The leukocidin pore: evidence for an octamer with four LukF subunits and four LukS subunits alternating around a central axis

The staphylococcal alpha-hemolysin (alphaHL) and leukocidin (Luk) polypeptides are members of a family of related beta-barrel pore-forming toxins. Upon binding to susceptible cells, alphaHL forms water-filled homoheptameric transmembrane pores. By contrast, Luk pores are formed by two classes of subunit, F and S, rendering a heptameric structure displeasing on symmetry grounds at least. Both the subunit stoichiometry and arrangement within the Luk pore have been contentious issues. Here we use chemical and genetic approaches to show that (1) the predominant, or perhaps the only, form of the Luk pore is an octamer; (2) the subunit stoichiometry is 1:1; and (3) the subunits are arranged in an alternating fashion about a central axis of symmetry, at least when a fused LukS-LukF construct is used. The experimental approaches we have used also open up new avenues for engineering the arrangement of the subunits of beta-barrel pore-forming toxins.     

Arresting and releasing Staphylococcal alpha-hemolysin at intermediate stages of pore formation by engineered disulfide bonds

alpha-Hemolysin (alphaHL) is secreted by Staphylococcus aureus as a water-soluble monomer that assembles into a heptamer to form a transmembrane pore on a target membrane. The crystal structures of the LukF water-soluble monomer and the membrane-bound alpha-hemolysin heptamer show that large conformational changes occur during assembly. However, the mechanism of assembly and pore formation is still unclear, primarily because of the difficulty in obtaining structural information on assembly intermediates. Our goal is to use disulfide bonds to selectively arrest and release alphaHL from intermediate stages of the assembly process and to use these mutants to test mechanistic hypotheses. To accomplish this, we created four double cysteine mutants, D108C/K154C (alphaHL-A), M113C/K147C (alphaHL-B), H48C/ N121C (alphaHL-C), I5C/G130C (alphaHL-D), in which disulfide bonds may form between the pre-stem domain and the beta-sandwich domain to prevent pre-stem rearrangement and membrane insertion. Among the four mutants, alphaHL-A is remarkably stable, is produced at a level at least 10-fold greater than that of the wild-type protein, is monomeric in aqueous solution, and has hemolytic activity that can be regulated by the presence or absence of reducing agents. Cross-linking analysis showed that alphaHL-A assembles on a membrane into an oligomer, which is likely to be a heptamer, in the absence of a reducing agent, suggesting that oxidized alphaHL-A is halted at a heptameric prepore state. Therefore, conformational rearrangements at positions 108 and 154 are critical for the completion of alphaHL assembly but are not essential for membrane binding or for formation of an oligomeric prepore intermediate.     

Asymmetric amino acid compositions of transmembrane beta-strands

In contrast to water-soluble proteins, membrane proteins reside in a heterogeneous environment, and their surfaces must interact with both polar and apolar membrane regions. As a consequence, the composition of membrane proteins' residues varies substantially between the membrane core and the interfacial regions. The amino acid compositions of helical membrane proteins are also known to be different on the cytoplasmic and extracellular sides of the membrane. Here we report that in the 16 transmembrane beta-barrel structures, the amino acid compositions of lipid-facing residues are different near the N and C termini of the individual strands. Polar amino acids are more prevalent near the C termini than near the N termini, and hydrophobic amino acids show the opposite trend. We suggest that this difference arises because it is easier for polar atoms to escape from the apolar regions of the bilayer at the C terminus of a beta-strand. This new characteristic of beta-barrel membrane proteins enhances our understanding of how a sequence encodes a membrane protein structure and should prove useful in identifying and predicting the structures of trans-membrane beta-barrels.     

alpha-Hemolysin, gamma-hemolysin, and leukocidin from Staphylococcus aureus: distant in sequence but similar in structure.

alpha-Hemolysin from Staphylococcus aureus assembles from a water-soluble, monomeric species to a membrane-bound heptamer on the surface of target cells, creating water-filled channels that lead to cell death and lysis. Staphylococcus aureus also produces the gamma-hemolysin and leukocidin toxins, which function as two component toxins in the disruption and lysis of erythrocytes and leukocytes. Analysis of the aligned sequences of alpha-hemolysin, gamma-hemolysin, and leukocidin in the context of the alpha-hemolysin heptamer structure supports the conclusion that even though the level of sequence identity between alpha-hemolysin and the gamma-hemolysin and leukocidin toxins is in the so-called twilight zone, the three-dimensional structures of the protomers are probably conserved. By analogy with alpha-hemolysin, gamma-hemolysin and leukocidin may also form oligomeric, transmembrane channels in which an antiparallel beta-barrel constitutes the primary membrane-embedded domain.     

A functional protein pore with a "retro" transmembrane domain.

Extended retro (reversed) peptide sequences have not previously been accommodated within functional proteins. Here, we show that the entire transmembrane portion of the beta-barrel of the pore-forming protein alpha-hemolysin can be formed by retrosequences comprising a total of 175 amino acid residues, 25 contributed by the central sequence of each subunit of the heptameric pore. The properties of wild-type and retro heptamers in planar bilayers are similar. The single-channel conductance of the retro pore is 15% less than that of the wild-type heptamer and its current-voltage relationship denotes close to ohmic behavior, while the wild-type pore is weakly rectifying. Both wild-type and retro pores are very weakly anion selective. These results and the examination of molecular models suggest that beta-barrels may be especially accepting of retro sequences compared to other protein folds. Indeed, the ability to form a retro domain could be diagnostic of a beta-barrel, explaining, for example, the activity of the retro forms of many membrane-permeabilizing peptides. By contrast with the wild-type subunits, monomeric retro subunits undergo premature assembly in the absence of membranes, most likely because the altered central sequence fails to interact with the remainder of the subunit, thereby initiating assembly. Despite this difficulty, a technique was devised for obtaining heteromeric pores containing both wild-type and retro subunits. Most probably as a consequence of unfavorable interstrand side-chain interactions, the heteromeric pores are less stable than either the wild-type or retro homoheptamers, as judged by the presence of subconductance states in single-channel recordings. Knowledge about the extraordinary plasticity of the transmembrane beta-barrel of alpha-hemolysin will be very useful in the de novo design of functional membrane proteins based on the beta-barrel motif.     

Location of a constriction in the lumen of a transmembrane pore by targeted covalent attachment of polymer molecules

Few methods exist for obtaining the internal dimensions of transmembrane pores for which 3-D structures are lacking or for showing that structures determined by crystallography reflect the internal dimensions of pores in lipid bilayers. Several approaches, involving polymer penetration and transport, have revealed limiting diameters for various pores. But, in general, these approaches do not indicate the locations of constrictions in the channel lumen. Here, we combine cysteine mutagenesis and chemical modification with sulfhydryl-reactive polymers to locate the constriction in the lumen of the staphylococcal alpha-hemolysin pore, a model protein of known structure. The rates of reaction of each of four polymeric reagents (MePEG-OPSS) of different masses towards individual single cysteine mutants, comprising a set with cysteines distributed over the length of the lumen of the pore, were determined by macroscopic current recording. The rates for the three larger polymers (1.8, 2.5, and 5.0 kD) were normalized with respect to the rates of reaction with a 1.0-kD polymer for each of the seven positions in the lumen. The rate of reaction of the 5.0-kD polymer dropped dramatically at the centrally located Cys-111 residue and positions distal to Cys-111, whether the reagent was applied from the trans or the cis side of the bilayer. This semi-quantitative analysis sufficed to demonstrate that a constriction is located at the midpoint of the pore lumen, as predicted by the crystal structure, and although the constriction allows a 2.5-kD polymer to pass, transport of a 5.0-kD molecule is greatly restricted. In addition, PEG chains gave greater reductions in pore conductance when covalently attached to the narrower regions of the lumen, permitting further definition of the interior of the pore. The procedures described here should be applicable to other pores and to related structures such as the vestibules of ion channels.     

PFIT and PFRIT: bioinformatic algorithms for detecting glycosidase function from structure and sequence

The identification of the enzymes involved in the metabolism of simple and complex carbohydrates presents one bioinformatic challenge in the post-genomic era. Here, we present the PFIT and PFRIT algorithms for identifying those proteins adopting the alpha/beta barrel fold that function as glycosidases. These algorithms are based on the observation that proteins adopting the alpha/beta barrel fold share positions in their tertiary structures having equivalent sets of atomic interactions. These are conserved tertiary interaction positions, which have been implicated in both structure and function. Glycosidases adopting the alpha/beta barrel fold share more conserved tertiary interactions than alpha/beta barrel proteins having other functions. The enrichment pattern of conserved tertiary interactions in the glycosidases is the information that PFIT and PFRIT use to predict whether any given alpha/beta barrel will function as a glycosidase or not. Using as a test set a database of 19 glycosidase and 45 nonglycosidase alpha/beta barrel proteins with low sequence similarity, PFIT and PFRIT can correctly predict glycosidase function for 84% of the proteins known to function as glycosidases. PFIT and PFRIT incorrectly predict glycosidase function for 25% of the nonglycosidases. The program PSI-BLAST can also correctly identify 84% of the 19 glycosidases, however, it incorrectly predicts glycosidase function for 50% of the nonglycosidases (twofold greater than PFIT and PFRIT). Overall, we demonstrate that the structure-based PFIT and PFRIT algorithms are both more selective and sensitive for predicting glycosidase function than the sequence-based PSI-BLAST algorithm.     

Solution structure of Pi4, a short four-disulfide-bridged scorpion toxin specific of potassium channels

Pi4 is a short toxin found at very low abundance in the venom of Pandinus imperator scorpions. It is a potent blocker of K(+) channels. Like the other members of the alpha-KTX6 subfamily to which it belongs, it is cross-linked by four disulfide bonds. The synthetic analog (sPi4) and the natural toxin (nPi4) have been obtained by solid-phase synthesis or from scorpion venom, respectively. Analysis of two-dimensional (1)H NMR spectra of nPi4 and sPi4 indicates that both peptides have the same structure. Moreover, electrophysiological recordings of the blocking of Shaker B K(+) channels by sPi4 (K(D) = 8.5 nM) indicate that sPi4 has the same blocking activity of nPi4 (K(D) = 8.0 nM), previously described. The disulfide bonds have been independently determined by NMR and structure calculations, and by Edman-degradation/mass-spectrometry identification of peptides obtained by proteolysis of nPi4. Both approaches indicate that the pairing of the half-cystines is (6)C-(27)C, (12)C-(32)C, (16)C-(34)C, and (22)C-(37)C. The structure of the toxin has been determined by using 705 constraints derived from NMR data on sPi4. The structure, which is well defined, shows the characteristic alpha/beta scaffold of scorpion toxins. It is compared to the structure of the other alpha-KTX6 subfamily members and, in particular, to the structure of maurotoxin, which shows a different pattern of disulfide bridges despite its high degree of sequence identity (76%) with Pi4. The structure of Pi4 and the high amounts of synthetic peptide available, will enable the detailed analysis of the interaction of Pi4 with K(+) channels.     

Assembly of Staphylococcal Leukocidin into a Pore-Forming Oligomer on Detergent-Resistant Membrane Microdomains,Lipid Rafts,in Human Polymorphonuclear Leukocytes

Staphylococcal leukocidin (Luk) consists of LukS and LukF, which cooperatively lyse human polymorphonuclear leukocytes (HPMNLs), monocytes, and macrophages. Here we found that LukS and LukF assembles into hetero-oligomeric pore complexes on the detergent-resistant membrane microdomains, lipid rafts of HPMNLs. When HPMNLs were treated with LukS alone, 24% of the added LukS was localized in lipid rafts. Furthermore, in HPMNLs treated with both LukS and LukF simultaneously, about 90% of high molecular-mass complexes of 100 kDa, which consists of LukS and LukF, were detected in the lipid raft fractions. In contrast, in HPMNLs treated with LukF alone, LukF was not localized in lipid rafts despite binding to the target cell membranes. Ten mM methyl-β-cyclodextrin, a dysfunctioning agent of lipid rafts, completely inhibited assembly of Luk on lipid rafts, and resulted in null leukocytolytic activity of Luk. Hence, we concluded that assembly of LukS and LukF into the pore-complex occurs in lipid rafts in HPMNLs and that LukF can bind to LukS, which had already bound to lipid rafts, to assemble into hetero-oligomers.     

Vibrio cholerae cytolysin is composed of an alpha-hemolysin-like core

The enteric pathogen Vibrio cholerae secretes a water-soluble 80-kD cytolysin, Vibrio cholerae cytolysin (VCC) that assembles into pentameric channels following proteolytic activation by exogenous proteases. Until now, VCC has been placed in a unique class of pore-forming toxins, distinct from paradigms such as Staphyloccal alpha-hemolysin. However, as reported here, amino acid sequence analysis and three-dimensional structure modeling indicate that the core component of the VCC toxin is related in sequence and structure to a family of hemolysins from Staphylococcus aureus that include leukocidin F and alpha-hemolysin. Furthermore, our analysis has identified the channel-forming region of VCC and a potential lipid head-group binding site, and suggests a conserved mechanism of assembly and lysis. An additional domain in the VCC toxin is related to plant lectins, conferring additional target cell specificity to the toxin.     

Stoichiometry of lipid interactions with transmembrane proteins--Deduced from the 3D structures

The stoichiometry of the first shell of lipids interacting with a transmembrane protein is defined operationally by the population of spin-labeled lipid chains whose motion is restricted directly by the protein. Interaction stoichiometries have been determined experimentally for a wide range of alpha-helical integral membrane proteins by using spin-label ESR spectroscopy. Here, we determine the spatially defined number of first-shell lipids at the hydrophobic perimeter of integral membrane proteins whose 3D structure has been determined by X-ray crystallography and lipid-protein interactions characterized by spin-labeling. Molecular modeling is used to build a single shell of lipids surrounding transmembrane structures derived from the PDB. Constrained energy optimization of the protein-lipid assemblies is performed by molecular mechanics. For relatively small proteins (up to 7-12 transmembrane helices), the geometrical first shell corresponds to that defined experimentally by perturbation of the lipid-chain dynamics. For larger, multi-subunit alpha-helical proteins, the lipids perturbed directly by the protein may either exceed or be less in number than those that can be accommodated at the intramembranous perimeter. In these latter cases, the motionally restricted spin-labeled lipids can be augmented by intercalation, or can correspond to a specific subpopulation at the protein interface, respectively. For monomeric beta-barrel proteins, the geometrical lipid stoichiometry corresponds to that determined from lipid mobility for a 22-stranded barrel, but fewer lipids are motionally restricted than can be accommodated around an eight-stranded barrel. Deviations from the geometrical first shell, in the beta-barrel case, are for the smaller protein with a highly curved barrel.     

EstB from Burkholderia gladioli: a novel esterase with a beta-lactamase fold reveals steric factors to discriminate between esterolytic and beta-lactam cleaving activity

Esterases form a diverse class of enzymes of largely unknown physiological role. Because many drugs and pesticides carry ester functions, the hydrolysis of such compounds forms at least one potential biological function. Carboxylesterases catalyze the hydrolysis of short chain aliphatic and aromatic carboxylic ester compounds. Esterases, D-alanyl-D-alanine-peptidases (DD-peptidases) and beta-lactamases can be grouped into two distinct classes of hydrolases with different folds and topologically unrelated catalytic residues, the one class comprising of esterases, the other one of beta-lactamases and DD-peptidases. The chemical reactivities of esters and beta-lactams towards hydrolysis are quite similar, which raises the question of which factors prevent esterases from displaying beta-lactamase activity and vice versa. Here we describe the crystal structure of EstB, an esterase isolated from Burkholderia gladioli. It shows the protein to belong to a novel class of esterases with homology to Penicillin binding proteins, notably DD-peptidase and class C beta-lactamases. Site-directed mutagenesis and the crystal structure of the complex with diisopropyl-fluorophosphate suggest Ser75 within the "beta-lactamase" Ser-x-x-Lys motif to act as catalytic nucleophile. Despite its structural homology to beta-lactamases, EstB shows no beta-lactamase activity. Although the nature and arrangement of active-site residues is very similar between EstB and homologous beta-lactamases, there are considerable differences in the shape of the active site tunnel. Modeling studies suggest steric factors to account for the enzyme's selectivity for ester hydrolysis versus beta-lactam cleavage.     

Importance of secondary structural specificity determinants in protein folding: insertion of a native beta-sheet sequence into an alpha-helical coiled-coil

To examine how a short secondary structural element derived from a native protein folds when in a different protein environment, we inserted an 11-residue beta-sheet segment (cassette) from human immunoglobulin fold, Fab new, into an alpha-helical coiled-coil host protein (cassette holder). This de novo design protein model, the structural cassette mutagenesis (SCM) model, allows us to study protein folding principles involving both short- and long-range interactions that affect secondary structure stability and conformation. In this study, we address whether the insertion of this beta-sheet cassette into the alpha-helical coiled-coil protein would result in conformational change nucleated by the long-range tertiary stabilization of the coiled-coil, therefore overriding the local propensity of the cassette to form beta-sheet, observed in its native immunoglobulin fold. The results showed that not only did the nucleating helices of the coiled-coil on either end of the cassette fail to nucleate the beta-sheet cassette to fold with an alpha-helical conformation, but also the entire chimeric protein became a random coil. We identified two determinants in this cassette that prevented coiled-coil formation: (1) a tandem dipeptide NN motif at the N-terminal of the beta-sheet cassette, and (2) the hydrophilic Ser residue, which would be buried in the hydrophobic core if the coiled-coil structure were to fold. By amino acid substitution of these helix disruptive residues, that is, either the replacement of the NN motif with high helical propensity Ala residues or the substitution of Ser with Leu to enhance hydrophobicity, we were able to convert the random coil chimeric protein into a fully folded alpha-helical coiled-coil. We hypothesized that this NN motif is a "secondary structural specificity determinant" which is very selective for one type of secondary structure and may prevent neighboring residues from adopting an alternate protein fold. These sequences with secondary structural specificity determinants have very strong local propensity to fold into a specific secondary structure and may affect overall protein folding by acting as a folding initiation site.     

A plant kinase plays roles in defense response against geminivirus by phosphorylation of a viral pathogenesis protein

The plant SNF1-related kinase (SnRK1) is the α-subunit of the SnRK1 heterotrimeric compleses. Although SnRK1 is widely known as a key regulator of plant response to various physiological processes including nutrient- and energy-sensing, regulation of global metabolism, and control of cell cycle, development, as well as abiotics stress, less is known about the function of SnRK1 during pathogen infection. Our previous work has demonstrated that a tomato SNF1-related kinase (SlSnRK1) can interact with and phosphorylate βC1, a pathogenesis protein encoded by tomato yellow leaf curl China betasatellite. Our results also showed that the plant SnRK1 can affect genimivirus infection in plant and reduce viral DNA accumulation. Phosphorylation of βC1 protein negatively impacts its function as a pathogenicity determinant. Here we provide more information on interaction between βC1 and SlSnRK1 and propose a mechanistic model for the SlSnRK1-mediated defense responses against geminiviruses and the potential role of SnRK1 in plant resistance to geminivirus.     

Long-range side-chain-main-chain interactions play crucial roles in stabilizing the (betaalpha)8 barrel motif of the alpha subunit of tryptophan synthase

The role of hither-to-fore unrecognized long-range hydrogen bonds between main-chain amide hydrogens and polar side chains on the stability of a well-studied (betaalpha)8, TIM barrel protein, the alpha subunit of tryptophan synthase (alphaTS), was probed by mutational analysis. The F19-D46 and I97-D124 hydrogen bonds link the N terminus of a beta-strand with the C terminus of the succeeding antiparallel alpha-helix, and the A103-D130 hydrogen bond links the N terminus of an alpha-helix with the C terminus of the succeeding antiparallel beta-strand, forming clamps for the respective betaalpha or alphabeta hairpins. The individual replacement of these aspartic acid side chains with alanine leads to what appear to be closely related partially folded structures with significantly reduced far-UV CD ellipticity and thermodynamic stability. Comparisons with the effects of eliminating another main-chain-side-chain hydrogen bond, G26-S33, and two electrostatic side-chain-side-chain hydrogen bonds, D38-H92 and D112-H146, all in the same N-terminal folding unit of alphaTS, demonstrated a unique role for the clamp interactions in stabilizing the native barrel conformation. Because neither the asparagine nor glutamic acid variant at position 46 can completely reproduce the spectroscopic, thermodynamic, or kinetic folding properties of aspartic acid, both size and charge are crucial to its unique role in the clamp hydrogen bond. Kinetic studies suggest that the three clamp hydrogen bonds act in concert to stabilize the transition state leading to the fully folded TIM barrel motif.     

Analysis of the factors that stabilize a designed two-stranded antiparallel beta-sheet

Autonomously folding beta-hairpins (two-strand antiparallel beta-sheets) have become increasingly valuable tools for probing the forces that control peptide and protein conformational preferences. We examine the effects of variations in sequence and solvent on the stability of a previously designed 12-residue peptide (1). This peptide adopts a beta-hairpin conformation containing a two-residue loop (D-Pro-Gly) and a four-residue interstrand sidechain cluster that is observed in the natural protein GB1. We show that the conformational propensity of the loop segment plays an important role in beta-hairpin stability by comparing 1 with (D)P--> N mutant 2. In addition, we show that the sidechain cluster contributes both to conformational stability and to folding cooperativity by comparing 1 with mutant 3, in which two of the four cluster residues have been changed to serine. Thermodynamic analysis suggests that the high loop-forming propensity of the (D)PG segment decreases the entropic cost of beta-hairpin formation relative to the more flexible NG segment, but that the conformational rigidity of (D)PG may prevent optimal contacts between the sidechains of the GB1-derived cluster. The enthalpic favorability of folding in these designed beta-hairpins suggests that they are excellent scaffolds for studying the fundamental mechanisms by which amino acid sidechains interact with one another in folded proteins.     

Beta2-microglobulin isoforms display an heterogeneous affinity for type I collagen

It has been claimed that beta2-microglobulin (beta2-m) interacts with type I and type II collagen, and this property has been linked to the tissue specificity of the beta2-m amyloid deposits that target the osteo-articular system. The binding parameters of the interaction between collagen and beta2-m were determined by band shift electrophoresis and surface plasma resonance by using bovine collagen of type I and type II and various isoforms of beta2-m. Wild-type beta2-m binds collagen type I with a Kd of 4.1 x 10(-4) M and type II with 2.3 x 10(-3) M. By the BIAcore system we monitored the binding properties of the conformers of the slow phase of folding of beta2-m. The folding intermediates during the slow phase of folding do not display any significant difference with respect to the binding properties of the fully folded molecule. The affinity of beta2-m truncated at the third N-terminal residue does not differ from that reported for the wild-type protein. Increased affinity for collagen type I is found in the case of N-terminal truncated species lacking of six residues. The Kd of this species is 3.4 x 10 (-5) M at pH 7.4 and its affinity increases to 4.9 x 10(-6) M at pH 6.4. Fluctuations of the affinity caused by beta2-m truncation and pH change can cause modifications of protein concentration in the solvent that surrounds the collagen, and could contribute to generate locally a critical protein concentration able to prime the protein aggregation.     

Structure of palmitoylated BET3: insights into TRAPP complex assembly and membrane localization

BET3 is a component of TRAPP, a complex involved in the tethering of transport vesicles to the cis-Golgi membrane. The crystal structure of human BET3 has been determined to 1.55-A resolution. BET3 adopts an alpha/beta-plait fold and forms dimers in the crystal and in solution, which predetermines the architecture of TRAPP where subunits are present in equimolar stoichiometry. A hydrophobic pocket within BET3 buries a palmitate bound through a thioester linkage to cysteine 68. BET3 and yeast Bet3p are palmitoylated in recombinant yeast cells, the mutant proteins BET3 C68S and Bet3p C80S remain unmodified. Both BET3 and BET3 C68S are found in membrane and cytosolic fractions of these cells; in membrane extractions, they behave like tightly membrane-associated proteins. In a deletion strain, both Bet3p and Bet3p C80S rescue cell viability. Thus, palmitoylation is neither required for viability nor sufficient for membrane association of BET3, which may depend on protein-protein contacts within TRAPP or additional, yet unidentified modifications of BET3. A conformational change may facilitate palmitoyl extrusion from BET3 and allow the fatty acid chain to engage in intermolecular hydrophobic interactions.     

Homology modeling and molecular dynamics simulation of odonthubuthus doriae (Od1) scorpion toxin in comparison to the BmK M1

All of the α-subgroups share similarity in their sequence and structure but different in the toxicity to various voltage-gated sodium channels (VGSCs). We modeled the first 3D structural model of the Od1 based on BmK M1 using homology modeling. The reliability of model for more investigation and compare to BmK M1 has been examined and confirmed. Then the model structure is further refined by energy minimization and molecular dynamics methods. The purpose of this modeling and simulation is comparison toxicity of two mentioned toxins by investigation structural feature of functional regions including core domain, 5-turn and C-terminal which make NC domain. In the one hand, it is intriguing that Od1 in comparison to BmK M1 shows same solvent accessible surface area (SASA) in 5-turn region but a little more exposed and feasibility (more SASA) in C-terminal region and key functional residues of C-terminal such as positive residues Arg58, lys62 and Arg (His)64. These data suggested that Od1 has similarity with BmK M1 but has more toxicity to sodium channel. In the other hand 5-turn proximity of C-terminal to 5-turn in BmK M1with cis peptide bond is less than Od1 without cis peptide bond which is a confirmation with experimental data about BmK M1.A better understanding of the 3-D structure of Od1and comparison to BmK M1 will be helpful for more investigation of functional characters action of natural toxins with a specialized role for VGSCs.