

Two single amino acid mutant proteins of beta-lactamase PC1 from Staphylococcus aureus, P2 Thr40----Ile and P54 Asp146----Asn, have been investigated using urea-gradient polyacrylamide gel electrophoresis, circular dichroism and sedimentation velocity. Investigation of the folded states of the mutants has shown that compared to wild-type PC1 they are slightly more expanded, and have reduced aromatic circular dichroism, but the same content of secondary structure as PC1. The mutants exhibit fast refolding kinetics to the folded state, in contrast to PC1, which refolds only slowly. We conclude from these results that the folded mutants are in a state close to but distinct from the native state of PC1 and have certain properties in common with the compact intermediate in the folding of beta-lactamase. Therefore, these single amino acid substitutions result in a folding pathway blocked at a point located after collapse of the already folded structural units into a globular shape, and close to the final reshuffling step that leads to the native state of the wild-type enzyme.

Evolutionary conservation and variation of protein folding pathways. Two protease inhibitor homologues from black mamba venom.

The pathways of unfolding and refolding of three homologous proteins are shown to be closely related. This implies that folding pathways, as well as the final folded conformation, have been largely conserved during the presumed evolutionary divergence of these proteins from a common ancestor. The pathways of the homologous proteins I and K from black mamba venom were determined here, using the disulphide interaction between their six cysteine residues to trap and identify the intermediate states, and are compared with those determined previously in the same way for the homologous bovine pancreatic trypsin inhibitor. The major one- and two-disulphide intermediates are the same with all three proteins; their kinetic roles are similar, although there are differences in the rates at which they are interconverted and in the minor intermediates that accumulate. As a consequence, different pathways may predominate with another homologous protein, even though the various most favourable pathways are the same. The energetics of the folding transitions and the stabilities of the folded states differ substantially for the three proteins. The differences in stabilities of the fully folded states are primarily reflected kinetically in the rate-determining rearrangements of the native-like conformation; the rates and equilibria of the other steps are not affected markedly. With the less stable proteins, the direct folding pathway of sequential formation of the three correct disulphide bonds becomes significant and is the most facile when considered on a solely intramolecular basis.

Effect on protein stability of reversing the charge on amino groups.

The amino groups of beta-lactoglobulins A and B, cytochrome c and ribonuclease were progressively converted to acidic groups by reaction with succinic anhydride. The mixtures of modified proteins generated in this way were analyzed by urea-gradient electrophoresis, which separates the molecules on the basis of their net charge and demonstrates visually their urea-induced unfolding transitions. Molecules succinylated to varying extents were resolved by the electrophoresis, so purification of the many modified species was not required. It is demonstrated that accurate estimates of the stability of the folded state of an individual species may be estimated very easily from its urea-gradient electrophoretic pattern. Changes in ionization of the protein upon unfolding may also be detected. The general electrostatic effect of varying the net charge on these proteins was small. Converting the normally basic ribonuclease and cytochrome c to neutral and then to acidic proteins caused the net stabilities of their folded states to vary by no more than a few kJ/mol. However, specific interactions between a few ionized groups appear to be more important in some instances. Succinylation of the 19th, and final, lysine residue of cytochrome c produced unfolding even in the absence of urea, whereas reaction of the first 18 had very little effect. Reaction of the initial amino groups of beta-lactoglobulins A and B produced a small increase in stability in a few instances, a decrease in others; modification of more than about ten groups abruptly caused unfolding in the absence of urea.