The biological potency of a series of analogues of human calcitonin correlates with their interactions with phospholipids

The conformational and lipid binding properties of several calcitonin analogs were compared. These analogs were designed to have the central amphipathic helical region of salmon calcitonin and N- and C-terminal segments similar to human calcitonin. The various analogs differed from one another either by removal of Leu19 from this hybrid analog, replacement of Leu19 with Gly19 or having a carboxyl terminus more closely related to salmon calcitonin. It had been found that replacement of Leu19 with Gly19 caused a marked reduction in the hypocalemic activity of the analog. The ability of the analogs to form helical structures in the presence of dimyristoylphosphatidylglycerol as well as their ability to lower the enthalpy of the calorimetric phase transition of this phospholipid correlates well with the hypocalcemic potency of the peptide.     

Structural determinants of salmon calcitonin bioactivity: the role of the Leu-based amphipathic alpha-helix

Salmon calcitonin (sCT) forms an amphipathic helix in the region 9-19, with the C-terminal decapeptide interacting with the helix (Amodeo, P., Motta, A., Strazzullo, G., Castiglione Morelli, M. A. (1999) J. Biomol. NMR 13, 161-174). To uncover the structural requirements for the hormone bioactivity, we investigated several sCT analogs. They were designed so as to alter the length of the central helix by removal and/or replacement of flanking residues and by selectively mutating or deleting residues inside the helix. The helix content was assessed by circular dichroism and NMR spectroscopies; the receptor binding affinity in human breast cancer cell line T 47D and the in vivo hypocalcemic activity were also evaluated. In particular, by NMR spectroscopy and molecular dynamics calculations we studied Leu(23),Ala(24)-sCT in which Pro(23) and Arg(24) were replaced by helix inducing residues. Compared with sCT, it assumes a longer amphipathic alpha-helix, with decreased binding affinity and one-fifth of the hypocalcemic activity, therefore supporting the idea of a relationship between a definite helix length and bioactivity. From the analysis of other sCT mutants, we inferred that the correct helix length is located in the 9-19 region and requires long range interactions and the presence of specific regions of residues within the sequence for high binding affinity and hypocalcemic activity. Taken together, the structural and biological data identify well defined structural parameters of the helix for sCT bioactivity.     

Sequence analysis of phospholamban. Identification of phosphorylation sites and two major structural domains

Phospholamban is a regulatory protein in cardiac sarcoplasmic reticulum that is phosphorylated by cAMP- and Ca2+/calmodulin-dependent protein kinase activities. In this report, we present the partial amino acid sequence of canine cardiac phospholamban and the identification of the sites phosphorylated by these two protein kinases. Gas-phase protein sequencing was used to identify 20 NH2-terminal residues. Overlap peptides produced by trypsin or papain digestion extended the sequence 16 residues to give the following primary structure: Ser-Ala-Ile-Arg-Arg-Ala-Ser-Thr-Ile-Glu-Met-Pro-Gln-Gln-Ala- Arg-Gln-Asn-Leu-Gln-Asn-Leu-Phe-Ile-Asn-Phe-(Cys)-Leu-Ile-Leu-Ile-(Cys)- Leu-Leu-Leu-Ile-. Phospholamban phosphorylated by either cAMP-dependent or Ca2+/calmodulin-dependent protein kinase was cleaved with trypsin, and the major phosphorylated peptide (comprising greater than 70% of the incorporated 32P label) was purified by reverse-phase high performance liquid chromatography. The identical sequence was revealed for the radioactive peptide obtained from phospholamban phosphorylated by either kinase: Arg-Ala-Ser-Thr-Ile-Glu-Met-Pro-Gln-Gln-. The adjacent residues Ser7 and Thr8 of phospholamban were identified as the unique sites phosphorylated by cAMP- and Ca2+/calmodulin-dependent protein kinases, respectively. These results establish that phospholamban is an oligomer of small, identical polypeptide chains. A hydrophilic, cytoplasmically oriented NH2-terminal domain on each monomer contains the unique, adjacent residues phosphorylated by cAMP- and Ca2+/calmodulin-dependent protein kinase activities. Analysis by hydropathic profiling and secondary structure prediction suggests that phospholamban monomers also contain a hydrophobic domain, which could form amphipathic helices sufficiently long to traverse the sarcoplasmic reticulum membrane. A model of phospholamban as a pentamer is presented in which the amphipathic alpha-helix of each monomer is a subunit of the pentameric membrane-anchored domain, which is comprised of an exterior hydrophobic surface and an interior hydrophilic region containing polar side chains.     

Design, synthesis, and characterization of a model peptide having potent calcitonin-like biological activity: implications for calcitonin structure/activity

A calcitonin analogue, MCT-II, having the potential to form an amphiphilic alpha-helix from residue 8 to residue 22 with a continuous surface of aliphatic leucine side chains on the hydrophobic face of the helix has been synthesized, and its physical and biological properties have been characterized. Properties exhibited by this peptide, including self-association in the micromolar concentration range with a concomitant increase in the percentage of alpha-helical structure, formation of stable monolayers at the air-water interface, and adsorption to the surface of egg lecithin single-bilayer vesicles, demonstrate that MCT-II can readily form an amphiphilic alpha-helical structure. Though MCT-II has minimal sequence homology to any particular natural analogue from residue 8 to residue 22, it has biological activity similar to that of salmon calcitonin I for receptor binding in brain and kidney membranes, for activation of adenylate cyclase, and in hypocalcemic potency in vivo. The amphiphilic alpha-helical structure of MCT-II, therefore, is important for binding to calcitonin receptors. It is also apparent that a hydrophilic residue commonly occurring on the hydrophobic face (position 15) in the natural calcitonins is not required for high biological activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution structure of the osteogenic 1-31 fragment of the human parathyroid hormone

The solution conformations of a selectively osteogenic 1-31 fragment of the human parathyroid hormone (hPTH), hPTH(1-31)NH(2), have been characterized by use of very high field NMR spectroscopy at 800 MHz. The combination of the CalphaH proton and (13)Calpha chemical shifts, (3)J(NH)(alpha) coupling constants, NH proton temperature coefficients, and backbone NOEs reveals that the hPTH(1-31)NH(2) peptide has well-formed helical structures localized in two distinct segments of the polypeptide backbone. There are also many characteristic NOEs defining specific side-chain/backbone and side-chain/side-chain contacts within both helical structures. The solution structure of hPTH(1-31)NH(2) contains a short N-terminal helical segment for residues 3-11, including the helix capping residues 3 and 11 and a long C-terminal helix for residues 16-30. The two helical structures are reinforced by well-defined capping motifs and side-chain packing interactions within and at both ends of these helices. On one face of the C-terminal helix, there are side-chain pairs of Glu22-Arg25, Glu22-Lys26, and Arg25-Gln29 that can form ion-pair and/or hydrogen bonding interactions. On the opposite face of this helix, there are characteristic hydrophobic interactions involving the aromatic side chain of Trp23 packing against the aliphatic side chains of Leu15, Leu24, Lys27, and Leu28. There is also a linear array of hydrophobic residues from Val2, to Leu7, to Leu11 and continuing on to residues His14 and Leu15 in the hinge region and to Trp23 in the C-terminal helix. Capping and hydrophobic interactions at the end of the N-terminal and at the beginning of the C-terminal helix appear to consolidate the helical structures into a V-shaped overall conformation for at least the folded population of the hPTH(1-31)NH(2) peptide. Stabilization of well-folded conformations in this linear 1-31 peptide fragment and possibly other analogues of human PTH may have a significant impact on the biological activities of the PTH peptides in general and specifically for the osteogenic/anabolic activities of bone-building PTH analogues.     

Molecular mapping of a histocompatibility-restricted immunodominant T cell epitope with synthetic and natural peptides: implications for T cell antigenic structure

We have prepared synthetic and natural peptides that have allowed delineation of a major antigenic site of myoglobin recognized by histocompatibility (I-Ed)-restricted T cell clones. The smallest peptide capable of stimulating T cell proliferation consisted of residues 136-146. Residues Glu 136, Lys 140, and Lys 145 were essential for antigenicity, whereas Lys 133 and Tyr 146 added potency but were not required for antigenicity. The periodicity of these residues suggests that folding the peptide into its native alpha-helical structure may be essential for antigenicity either by forming a hydrophilic binding site for the T cell receptor or by participating in antigen presentation. The same folding could also produce a hydrophobic site on the opposite side of the alpha-helix that could participate in hydrophobic interactions. The extrapolation of these findings to other known peptide antigens suggests that this tendency to form an amphipathic alpha-helix may be a general property of antigenic sites recognized by T cells, perhaps due to a different functional role of each type of site in eliciting T cell responses.     

Biologically potent analogues of salmon calcitonin which do not contain an N-terminal disulfide-bridged ring structure

The disulfide bridge formed between the cysteine residues at positions 1 and 7 of salmon calcitonin (sCT) is not required for biological activity. The analogues [Ala1,7]sCT,[AcmCys1,7]sCT and [AmcCys1,Ala7]sCT (AcmC = S-acetamido-methylcysteine) are linear sequences which retain full hypocalcemic activity in the intact rat and ability to activate adenylate cyclase of rat renal membranes. The secondary structure of these peptides in aqueous solution in the presence or absence of lipid is not greatly perturbed by the opening of the disulfide ring. In contrast with salmon calcitonin, substitution of Cys by AcmCys in human calcitonin results in greatly reduced hypocalcemic activity but no loss in the ability of the peptide to activate renal adenylate cyclase. Thus in vitro activation of adenylate cyclase by human calcitonin analogues is not always correlated with in vivo hypocalcemic potency.     

Design of potent, non-toxic antimicrobial agents based upon the structure of the frog skin peptide, pseudin-2

Pseudin-2, a naturally occurring 24 amino-acid-residue antimicrobial peptide first isolated from the skin of the South American paradoxical frog Pseudis paradoxa, has weak hemolytic and cytolytic activity but also relatively low potency against microorganisms. In a membrane-mimetic environment, the peptide exists in an amphipathic alpha-helical conformation. Analogs of the peptide with increased cationicity and alpha-helicity were chemically synthesized by progressively substituting neutral and acidic amino acid residues on the hydrophilic face of the alpha-helix by lysine. Analogs with up to three L-lysine substitutions showed increased potency against a range of gram-negative and gram-positive bacteria (up to 16-fold) whilst retaining low hemolytic activity. The analog [D-Lys3, D-Lys10, D-Lys14]pseudin-2 showed potent activity against gram-negative bacteria (minimum inhibitory concentration, MIC=5 microM against several antibiotic-resistant strains of Escherichia coli) but very low hemolytic activity (HC50>500 microM) and cytolytic activity against L929 fibroblasts (LC50=215 microM). Increasing the number of l-lysines to four and five did not enhance antimicrobial potency further but increased hemolytic activity towards human erythrocytes. Time-kill studies demonstrated that the analog [Lys3, Lys10, Lys14, Lys21]pseudin-2 at a concentration of 1 x MIC was bacteriocidal against E. coli (99.9% cell death after 96 min) but was bacteriostatic against S. aureus. Increasing the hydrophobicity of pseudin-2, while maintaining the amphipathic character of the molecule, by substitution of neutral amino acids on the hydrophobic face of the alpha-helix by L-phenylalanine, had only minor effects on antimicrobial and hemolytic activities.     

Influence of lipids on membrane assembly and stability of the potassium channel KcsA

A novel antibacterial peptide, moricin, isolated from the silkworm Bombyx mori, consists of 42 amino acids. It is highly basic and the amino acid sequence has no significant similarity to those of other antibacterial peptides. The 20 structures of moricin in methanol have been determined from two-dimensional 1H-nuclear magnetic resonance spectroscopic data. The solution structure reveals an unique structure comprising of a long alpha-helix containing eight turns along nearly the full length of the peptide except for four N-terminal residues and six C-terminal residues. The electrostatic surface map shows that the N-terminal segment of the alpha-helix, residues 5-22, is an amphipathic alpha-helix with a clear separation of hydrophobic and hydrophilic faces, and that the C-terminal segment of the alpha-helix, residues 23-36, is a hydrophobic alpha-helix except for the negatively charged surface at the position of Asp30. The results suggest that the amphipathic N-terminal segment of the alpha-helix is mainly responsible for the increase in permeability of the membrane to kill the bacteria.     

Spatial proximity between a photolabile residue in position 19 of salmon calcitonin and the amino terminus of the human calcitonin receptor

Calcitonins are 32-amino acid peptide hormones with both peripheral and central actions mediated via specific cell surface receptors, which belong to the class II subfamily of G protein-coupled receptors. Understanding receptor function, particularly in terms of ligand recognition by calcitonin receptors, may aid in the rational design of calcitonin analogs with increased potency and improved selectivity. To directly identify sites of proximity between calcitonin and its receptor, we carried out photoaffinity labeling studies followed by protein digestion and mapping of the radiolabeled photoconjugated receptor. A fully active salmon calcitonin analog [Arg(11,18),Bpa19]sCT, incorporating a photolabile p-benzoyl-L-phenylalanine into position 19 of the ligand, has been used to demonstrate spatial proximity between residue 19 of the peptide and the amino-terminal extracellular domain of the receptor. Cyanogen bromide cleavage together with endoproteinase Asp-N digestion indicated that binding was predominantly to the region delimited by receptor residues Cys134 and Met187. Binding to this fragment was supported further by cyanogen bromide-digestion of receptors that were mutated to remove the predicted cleavage site at Met133 (M133A, M133L). Binding within the 54-amino acid fragment was refined further by digestion with endoproteinase Lys-C to the 8-amino acid region corresponding to Cys134-Lys141. These results provide the first direct demonstration of a contact domain between salmon calcitonin and its receptor and will contribute toward modeling of the calcitonin-receptor interface.     

Self-assembly of amphipathic β-sheet peptides: insights and applications

Amphipathic peptides composed of alternating polar and nonpolar residues have a strong tendency to self-assemble into one-dimensional, amyloid-like fibril structures. Fibrils derived from peptides of general (XZXZ)(n) sequence in which X is hydrophobic and Z is hydrophilic adopt a putative β-sheet bilayer. The bilayer configuration allows burial of the hydrophobic X side chain groups in the core of the fibril and leaves the polar Z side chains exposed to solvent. This architectural arrangement provides fibrils that maintain high solubility in water and has facilitated the recent exploitation of self-assembled amphipathic peptide fibrils as functional biomaterials. This article is a critical review of the development and application of self-assembling amphipathic peptides with a focus on the fundamental insight these types of peptides provide into peptide self-assembly phenomena.     

Solution structure of a bent alpha-helix

We present the solution structure determination of a peptide with sequence Ac-WEAQAREALAKEAQARA-NH2, using NMR data. The peptide has a high population of a stable alpha-helical structure in the middle with fraying ends. In addition, our data are consistent with the presence of several other transient and interconverting conformers. The peptide sequence was designed using amino acids that have propensity for alpha-helix specificity. The presence of E-R/K (i, i + 4) ion pairs was expected to enhance the stability of the alpha-helix by introducing favorable electrostatic interactions at the side chain level, in addition to the characteristic backbone (i, i + 4) hydrogen bonds. The NMR structural ensemble demonstrates competition between E-R/K (i, i + 4) and (i, i - 1) ion pair formation, with the (i, i - 1) interactions being dominant. The relative topologies of the charged side chains demonstrate flexibility and overall compromised favorable medium/long-range electrostatic interactions. The peptide alpha-helix is bent despite the lack of an amphipathic sequence. Bending is introduced by a strong (i, i + 8) hydrophobic interaction between the side chains of N-terminal tryptophan and leucine at the middle of the peptide sequence.     

The effects of the side chains of hydrophobic aliphatic amino acid residues in an amphipathic polypeptide on the formation of alpha helix and its association

The polypeptide alpha3, which was synthesized by us to produce an amphipathic helix structure, contains the regular three times repeated sequence (LETLAKA)(3), and alpha3 forms a fibrous assembly. To clarify how the side chains of amino acid residues affect the formation of alpha helix, Leu residues, which are located in the hydrophobic surface of an amphipathic helix, were replaced by other hydrophobic aliphatic amino acid residues systematically, and the characters of the resulting polypeptides were studied. According to the circular dichroism (CD) spectra, the Ile-substituted polypeptides formed alpha helix like alpha3. However, their helix formation ability was weaker than that of alpha3 under some conditions. The Val-substituted polypeptides formed alpha helix only under restricted condition. The Ala-substituted polypeptides did not form alpha helix under any condition. Thus, it is clear that the order of the alpha helix formation ability is as follows: Leu >or= Ile > Val > Ala. The formation of alpha helix was confirmed by Fourier Transform Infrared (FTIR) spectra. Through electron microscopic observation, it was clarified that the formation of the alpha helix structure correlates with the formation of a fibrous assembly. The amphipathic alpha helix structure would be stabilized by the formation of the fibrous assembly.     

Mouse interleukin-2 structure-function studies: substitutions in the first alpha-helix can specifically inactivate p70 receptor binding and mutations in the fifth alpha-helix can specifically inactivate p55 receptor binding.

The function of two alpha-helical regions of mouse interleukin-2 were analyzed by saturation substitution analysis. The functional parts of the first alpha-helix (A) was defined as residues 31-39 by the observation that proline substitutions within this region inactivate the protein. Four residues within alpha-helix A, Leu31, Asp34, Leu35 and Leu38, were found to be crucial for biological activity. Structural modeling suggested that these four residues are clustered on one face of alpha-helix A. Residues 31 and 35 had to remain hydrophobic for the molecule to be functional. At residue 38 there was a preference for hydrophobic side chain residues, while at residue 34 some small side chain residues as well as acidic or amide side chain residues were functionally acceptable. Inactivating changes at residue 34 had no effect upon the ability of the protein to interact with the p55 receptor. Disruption of the fifth alpha-helix (E), which had little effect upon biological activity, resulted in an inability of the protein to interact with the p55 receptor. Mutagenesis of the alpha-helix E region demonstrated that alpha-helicity and the nature of the side chain residues in this region were unimportant for biological activity. The region immediately proximal to alpha-helix E was important only for the single intramolecular disulfide linkage.     

Stability of protein-bound conformations of bioactive peptides: the folded conformation of an epidermal growth factor-like thrombomodulin fragment is similar to that recognized by thrombin

The relationship between the free and bound conformations of bioactive peptides is explored using the epidermal growth factor (EGF)-like thrombomodulin fragment hTM409-426 as a model system. The hTM409-426 peptide has a sequence of C(409)PEGYILDDGFIC(421)TDIDE (with a disulfide bond between Cys409 and Cys421) and is a selective inhibitor of thrombin. Upon binding to thrombin, hTM409-426 adopts a well-defined conformation-namely, a beta-turn followed by an antiparallel beta-sheet, similar to those found in all other EGF-like protein repeats (Hrabal et al., Protein Science, 1996, Vol. 5, 195-203). Here we demonstrate that, at pH 6.8 and at 25 degrees C, the hTM409-426 peptide in the free state is very flexible, but still populates a type II beta-turn over residues Pro410-Glu411-Gly412-Tyr413 and the clustering of some hydrophobic side chains, both of which are present in the thrombin-bound conformation. At a lower temperature of 5 degrees C, significant conformational shifts of the C alpha H proton resonances and extensive medium- and long-range NOEs are observed, indicating the presence of folded conformations with unique backbone-backbone and side-chain interactions. A comparison of the NOE patterns in the free state with transferred NOEs shows that the free-state folded and the thrombin-bound conformations of the hTM409-426 peptide are very similar, particularly over residues Pro410-Ile424. The folded conformation of hTM409-426 appears to be stabilized by two hydrophobic clusters, one formed by the side chains of residues Pro410, Tyr413, Leu415, and Phe419 and the Cys409-Cys421 disulfide bond, the second involving residues Ile414 and Ile424. These results indicate that the overall topology of the thrombin-bound conformation of the hTM409-426 peptide is prefolded in the free state and the primary sequence (including the disulfide bond) may be selective for an ensemble of conformations similar to that recognized by thrombin.     

Novel lactoferrampin antimicrobial peptides derived from human lactoferrin

Human lactoferrampin is a novel antimicrobial peptide found in the cationic N-terminal lobe of the iron-binding human lactoferrin protein. The amino acid sequence that directly corresponds to the previously characterized bovine lactoferrin-derived lactoferrampin peptide is inactive on its own (WNLLRQAQEKFGKDKSP, residues 269-285). However, by increasing the net positive charge near the C-terminal end of human lactoferrampin, a significant increase in its antibacterial and Candidacidal activity was obtained. Conversely, the addition of an N-terminal helix cap (sequence DAI) did not have any appreciable effect on the antibacterial or antifungal activity of human lactoferrampin peptides, even though it markedly influenced that of bovine lactoferrampin. The solution structure of five human lactoferrampin variants was determined in SDS micelles and all of the structures display a well-defined amphipathic N-terminal helix and a flexible cationic C-terminus. Differential scanning calorimetry studies indicate that this peptide is capable of inserting into the hydrophobic core of a membrane, while fluorescence spectroscopy results suggest that a hydrophobic patch encompassing the single Trp and Phe residues as well as Leu, Ile and Ala side chains mediates the interaction between the peptide and the hydrophobic core of a phospholipid bilayer.     

Effects of glycosylation on the structure and dynamics of eel calcitonin in micelles and lipid bilayers determined by nuclear magnetic resonance spectroscopy.

The three-dimensional structures of eel calcitonin (CT) and two glycosylated CT derivatives, [Asn(GlcNAc)3]-CT (CT-GlcNAc) and [Asn(Man6-GlcNAc2)3]-CT (CT-M6), in micelles were determined by solution NMR spectroscopy. The topologies of these peptides associated with oriented lipid bilayers were determined with solid-state NMR. All of the peptides were found to have an identical conformation in micelles characterized by an amphipathic alpha-helix consisting of residues Ser5 through Leu19 followed by an unstructured region at the C-terminus. The overall conformation of the peptide moiety was not affected by the glycosylation. Nevertheless, comparison of the relative exchange rates of the Leu12 amide proton might suggest the possibility that fluctuations of the alpha-helix are reduced by glycosylation. The presence of NOEs between the carbohydrate and the peptide moieties of CT-GlcNAc and CT-M6 and the amide proton chemical shift data suggested that the carbohydrate interacted with the peptide, and this might account for the conformational stabilization of the alpha-helix. Both the unmodified CT and the glycosylated CT were found to have orientations with their helix axes parallel to the plane of the lipid bilayers by solid-state NMR spectroscopy.     

The effects of selective amino acid substitution upon neuropeptide Y antisecretory potency in rat jejunum mucosa

The antisecretory potency of NPY and a series of truncated and structural analogues of NPY have been tested upon mucosal preparations of rat small intestine. Single amino acid substitutions, i.e., [Ile34]NPY, [Pro34]NPY, resulted in severe attenuation and loss of biological activity, respectively, and neither peptide affected NPY responses. An agonist order of potency: NPY greater than or equal to [Glu16,Ser18,Ala22,Leu28,31]NPY (ESALL-NPY) greater than [Cys2,Aoc5-24,DCys27]NPY (C2-NPY) greater than [Aoc5-24]NPY greater than [Des-Ser3,Des- Lys4]C2-NPY much greater than [Cys5,Aoc7-20,DCys24]NPY (C5-NPY) greater than equal to [DCys7,Aoc8-17, Cys20]NPY (C7-NPY) greater than [Aoc8-17]NPY greater than or equal to [Ile34]C7-NPY much greater than [Aoc2-27]NPY much greater than [Pro34]C2-NPY was obtained. The use of analogues based upon the tertiary structural model of NPY with varying amounts of N- and C-terminal helical regions removed and replaced with a single 8-aminooctanoic acid residue (Aoc) has allowed us to assess the structural requirements for activation of the regions in close apposition to each other. The polyproline helix, beta-turn and majority of the amphipathic alpha-helix serve a structural role bringing N- and C-terminal residues together for optimal receptor recognition and activation.     

Sequential 1H NMR assignment and secondary structure determination of salmon calcitonin in solution

Salmon calcitonin (sCT) has been investigated by NMR at 500 MHz in a 90% DMSOd6-10% 1H2O (v/v) mixture at 278 K. All backbone and side-chain resonances of the hormone have been assigned by using high-resolution phase-sensitive two-dimensional techniques. Analysis of the type and magnitude of the observed sequential nuclear Overhauser effects, the NH-alpha CH spin-spin coupling constants, and the 1H/2H exchange kinetics measured in 80% DMSOd6-20% 2H2O (v/v) at 278 K enabled prediction of the secondary structure. Overall, an extended conformation is the dominant feature of the solution, but there are clear indications for a short double-stranded antiparallel beta sheet in the central region comprising residues 12-18, connected by a three-residue hairpin loop formed by residues 14-16. Two tight turns, made by residues 6-9 and 25-28, were also identified, but no evidence was found for the presence of a regular helical segment. The beta sheet favors an amphipathic distribution of the residues, orienting the predominantly hydrophilic Ser13, Glu15, and His17 side chains above the plane of the sheet, and the predominantly hydrophobic Leu12, Gln14, and Leu16 below it. This is interpreted as the "seed" of the amphipathic alpha helix postulated to be responsible for the interaction of sCT with lipids, a situation reminiscent of the folding mechanism of signal peptides in the interaction with membranes. The possible significance of the cis-trans Pro23 isomerism is discussed.     

Analysis of side-chain organization on a refined model of charybdotoxin: structural and functional implications.

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.