Localization of prefoldin interaction sites in the hyperthermophilic group II chaperonin and correlations between binding rate and protein transfer rate

Prefoldin is a molecular chaperone that captures a protein-folding intermediate and transfers it to a group II chaperonin for correct folding. The manner by which prefoldin interacts with a group II chaperonin is poorly understood. Here, we have examined the prefoldin interaction site in the archaeal group II chaperonin, comparing the interaction of two Thermococcus chaperonins and their mutants with Pyrococcus prefoldin by surface plasmon resonance. We show that the mutations of Lys250 and Lys256 of Thermococcus alpha chaperonin residues to Glu residues increase the affinity to Pyrococcus prefoldin to the level of Thermococcus beta chaperonin and Pyrococcus chaperonin, indicating that their Glu250 and Glu256 residues of the helical protrusion region are responsible for relatively stronger binding to Pyrococcus prefoldin than Thermococcus alpha chaperonin. Since the putative chaperonin binding sites in the distal ends of Pyrococcus prefoldin are rich in basic residues, electrostatic interaction seems to be important for their interaction. The substrate protein transfer rate from prefoldin correlates well with its affinity for chaperonin.     

Cryo-EM structure of a group II chaperonin in the prehydrolysis ATP-bound state leading to lid closure

Chaperonins are large ATP-driven molecular machines that mediate cellular protein folding. Group II chaperonins use their "built-in lid" to close their central folding chamber. Here we report the structure of an archaeal group II chaperonin in its prehydrolysis ATP-bound state at subnanometer resolution using single particle cryo-electron microscopy (cryo-EM). Structural comparison of Mm-cpn in ATP-free, ATP-bound, and ATP-hydrolysis states reveals that ATP binding alone causes the chaperonin to close slightly with a ～45° counterclockwise rotation of the apical domain. The subsequent ATP hydrolysis drives each subunit to rock toward the folding chamber and to close the lid completely. These motions are attributable to the local interactions of specific active site residues with the nucleotide, the tight couplings between the apical and intermediate domains within the subunit, and the aligned interactions between two subunits across the rings. This mechanism of structural changes in response to ATP is entirely different from those found in group I chaperonins.     

A thermophilic mini-chaperonin contains a conserved polypeptide-binding surface: combined crystallographic and NMR studies of the GroEL apical domain with implications for substrate interactions

A homologue of the Escherichia coli GroEL apical domain was obtained from thermophilic eubacterium Thermus thermophilus. The domains share 70 % sequence identity (101 out of 145 residues). The thermal stability of the T. thermophilus apical domain (Tm>100 degrees C as evaluated by circular dichroism) is at least 35 degrees C greater than that of the E. coli apical domain (Tm=65 degrees C). The crystal structure of a selenomethione-substituted apical domain from T. thermophilus was determined to a resolution of 1.78 A using multiwavelength-anomalous-diffraction phasing. The structure is similar to that of the E. coli apical domain (root-mean-square deviation 0.45 A based on main-chain atoms). The thermophilic structure contains seven additional salt bridges of which four contain charge-stabilized hydrogen bonds. Only one of the additional salt bridges would face the "Anfinsen cage" in GroEL. High temperatures were exploited to map sites of interactions between the apical domain and molten globules. NMR footprints of apical domain-protein complexes were obtained at elevated temperature using 15N-1H correlation spectra of 15N-labeled apical domain. Footprints employing two polypeptides unrelated in sequence or structure (an insulin monomer and the SRY high-mobility-group box, each partially unfolded at 50 degrees C) are essentially the same and consistent with the peptide-binding surface previously defined in E. coli GroEL and its apical domain-peptide complexes. An additional part of this surface comprising a short N-terminal alpha-helix is observed. The extended footprint rationalizes mutagenesis studies of intact GroEL in which point mutations affecting substrate binding were found outside the "classical" peptide-binding site. Our results demonstrate structural conservation of the apical domain among GroEL homologues and conservation of an extended non-polar surface recognizing diverse polypeptides.     

Gene duplication and the evolution of group II chaperonins: implications for structure and function

Chaperonins are multisubunit protein-folding assemblies. They are composed of two distinct structural classes, which also have a characteristic phylogenetic distribution. Group I chaperonins (called GroEL/cpn60/hsp60) are present in Bacteria and eukaryotic organelles while group II chaperonins are found in Archaea (called the thermosome or TF55) and the cytoplasm of eukaryotes (called CCT or TriC). Gene duplication has been an important force in the evolution of group II chaperonins: Archaea possess one, two, or three homologous chaperonin subunit-encoding genes, and eight distinct CCT gene families (paralogs) have been described in eukaryotes. Phylogenetic analyses indicate that while the duplications in archaeal chaperonin genes have occurred numerous times independently in a lineage-specific fashion, the eight different CCT subunits found in eukaryotes are the products of duplications that occurred early and very likely only once in the evolution of the eukaryotic nuclear genome. Analyses of CCT sequences from diverse eukaryotic species reveal that each of the CCT subunits possesses a suite of invariant subunit-specific amino acid residues ("signatures"). When mapped onto the crystal structure of the archaeal chaperonin from Thermoplasma acidophilum, these signatures are located in the apical, intermediate, and equatorial domains. Regions that were found to be variable in length and/or amino acid sequence were localized primarily to the exterior of the molecule and, significantly, to the extreme tip of the apical domain (the "helical protrusion"). In light of recent biochemical and electron microscopic data describing specific CCT-substrate interactions, our results have implications for the evolution of subunit-specific functions in CCT.     

ATP binding is critical for the conformational change from an open to closed state in archaeal group II chaperonin

Group II chaperonins, found in archaea and in eukaryotic cytosol, do not have a co-chaperonin corresponding to GroES. Instead, it is suggested that the helical protrusion extending from the apical domain acts as a built-in lid for the central cavity and that the opening and closing of the lid is regulated by ATP binding and hydrolysis. However, details of this conformational change remain unclear. To investigate the conformational change associated with the ATP-driven cycle, we conducted protease sensitivity analyses and tryptophan fluorescence spectroscopy of alpha-chaperonin from a hyperthermophilic archaeum, Thermococcus strain KS-1. In the nucleotide-free or ADP-bound state, the chaperonin, especially in the helical protrusion region, was highly sensitive to proteases. Addition of ATP and ammonium sulfate induced the transition to the relatively protease-resistant form. The fluorescence intensity of the tryptophan residue introduced at the tip of the helical protrusion was enhanced by the presence of ATP or ammonium sulfate. We conclude that ATP binding induces the conformational change from the lid-open to lid-closed form in archaeal group II chaperonin.     

Structural and functional conservation of Mycobacterium tuberculosis GroEL paralogs suggests that GroEL1 Is a chaperonin

GroEL is a group I chaperonin that facilitates protein folding and prevents protein aggregation in the bacterial cytosol. Mycobacteria are unusual in encoding two or more copies of GroEL in their genome. While GroEL2 is essential for viability and likely functions as the general housekeeping chaperonin, GroEL1 is dispensable, but its structure and function remain unclear.Here, we present the 2.2-Å resolution crystal structure of a 23-kDa fragment of Mycobacterium tuberculosis GroEL1 consisting of an extended apical domain. Our X-ray structure of the GroEL1 apical domain closely resembles those of Escherichia coli GroEL and M. tuberculosis GroEL2, thus highlighting the remarkable structural conservation of bacterial chaperonins. Notably, in our structure, the proposed substrate-binding site of GroEL1 interacts with the N-terminal region of a symmetry-related neighboring GroEL1 molecule. The latter is consistent with the known GroEL apical domain function in substrate binding and is supported by results obtained from using peptide array technology. Taken together, these data show that the apical domains of M. tuberculosis GroEL paralogs are conserved in three-dimensional structure, suggesting that GroEL1, like GroEL2, is a chaperonin.     

Determination of the spatial structure of insectotoxin 15A from Buthus erpeus by (1)H-NMR spectroscopy data]

The solution structure of insectotoxin 15A (35 residues) from scorpion Buthus eupeus was determined on the basis of 386 interproton distance restraints 12 hydrogen-bonding restraints and 113 dihedral angle restraints derived from 1H NMR experiments. A group of 20 structures was calculated with the distance geometry program DIANA followed by the restrained energy minimization with the program CHARMM. The atomic RMS distribution about the mean coordinate position is 0.64 +/- 0.11 A for the backbone atoms and 1.35 +/- 0.20 A for all atoms. The structure contains an alpha-helix (residues 10-20) and a three-stranded antiparallel beta-sheet (residues 2-5, 24-28 and 29-33). A pairing of the eight cysteine residues of insectotoxin 15A was established basing on NMR data. Three disulfide bridges (residues 2-19, 16-31 and 20-33) connect the alpha-helix with the beta-sheet, and the fourth one (5-26) joins beta-strands together. The spatial fold of secondary structure elements (the alpha-helix and the beta-sheet) of the insectotoxin 15A is very similar to those of the other short and long scorpion toxins in spite of a low (about 20%) sequence homology.     

GroEL recognises sequential and non-sequential linear structural motifs compatible with extended beta-strands and alpha-helices.

The chaperonin GroEL binds a variety of polypeptides that share no obvious sequence similarity. The precise structural, chemical and dynamic features that are recognised remain largely unknown. Structural models of the complex between GroEL and its co-chaperonin GroES, and of the isolated apical domain of GroEL (minichaperone; residues 191-376) with a 17 residue N-terminal tag show that a linear sequential sequence (extended beta-strand) can be bound. We have analysed characteristics of the motifs that bind to GroEL by using affinity panning of immobilised GroEL minichaperones for a library of bacteriophages that display the fungal cellulose-binding domain of the enzyme cellobiohydrolase I. This protein has seven non-sequential residues in its binding site that form a linear binding motif with similar dimensions and characteristics to the peptide tag that was bound to the minichaperone GroEL(191-376). The seven residues thus form a constrained scaffold. We find that GroEL does bind suitable mutants of these seven residues. The side-chains recognised do not have to be totally hydrophobic, but polar and positively charged chains can be accommodated. Further, the spatial distribution of the side-chains is also compatible with those in an alpha-helix. This implies that GroEL can bind a wide range of structures, from extended beta-strands and alpha-helices to folded states, with exposed side-chains. The binding site can accommodate substrates of approximately 18 residues when in a helical or seven when in an extended conformation. The data support two activities of GroEL: the ability to act as a temporary parking spot for sticky intermediates by binding many motifs; and an unfolding activity of GroEL by binding an extended sequential conformation of the substrate.     

Structure of the membrane domain of subunit b of the Escherichia coli F0F1 ATP synthase.

The structure of the N-terminal transmembrane domain (residues 1-34) of subunit b of the Escherichia coli F0F1-ATP synthase has been solved by two-dimensional 1H NMR in a membrane mimetic solvent mixture of chloroform/methanol/H2O (4:4:1). Residues 4-22 form an alpha-helix, which is likely to span the hydrophobic domain of the lipid bilayer to anchor the largely hydrophilic subunit b in the membrane. The helical structure is interrupted by a rigid bend in the region of residues 23-26 with alpha-helical structure resuming at Pro-27 at an angle offset by 20 degrees from the transmembrane helix. In native subunit b, the hinge region and C-terminal alpha-helical segment would connect the transmembrane helix to the cytoplasmic domain. The transmembrane domains of the two subunit b in F0 were shown to be close to each other by cross-linking experiments in which single Cys were substituted for residues 2-21 of the native subunit and b-b dimer formation tested after oxidation with Cu(II)(phenanthroline)2. Cys residues that formed disulfide cross-links were found with a periodicity indicative of one face of an alpha-helix, over the span of residues 2-18, where Cys at positions 2, 6, and 10 formed dimers in highest yield. A model for the dimer is presented based upon the NMR structure and distance constraints from the cross-linking data. The transmembrane alpha-helices are positioned at a 23 degrees angle to each other with the side chains of Thr-6, Gln-10, Phe-14, and Phe-17 at the interface between subunits. The change in direction of helical packing at the hinge region may be important in the functional interaction of the cytoplasmic domains.     

MgATP binding to the nucleotide-binding domains of the eukaryotic cytoplasmic chaperonin induces conformational changes in the putative substrate-binding domains.

The eukaryotic cytosolic chaperonins are large heterooligomeric complexes with a cylindrical shape, resembling that of the homooligomeric bacterial counterpart, GroEL. In analogy to GroEL, changes in shape of the cytosolic chaperonin have been detected in the presence of MgATP using electron microscopy but, in contrast to the nucleotide-induced conformational changes in GroEL, no details are available about the specific nature of these changes. The present study identifies the structural regions of the cytosolic chaperonin that undergo conformational changes when MgATP binds to the nucleotide binding domains. It is shown that limited proteolysis with trypsin in the absence of MgATP cleaves each of the eight subunits approximately in half, generating two fragments of approximately 30 kDa. Using mass spectrometry (MS) and N-terminal sequence analysis, the cleavage is found to occur in a narrow span of the amino acid sequence, corresponding to the peptide binding regions of GroEL and to the helical protrusion, recently identified in the structure of the substrate binding domain of the archeal group II chaperonin. This proteolytic cleavage is prevented by MgATP but not by ATP in the absence of magnesium, ATP analogs (MgATPyS and MgAMP-PNP) or MgADP. These results suggest that, in analogy to GroEL, binding of MgATP to the nucleotide binding domains of the cytosolic chaperonin induces long range conformational changes in the polypeptide binding domains. It is postulated that despite their different subunit composition and substrate specificity, group I and group II chaperonins may share similar, functionally-important, conformational changes. Additional conformational changes are likely to involve a flexible helix-loop-helix motif, which is characteristic for all group II chaperonins.     

Single-molecule fluorescence polarization study of conformational change in archaeal group II chaperonin

Group II chaperonins found in archaea and in eukaryotic cytosol mediate protein folding without a GroES-like cofactor. The function of the cofactor is substituted by the helical protrusion at the tip of the apical domain, which forms a built-in lid on the central cavity. Although many studies on the change in lid conformation coupled to the binding and hydrolysis of nucleotides have been conducted, the molecular mechanism of lid closure remains poorly understood. Here, we performed a single-molecule polarization modulation to probe the rotation of the helical protrusion of a chaperonin from a hyperthermophilic archaeum, Thermococcus sp. strain KS-1. We detected approximately 35° rotation of the helical protrusion immediately after photorelease of ATP. The result suggests that the conformational change from the open lid to the closed lid state is responsible for the approximately 35° rotation of the helical protrusion.     

Three conformations of an archaeal chaperonin, TF55 from Sulfolobus shibatae

Chaperonins are cylindrical, oligomeric complexes, essential for viability and required for the folding of other proteins. The GroE (group I) subfamily, found in eubacteria, mitochondria and chloroplasts, have 7-fold symmetry and provide an enclosed chamber for protein subunit folding. The central cavity is transiently closed by interaction with the co-protein, GroES. The most prominent feature specific to the group II subfamily, found in archaea and in the eukaryotic cytosol, is a long insertion in the substrate-binding region. In the archaeal complex, this forms an extended structure acting as a built-in lid, obviating the need for a GroES-like co-factor. This extension occludes a site known to bind non-native polypeptides in GroEL. The site and nature of substrate interaction are not known for the group II subfamily. The atomic structure of the thermosome, an archaeal group II chaperonin, has been determined in a fully closed form, but the entry and exit of protein substrates requires transient opening. Although an open form has been investigated by electron microscopy, conformational changes in group II chaperonins are not well characterized. Using electron cryo-microscopy and three-dimensional reconstruction, we describe three conformations of a group II chaperonin, including an asymmetric, bullet-shaped form, revealing the range of domain movements in this subfamily.     

Novel mode of ligand recognition by the Erbin PDZ domain

Erbin contains a class I PDZ domain that binds to the C-terminal region of the receptor tyrosine kinase ErbB2, a class II ligand. The crystal structure of the human Erbin PDZ bound to the peptide EYLGLDVPV corresponding to the C-terminal residues 1247-1255 of human ErbB2 has been determined at 1.25-A resolution. The Erbin PDZ deviates from the canonical PDZ fold in that it contains a single alpha-helix. The isopropyl group of valine at position -2 of the ErbB2 peptide interacts with the Erbin Val(1351) and displaces the peptide backbone away from the alpha-helix, elucidating the molecular basis of class II ligand recognition by a class I PDZ domain. Strikingly, the phenolic ring of tyrosine -7 enters into a pocket formed by the extended beta 2-beta 3 loop of the Erbin PDZ. Phosphorylation of tyrosine -7 abolishes this interaction but does not affect the binding of the four C-terminal peptidic residues to PDZ, as revealed by the crystal structure of the Erbin PDZ complexed with a phosphotyrosine-containing ErbB2 peptide. Since phosphorylation of tyrosine -7 plays a critical role in ErbB2 function, the selective binding and sequestration of this residue in its unphosphorylated state by the Erbin PDZ provides a novel mechanism for regulation of the ErbB2-mediated signaling and oncogenicity.     

NMR solution structure of the mitochondrial F1beta presequence from Nicotiana plumbaginifolia

We have isolated, characterized and determined the three-dimensional NMR solution structure of the presequence of ATPsynthase F1beta subunit from Nicotiana plumbaginifolia. A general method for purification of presequences is presented. The method is based on overexpression of a mutant precursor containing a methionine residue introduced at the processing site, followed by CNBr-cleavage and purification of the presequence on a cation-exchange column. The F1beta presequence, 53 amino acid residues long, retained its native properties as evidenced by inhibition of in vitro mitochondrial import and processing at micromolar concentrations. CD spectroscopy revealed that the F1beta presequence formed an alpha-helical structure in membrane mimetic environments such as SDS and DPC micelles (approximately 50% alpha-helix), and in acidic phospholipid bicelles (approximately 60% alpha-helix). The NMR solution structure of the F1beta presequence in SDS micelles was determined on the basis of 518 distance and 21 torsion angle constraints. The structure was found to contain two helices, an N-terminal amphipathic alpha-helix (residues 4-15) and a C-terminal alpha-helix (residues 43-53), separated by a largely unstructured 27 residue long internal domain. The N-terminal amphipathic alpha-helix forms the putative Tom20 receptor binding site, whereas the C-terminal alpha-helix is located upstream of the mitochondrial processing peptidase cleavage site.     

Structural elucidation of the protein- and membrane-binding properties of the N-terminal tail domain of human annexin II

The conformational preferences and the solution structure of AnxII(N31), a peptide corresponding to the full-length sequence (residues 1-31) of the human annexin II N-terminal tail domain, were investigated by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. CD results showed that AnxII(N31) adopts a mainly alpha-helical conformation in hydrophobic or membrane-mimetic environments, while a predominantly random structure is adopted in aqueous buffer. In contrast to previous results of the annexin I N-terminal domain peptide [Yoon et al. (2000) FEBS Lett. 484, 241-245], calcium ions showed no effect on the structure of AnxII(N31). The NMR-derived structure of AnxII(N31) in 50% TFE/water mixture showed a horseshoe-like fold comprising the N-terminal amphipathic alpha-helix, the following loop, and the C-terminal helical region. Together, the results establish the first detailed structural data on the N-terminal tail domain of annexin II, and suggest the possibility of the domain to undergo Ca(2+)-independent membrane-binding.     

On the evolutionary origin of the chaperonins

An analysis of the apical domain of the Group-I and Group-II chaperonins shows that they have structural similarities to two different protein folds: a "swivel-domain" phosphotransferase and a thioredoxin-like peroxiredoxin. There is no significant sequence similarity that supports either similarity and the degree of similarity based on structure is comparable but weak for both relationships. Based on possible evolutionary transitions, we deduced that a phosphotransferase origin would require both a large insertion and deletion of structure whereas a peroxiredoxin origin requires only a peripheral rearrangement, similar to an internal domain-swap. We postulate that this change could have been triggered by the insertion of a peroxiredoxin into the ATPase domain that led to the modern chaperonin domain arrangement. The peroxidoxin fold is the most highly embellished member of the thioredoxin super-family and the insertion event may have "overloaded" the core, leading to a rearrangement. A peroxiredoxin origin for the domain also provides a functional explanation, as the peroxiredoxins can act as chaperones when they adopt a multimeric ring complex, similar to the chaperonin subunit configuration. In addition, several of the GroEL apical domain hydrophobic residues which interact with the unfolded protein are located in a position that corresponds to the protein substrate binding region of the peroxiredoxin fold. We suggest that the origin of the ur-chaperonin from a thioredoxin/peroxiredoxin fold might also account for the number of thioredoxin-fold containing proteins that interact with chaperonins, such as tubulin and phosducin-like proteins.     

Crystal structure of calmodulin

The crystal structure of calmodulin has been determined to 3.6 A resolution. At this resolution the polypeptide chain can be traced. Some of the side chains have tentatively been identified. Refinement of the structure with x-ray diffraction data measured to 1.65 A resolution is continuing. As reported by Babu et al. calmodulin is about 65 A long and 30 A in diameter. Homolog domains 1 and 2 are related by a local twofold axis, as in parvalbumin and in troponin C, and form one end of the molecule. Domains 3 and 4 form the other end. The second alpha-helix of domain 2 and a short interdomain region are continuous with the first helix of domain 3, thereby forming a single helix from residues 67-93. The central region, residues 75-84, of this long helix forms a handle connecting the two pairs of homolog domains. Exclusive of the residues, 75-84, in the handle the closet approach of side chains of pair 1, 2 to pair 3, 4 is 12 A. The spatial relationship of pair 1, 2 to pair 3, 4 is similar in calmodulin to the relationship of the corresponding pairs in troponin C. However, in troponin C there are three additional residues in the handle region of the long alpha-helix and the two pairs are about 5.0 A further apart. On the surface of pair 1, 2 in calmodulin there is one extended region with many hydrophobic side chains from both domain 1 and domain 2. This hydrophobic patch is bounded by two distinct clusters of anionic side chains, one from the beginning of the first helix of domain 1 and on the other side of the hydrophobic surface one from the beginning of the first helix of domain 2. Homologously, the hydrophobic patch on the surface of pair 3, 4 is bounded by two clusters of aspartate and glutamate residues. Either or both of these hydrophobic surfaces may be sites to which calmodulin target proteins bind.     

Role of the helical protrusion in the conformational change and molecular chaperone activity of the archaeal group II chaperonin

To elucidate the exact role of the helical protrusion of a group II chaperonin in its molecular chaperone function, three deletion mutants of the chaperonin from a hyperthermophilic archaeum (Thermococcus sp. strain KS-1) lacking one-third, two-thirds, and the whole of the helical protrusion were constructed. The helical protrusion is thought to be substituted for the co-chaperonin GroES of a group I chaperonin and to be important for binding to unfolded proteins. Protease sensitivity assays and small angle x-ray scattering experiments were performed to demonstrate the conformation change of the wild type protein and the deletion mutants by adenine nucleotides. Whereas the binding of ATP to the wild type protein induced a structural transition corresponding to the closure of the built-in lid, it did not cause significant structural changes in deletion mutants. Although the mutants effectively protected proteins from thermal aggregation, ATP-dependent protein folding ability was remarkably diminished. We conclude that the helical protrusion is not necessarily important for binding to unfolded proteins, but its ATP-dependent conformational change mediates folding of captured unfolded proteins.     

Solution structure of the ribosome recycling factor from Aquifex aeolicus

The solution structure of ribosome recycling factor (RRF) from hyperthermophilic bacterium, Aquifex aeolicus, was determined by heteronuclear multidimensional NMR spectroscopy. Fifteen structures were calculated using restraints derived from NOE, J-coupling, and T1/T2 anisotropies. The resulting structure has an overall L-shaped conformation with two domains and is similar to that of a tRNA molecule. The domain I (corresponding to the anticodon stem of tRNA) is a rigid three alpha-helix bundle. Being slightly different from usual coiled-coil arrangements, each helix of domain I is not twisted but straight and parallel to the main axis. The domain II (corresponding to the portion with the CCA end of tRNA) is an alpha/beta domain with an alpha-helix and two beta-sheets, that has some flexible regions. The backbone atomic root-mean-square deviation (rmsd) values of both domains were 0.7 A when calculated separately, which is smaller than that of the molecule as a whole (1.4 A). Measurement of 15N-[1H] NOE values show that the residues in the corner of the L-shaped molecule are undergoing fast internal motion. These results indicate that the joint region between two domains contributes to the fluctuation in the orientation of two domains. Thus, it was shown that RRF remains the tRNA mimicry in solution where it functions.     

Solution structure of BmP01 from the venom of scorpion Buthus martensii Karsch

From the venom of scorpion Buthus martensii Karsch,a short peptide (BmP01, 29 amino acid residues) was isolated and characterized as previously reported (Lebren, R. R., et al. (1997) Eur. J. Biochem. 245, 457-464). It was shown to reduce 33% outward K(+) channel (hippocampal neurons) currents at 10 microM. The solution structure of BmP01 was determined by 2D (1)H NMR spectroscopy. The NOEs, coupling constants, and H-D exchange obtained from NMR spectroscopy were used in structural calculations. The conformation of BmP01 is composed of a short alpha-helix (Cys 3-Thr 12) and a two-stranded antiparallel beta-sheet (Ala 15-Asp 20 and Lys 23-Pro 28). There are three disulfide bridges (Cys 3-Cys 19, Cys 6-Cys 24 and Cys 10-Cys 26) connecting the alpha-helix and beta-sheet. Asp 20 to Lys 23 form a type II turn linking the two strands. Structural and electrostatic potential comparison between BmP01 and its analogues are also presented.