Membrane-insertion fragments of Bcl-xL, Bax, and Bid

Apoptosis regulators of the Bcl-2 family associate with intracellular membranes from mitochondria and the endoplasmic reticulum, where they perform their function. The activity of these proteins is related to the release of apoptogenic factors, sequestered in the mitochondria, to the cytoplasm, probably through the formation of ion and/or protein transport channels. Most of these proteins contain a C-terminal putative transmembrane (TM) fragment and a pair of hydrophobic alpha helices (alpha5-alpha6) similar to the membrane insertion fragments of the ion-channel domain of diphtheria toxin and colicins. Here, we report on the membrane-insertion properties of different segments from antiapoptotic Bcl-x(L) and proapoptotic Bax and Bid, that correspond to defined alpha helices in the structure of their soluble forms. According to prediction methods, there are only two putative TM fragments in Bcl-x(L) and Bax (the C-terminal alpha helix and alpha-helix 5) and one in activated tBid (alpha-helix 6). The rest of their sequence, including the second helix of the pore-forming domain, displays only weak hydrophobic peaks, which are below the prediction threshold. Subsequent analysis by glycosylation mapping of single alpha-helix segments in a model chimeric system confirms the above predictions and allows finding an extra TM fragment made of helix alpha1 of Bax. Surprisingly, the amphipathic helices alpha6 of Bcl-x(L) and Bax and alpha7 of Bid do insert in membranes only as part of the alpha5-alpha6 (Bcl-x(L) and Bax) or alpha6-alpha7 (Bid) hairpins but not when assayed individually. This behavior suggests a synergistic insertion and folding of the two helices of the hairpin that could be due to charge complementarity and additional stability provided by turn-inducing residues present at the interhelical region. Although these data come from chimeric systems, they show direct potentiality for acquiring a membrane inserted state. Thus, the above fragments should be considered for the definition of plausible models of the active, membrane-bound species of Bcl-2 proteins.     

Ribosomal protein L7/L12 has a helix-turn-helix motif similar to that found in DNA-binding regulatory proteins.

Inspection of the structure of the C-terminal domain of ribosomal protein L7/L12 (1) reveals a helix-turn-helix motif similar to the one found in many DNA-binding regulatory proteins (2-5). The 19 alpha-carbon atoms of the L7/L12 alpha-helices superimpose on the DNA binding helices of CAP and cro with root-mean-square distances between corresponding alpha carbons of 1.45 and 1.55 A, respectively. These helices in L7/L12 are within a patch of highly conserved residues on the surface of L7/L12 whose role is as yet uncertain. We raise the possibility that they may constitute a binding site for nucleic acids, most probably RNA. Consistent with this hypothesis are calculations of the electrostatic charge potential surrounding the protein, which show a region of positive potential centered on the first of these helices.     

A novel dimeric structure of the RimL Nalpha-acetyltransferase from Salmonella typhimurium

RimL is responsible for converting the prokaryotic ribosomal protein from L12 to L7 by acetylation of its N-terminal amino group. We demonstrate that purified RimL is capable of posttranslationally acetylating L12, exhibiting a V(max) of 21 min(-1). We have also determined the apostructure of RimL from Salmonella typhimurium and its complex with coenzyme A, revealing a homodimeric oligomer with structural similarity to other Gcn5-related N-acetyltransferase superfamily members. A large central trough located at the dimer interface provides sufficient room to bind both L12 N-terminal helices. Structural and biochemical analysis indicates that RimL proceeds by single-step transfer rather than a covalent-enzyme intermediate. This is the first structure of a Gcn5-related N-acetyltransferase family member with demonstrated activity toward a protein N(alpha)-amino group and is a first step toward understanding the molecular basis for N(alpha)acetylation and its function in cellular regulation.     

Pi-turns in proteins and peptides: Classification, conformation, occurrence, hydration and sequence.

The i + 5-->i hydrogen bonded turn conformation (pi-turn) with the fifth residue adopting alpha L conformation is frequently found at the C-terminus of helices in proteins and hence is speculated to be a "helix termination signal." An analysis of the occurrence of i + 5-->i hydrogen bonded turn conformation at any general position in proteins (not specifically at the helix C-terminus), using coordinates of 228 protein crystal structures determined by X-ray crystallography to better than 2.5 A resolution is reported in this paper. Of 486 detected pi-turn conformations, 367 have the (i + 4)th residue in alpha L conformation, generally occurring at the C-terminus of alpha-helices, consistent with previous observations. However, a significant number (111) of pi-turn conformations occur with (i + 4)th residue in alpha R conformation also, generally occurring in alpha-helices as distortions either at the terminii or at the middle, a novel finding. These two sets of pi-turn conformations are referred to by the names pi alpha L and pi alpha R-turns, respectively, depending upon whether the (i + 4)th residue adopts alpha L or alpha R conformations. Four pi-turns, named pi alpha L'-turns, were noticed to be mirror images of pi alpha L-turns, and four more pi-turns, which have the (i + 4)th residue in beta conformation and denoted as pi beta-turns, occur as a part of hairpin bend connecting twisted beta-strands. Consecutive pi-turns occur, but only with pi alpha R-turns. The preference for amino acid residues is different in pi alpha L and pi alpha R-turns. However, both show a preference for Pro after the C-termini. Hydrophilic residues are preferred at positions i + 1, i + 2, and i + 3 of pi alpha L-turns, whereas positions i and i + 5 prefer hydrophobic residues. Residue i + 4 in pi alpha L-turns is mainly Gly and less often Asn. Although pi alpha R-turns generally occur as distortions in helices, their amino acid preference is different from that of helices. Poor helix formers, such as His, Tyr, and Asn, also were found to be preferred for pi alpha R-turns, whereas good helix former Ala is not preferred. pi-Turns in peptides provide a picture of the pi-turn at atomic resolution. Only nine peptide-based pi-turns are reported so far, and all of them belong to pi alpha L-turn type with an achiral residue in position i + 4. The results are of importance for structure prediction, modeling, and de novo design of proteins.     

Cooperative effects in hydrogen-bonding of protein secondary structure elements: a systematic analysis of crystal data using Secbase

A systematic analysis of the hydrogen-bonding geometry in helices and beta sheets has been performed. The distances and angles between the backbone carbonyl O and amide N atoms were correlated considering more than 1500 protein chains in crystal structures determined to a resolution better than 1.5 A. They reveal statistically significant trends in the H-bond geometry across the different secondary structural elements. The analysis has been performed using Secbase, a modular extension of Relibase (Receptor Ligand Database) which integrates information about secondary structural elements assigned to individual protein structures with the various search facilities implemented into Relibase. A comparison of the mean hydrogen-bond distances in alpha helices and 3(10) helices of increasing length shows opposing trends. Whereas in alpha helices the mean H-bond distance shrinks with increasing helix length and turn number, the corresponding mean dimension in 3(10) helices expands in a comparable series. Comparing similarly the hydrogen-bond lengths in beta sheets there is no difference to be found between the mean H-bond length in antiparallel and parallel beta sheets along the strand direction. In contrast, an interesting systematic trend appears to be given for the hydrogen bonds perpendicular to the strands bridging across an extended sheet. With increasing number of accumulated strands, which results in a growing number of back-to-back piling hydrogen bonds across the strands, a slight decrease of the mean H-bond distance is apparent in parallel beta sheets whereas such trends are obviously not given in antiparallel beta sheets. This observation suggests that cooperative effects mutually polarizing spatially well-aligned hydrogen bonds are present either in alpha helices and parallel beta sheets whereas such influences seem to be lacking in 3(10) helices and antiparallel beta sheets.     

A fifth protein subunit Ph1496p elevates the optimum temperature for the ribonuclease P activity from Pyrococcus horikoshii OT3

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5' leader sequence of precursor tRNA. We previously found that the reconstituted particle (RP) composed of RNase P RNA and four proteins (Ph1481p, Ph1601p, Ph1771p, and Ph1877p) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 exhibited the RNase P activity, but had a lower optimal temperature (around at 55 degrees C), as compared with 70 degrees C of the authentic RNase P from P. horikoshii [Kouzuma et al., Biochem. Biophys. Res. Commun. 306 (2003) 666-673]. In the present study, we found that addition of a fifth protein Ph1496p, a putative ribosomal protein L7Ae, to RP specifically elevated the optimum temperature to about 70 degrees C comparable to that of the authentic RNase P. Characterization using gel shift assay and chemical probing localized Ph1496p binding sites on two stem-loop structures encompassing nucleotides A116-G201 and G229-C276 in P. horikoshii RNase P RNA. Moreover, the crystal structure of Ph1496p was determined at 2.0 A resolution by the molecular replacement method using ribosomal protein L7Ae from Haloarcula marismortui as a search model. Ph1496p comprises five alpha-helices and a four stranded beta-sheet. The beta-sheet is sandwiched by three helices (alpha1, alpha4, and alpha5) at one side and two helices (alpha2 and alpha3) at other side. The archaeal ribosomal protein L7Ae is known to be a triple functional protein, serving as a protein component in ribosome and ribonucleoprotein complexes, box C/D, and box H/ACA. Although we have at present no direct evidence that Ph1496p is a real protein component in the P. horikoshii RNase P, the present result may assign an RNase P protein to L7Ae as a fourth function.     

Structural and sequence characteristics of long alpha helices in globular proteins.

Elucidation of the detailed structural features and sequence requirements for alpha helices of various lengths could be very important in understanding secondary structure formation in proteins and, hence, in the protein folding mechanism. An algorithm to characterize the geometry of an alpha helix from its C(alpha) coordinates has been developed and used to analyze the structures of long alpha helices (number of residues > or = 25) found in globular proteins, the crystal structure coordinates of which are available from the Brookhaven Protein Data Bank. All long alpha helices can be unambiguously characterized as belonging to one of three classes: linear, curved, or kinked, with a majority being curved. Analysis of the sequences of these helices reveals that the long alpha helices have unique sequence characteristics that distinguish them from the short alpha helices in globular proteins. The distribution and statistical propensities of individual amino acids to occur in long alpha helices are different from those found in short alpha helices, with amino acids having longer side chains and/or having a greater number of functional groups occurring more frequently in these helices. The sequences of the long alpha helices can be correlated with their gross structural features, i.e., whether they are curved, linear, or kinked, and in case of the curved helices, with their curvature.     

Major structural determinants of transmembrane proteins identified by principal component analysis

We identify amino acid characteristics important in determining the secondary structures of transmembrane proteins, and compare them with characteristics important for cytoplasmic proteins. Using information derived from multiple sequence alignments, we perform a principal component analysis (PCA) to identify the directions in the 20-dimensional amino acid frequency space that comprise the most variance within each protein secondary structure. These vectors represent the important position-specific properties of the amino acids for coils, turns, beta sheets, and alpha helices. As expected, the most important axis for most of the datasets was hydrophobicity. Additional axes, distinct from hydrophobicity, are surprising, especially in the case of transmembrane alpha helices, where the effects of aromaticity and beta-branching are the next two most significant characteristics. The axis representing beta-branching also has equal importance in cytoplasmic and transmembrane helices, a finding that contrasts with some experimental results in membrane-like environments. In a further analysis, we examine trends for some of the PCA axes over averaged transmembrane alpha helices, and find interesting results for aromaticity.     

A comparative study of peptide models of the alpha-domain of alpha-lactalbumin, lysozyme, and alpha-lactalbumin/lysozyme chimeras allows the elucidation of critical factors that contribute to the ability to form stable partially folded states

alpha-Lactalbumin (alpha LA) forms a well-populated equilibrium molten globule state, while the homologous protein hen lysozyme does not. alpha LA is a two-domain protein and the alpha-domain is more structured in the molten globule state than is the beta-domain. Peptide models derived from the alpha-subdomain that contain the A, B, D, and 3(10) helices of alpha LA are capable of forming a molten globule state in the absence of the remainder of the protein. Here we report comparative studies of a peptide model derived from the same region of hen lysozyme and a set of chimeric alpha-lactalbumin--lysozyme constructs. Circular dichroism, dynamic light scattering, sedimentation equilibrium, and fluorescence experiments indicate that the lysozyme construct does not fold. Chimeric constructs were prepared to probe the origins of the difference in the ability of the two isolated subdomains to fold. The first consists of the A and B helices of alpha LA cross-linked to the D and C-terminal 3(10) helices of lysozyme. This construct is highly helical, while a second construct that contains the A and B helices of lysozyme cross-linked to the D and 3(10) helices of alpha LA does not fold. Furthermore, the disulfide cross-linked homodimer of the alpha LA AB peptide is helical, while the homodimer of the lysozyme AB peptide is unstructured. Thus, the AB helix region of alpha LA appears to have an intrinsic ability to form structure as long as some relatively nonspecific interactions can be made with other regions of the protein. Our studies show that the A and B helices plays a key role in the ability of the respective alpha-subdomains to fold.     

Molecular basis for the structural instability of human DJ-1 induced by the L166P mutation associated with Parkinson's disease

DJ-1 is a dimeric protein of unknown function in vivo. A mutation in the human DJ-1 gene causing substitution of proline for leucine at residue 166 (L166P) has been linked to early onset Parkinson's disease. Lack of structural stability has precluded experimental determination of atomic-resolution structures of the L166P DJ-1 polymorph. We have performed multiple molecular dynamics (MD) simulations ( approximately 1/3 mus) of the wild-type and L166P DJ-1 polymorph at physiological temperature to predict specific structural effects of the L166P substitution. L166P disrupted helices alpha1, alpha5, alpha6 and alpha8 with alpha8 undergoing particularly severe disruption. Secondary structural elements critical for protein stability and dimerization were significantly disrupted across the entire dimer interface, as were extended hydrophobic surfaces involved in dimer formation. Relative to wild-type DJ-1, L166P DJ-1 populated a broader ensemble of structures, many of which corresponded to distorted conformations. In a L166P dimer model the substitution significantly destabilized the dimer interface, interrupting >100 intermolecular contacts that are important for dimer formation. The L166P substitution also led to major perturbations in the region of a highly conserved cysteine residue (Cys-106) that participates in dimerization and that is critical for a proposed chaperone function of DJ-1. Cys-106 is located approximately 16 A from the substitution site, demonstrating that structural disruptions propagate throughout the whole protein. Furthermore, L166P DJ-1 showed a significant increase in hydrophobic surface area relative to wild-type protein, possibly explaining the tendency of the mutant protein to aggregate. These simulations provide details about specific structural disturbances throughout L166P DJ-1 that previous studies have not revealed.     

Variants of 3(10)-helices in proteins

An analysis of the shortest 3(10)-helices, containing three helical residues and two flanking capping residues that participate in two consecutive i + 3 --> i hydrogen bonds, shows that not all helices belong to the classic 3(10)-helix, where the three central residues adopt the right-handed helical conformation (alpha(R)). Three variants identified are: 3L10-helix with all residues in the left-handed helical region (alpha(L)), 3EL10-helix where the first residue is in the extended region followed by two residues in the alpha(L) conformation, and its mirror-image, the 3E'R10-helix. In the context of these helices, as well as the equivalent variants of alpha-helices, the length dependence of the handedness of secondary structures in protein structure is discussed. There are considerable differences in the amino acid preferences at different positions in the various types of 3(10)-helices. Each type of 3(10)-helix can be thought to be made up of an extension of a particular type of beta-turn (made up of residues i to i + 3) such that the (i + 3)th residue assumes the same conformation as the preceding residue. Distinct residue preferences at i and i + 3 positions seem to decide whether a particular stretch of four residues will be a beta-turn or a 3(10)-helix in the folded structure.     

Nuclear magnetic resonance assignment and secondary structure of an ankyrin-like repeat-bearing protein: myotrophin.

Multidimensional heteronuclear NMR has been applied to the structural analysis of myotrophin, a novel protein identified from spontaneously hypertensive rat hearts and hypertrophic human hearts. Myotrophin has been shown to stimulate protein synthesis in myocytes and likely plays an important role in the initiation of cardiac hypertrophy, a major cause of mortality in humans. Recent cDNA cloning revealed that myotrophin has 11B amino acids containing 2.5 contiguous ANK repeats, a motif known to be involved in a wide range of macromolecular recognition. A series of two- and three-dimensional heteronuclear bond correlation NMR experiments have been performed on uniformly 15N-labeled or uniformly 15N/13C-labeled protein to obtain the 1H, 15N, and 13C chemical shift assignments. The secondary structure of myotrophin has been determined by a combination of NOEs, NH exchange data, 3JHN alpha coupling constants, and chemical shifts of 1H alpha, 13C alpha, and 13 C beta. The protein has been found to consist of seven helices, all connected by turns or loops. Six of the seven helices (all but the C-terminal helix) form three separate helix-turn-helix motifs. The two full ANK repeats in myotrophin are characteristic of multiple turns followed by a helix-turn-helix motif. A hairpin-like turn involving L32-R36 in ANK repeat #1 exhibits slow conformational averaging on the NMR time scale and appears dynamically different from the corresponding region (D65-169) of ANK repeat #2.     

The solution structure of FADD death domain. Structural basis of death domain interactions of Fas and FADD.

A signal of Fas-mediated apoptosis is transferred through an adaptor protein Fas-associated death domain protein (FADD) by interactions between the death domains of Fas and FADD. To understand the signal transduction mechanism of Fas-mediated apoptosis, we solved the solution structure of a murine FADD death domain. It consists of six helices arranged in a similar fold to the other death domains. The interactions between the death domains of Fas and FADD analyzed by site-directed mutagenesis indicate that charged residues in helices alpha2 and alpha3 are involved in death domain interactions, and the interacting helices appear to interact in anti-parallel pattern, alpha2 of FADD with alpha3 of Fas and vice versa.     

Crosslinking between alpha and beta subunits defines the orientation and spatial relationship of some of the transmembrane helices of the proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli

The proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli is composed of two types of subunits, alpha and beta, organized as an alpha(2)beta(2) tetramer. The protein contains three recognizable domains, of which domain II is the transmembrane region of the molecule containing the pathway for proton translocation. Domain II is composed of four transmembrane helices at the carboxyl-terminus of the alpha subunit and either eight or nine transmembrane helices at the amino-terminal region of the beta subunit. We have introduced pairs of cysteine residues into a cysteine-free transhydrogenase by site-directed mutagenesis. Disulfide bond formation between some of these cysteine residues occurred spontaneously or on treatment with cupric 1, 10-phenanthrolinate. Analysis of crosslinked products confirmed that there are nine transmembrane helices in the domain II region of the beta subunit. The proximity to one another of several of the transmembrane helices was determined. Thus, helices 2 and 4 are close to helix 6 (nomenclature of Meuller and Rydstr?m, J. Biol. Chem. 274, 19072-19080, 1999), and helix 3 and the carboxyl-terminal eight residues of the alpha subunit are close to helix 7. In the alpha(2)beta(2) tetramer, helices 2 and 4 of one alpha subunit are close to the same pair of transmembrane helices of the other alpha subunit, and helix 6 of one beta subunit is close to helix 6 of the other beta subunit.     

BCL-XL dimerization by three-dimensional domain swapping

Dimeric interactions among anti- and pro-apoptotic members of the BCL-2 protein family are dynamically regulated and intimately involved in survival and death functions. We report the structure of a BCL-X(L) homodimers a 3D-domain swapped dimer (3DDS). The X-ray crystal structure demonstrates the mutual exchange of carboxy-terminal regions including BH2 (Bcl-2 homology 2) between monomer subunits, with the hinge region occurring at the hairpin turn between the fifth and sixth alpha helices. Both BH3 peptide-binding hydrophobic grooves are unoccupied in the 3DDS dimer and available for BH3 peptide binding, as confirmed by sedimentation velocity analysis. BCL-X(L) 3DDS dimers have increased pore-forming activity compared to monomers, suggesting that 3DDS dimers may act as intermediates in membrane pore formation. Chemical crosslinking studies of Cys-substituted BCL-X(L) proteins demonstrate that 3DDS dimers form in synthetic lipid vesicles.     

Structural features of the scaffold interaction domain at the N terminus of the major capsid protein (VP5) of herpes simplex virus type 1

Protein-protein interactions drive the assembly of the herpes simplex virus type 1 capsid. A key interaction occurs between the C terminus of the scaffold protein and the N terminus of the major capsid protein (VP5). Results from alanine-scanning mutagenesis of hydrophobic residues in the N terminus of VP5 revealed seven residues (I27, L35, F39, L58, L65, L67, and L71) that reside in two predicted alpha helices (helix 1(22-42) and helix 2(58-72)) that are important for this bimolecular interaction. The goal of the present study was to further characterize the VP5 scaffold interaction domain (SID). Amino acids at the seven positions were replaced with L, M, V or P (I27); I, M, V, or P (L35, L58, L65, L67, and L71); and H, W, Y, or L (F39). Replacement with a hydrophobic side chain did not affect the interaction with scaffold protein in yeast cells or the ability of a virus specifying the mutation from replicating in cells. The mutation to the proline side chain abolished the interaction in all cases and was lethal for virus replication. Mutant viruses with proline substitutions in helix 1(22-42) at positions 27 and 35 assembled large open capsid shells that did not attain closure. Proline substitutions in helix 2(58-72) at either position 59, 65, or 67 abolished the accumulation of VP5 protein, and, at 58 and 71, although VP5 did accumulate, capsid shells were not assembled. Thus, the second SID, SID2, is highly structured, and this alpha helix (helix 2(58-72)) is likely involved in capsomere-capsomere interactions during shell accretion. Conserved glycine G59 in helix 2(58-72) was also mutated. G59 may act as a flexible "hinge" in helix 2(58-72) because decreasing the movement of this side chain by replacement with valine impaired capsid assembly. Thus, the N terminus of VP5 and the alpha helices embedded in this domain, as in the capsid shell proteins of some double-stranded DNA phages, are a key regulator of shell accretion and stabilization.     

Dissection of the de novo designed peptide alpha t alpha: stability and properties of the intact molecule and its constituent helices

Alpha t alpha is a de novo designed 38-residue peptide [Fezoui et al. (1995) Protein Sci. 4, 286-295] that adopts a helical hairpin conformation in solution [Fezoui et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3675-3679; Fezoui et al. (1997) Protein Sci. 6, 1869-1877]. Since alpha t alpha was developed as a model system for protein folding at the stage where secondary structures interact and become mutually stabilizing, it is of interest to investigate the increase in stability that occurs with helix association. alpha t alpha was dissected into its component helices and the relative stabilities of the individual helices and the parent molecule were assessed. The Delta G0 of unfolding of alpha t alpha measured by guanidine hydrochloride denaturation was determined to be 3.4 kcal/mol. The equilibrium constant for folding of alpha t alpha was estimated from the Delta G0 as 338 and from hydrogen exchange measurements as 259. The stability of the helices in intact alpha t alpha over the individual helices increased by a factor of at least 37 based on amide proton exchange measurements. Sedimentation equilibrium studies showed very little association of the peptides to form either homo- or heterodimers, suggesting that helix association is stabilized by the high effective concentration of the helices caused by the presence of the connecting turn. The effects of salt and pH on the helicity of the component peptides are largely reflected in the intact molecule, implying that short-range interactions still make important contributions to the conformation of the intact molecule even though significant stabilization is caused by helix association.     

Conformation of alpha zeins in solid state by Fourier transform IR

The major maize storage proteins (alpha zeins) are deposited as an insoluble mass in the protein bodies of the endosperm. Because they are insoluble in water, most structural studies are performed in alcohol solutions. To solve the question raised by several authors about denaturation of the alpha zein structure by alcohol, we analyze the secondary structure of alpha zeins prepared with and without solubilization in alcohol (corn gluten meal and protein bodies with high concentrations of alpha zeins and traces of beta zeins). The secondary structures of alpha zeins are analyzed in the solid state by Fourier transform IR spectroscopy (FTIR) in KBr pellets and solid-state 13C-NMR spectroscopy. The proportion of secondary structures obtained by FTIR of alpha zeins prepared with and without solubilization in alcohol yield almost identical proportions of alpha helices and beta sheets. The proportion of alpha helices (43%) agrees with that measured by circular dichroism in an alcohol solution. However, the proportion of beta sheets (28%) is higher than the one measured by the same technique. Gluten and protein body samples with high beta zein content showed higher beta sheet and lower alpha helix proportions than that obtained for alpha zein preparations. The solid-state 13C-NMR spectra show the carbonyl peak for the alpha zeins at delta 176 and for the sample rich in beta zeins at delta 172, which demonstrates the presence of a high content of alpha helices and beta sheets, respectively. These results indicate that alcohol solubilization does not affect the conformation of alpha zeins, validating the secondary structure measurements in solution.     

Insertion and organization within membranes of the delta-endotoxin pore-forming domain, helix 4-loop-helix 5, and inhibition of its activity by a mutant helix 4 peptide

The pore-forming domain of Bacillus thuringiensis Cry1Ac insecticidal protein comprises of a seven alpha-helix bundle (alpha1-alpha7). According to the "umbrella model," alpha4 and alpha5 helices form a hairpin structure thought to be inserted into the membrane upon binding. Here, we have synthesized and characterized the hairpin domain, alpha4-loop-alpha5, its alpha4 and alpha5 helices, as well as mutant alpha4 peptides based on mutations that increased or decreased toxin toxicity. Membrane permeation studies revealed that the alpha4-loop-alpha5 hairpin is extremely active compared with the isolated helices or their mixtures, indicating the complementary role of the two helices and the need for the loop for efficient insertion into membranes. Together with spectrofluorometric studies, we provide direct evidence for the role of alpha4-loop-alpha5 as the membrane-inserted pore-forming hairpin in which alpha4 and alpha5 line the lumen of the channel and alpha5 also participates in the oligomerization of the toxin. Strikingly, the addition of the active alpha4 mutant peptide completely inhibits alpha4-loop-alpha5 pore formation, thus providing, to our knowledge, the first example that a mutated helix within a pore can function as an "immunity protein" by directly interacting with the segments that form the pore. This presents a potential means of interfering with the assembly and function of other membrane proteins as well.