spider toxins comprising disulfide-rich and linear amphipathic domains: a new class of molecules...

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/febs.12547 This article is protected by copyright. All rights reserved.

Spider toxins comprising disulfide-rich and linear amphipathic domains: A

new class of molecules identified in the lynx spider Oxyopes takobius

Alexander A. Vassilevski#, Maria Y. Sachkova, Anastasija A. Ignatova, Sergey A. Kozlov,

Alexei V. Feofanov, Eugene V. Grishin

M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian

Academy of Sciences; ul. Miklukho-Maklaya, 16/10, 117997 Moscow, Russia

#Corresponding author. Tel.: 7-495-336-6540; Fax: 7-495-330-7301; E-mail: [email protected].

www.ibch.ru

Running title: Two-domain spider toxins

Article type : Original Article

Abbreviations: AMP, antimicrobial peptide; BCECF, 2′,7′-bis(2-carboxyethyl)-5(6)-

carboxyfluorescein acetoxymethyl ester; CLSM, confocal laser scanning microscopy; DOPC,

1,2-dioleoyl-glycero-3-phosphocholine; DOPE, 1,2-dioleoyl-glycero-3-

phosphoethanolamine; DOPG, 1,2-dioleoyl-glycero-3-phosphoglycerol; ESM, extra

structural motif; FBS, fetal bovine serum; GUV, giant unilamellar vesicle; ICK, inhibitor

cystine knot; LB, liquid broth; MAP, membrane-active peptide; MEM, minimum essential

medium; MIC, minimal inhibitory concentration; PI, propidium iodide; PQM, processing

quadruplet motif; PSM, principal structural motif; RP, reversed-phase; SEC, size-exclusion

chromatography; TFA, trifluoroacetic acid.

Keywords: spider venom, neurotoxin, cytotoxin, antimicrobial peptide, precursor protein,

inhibitor cystine knot, transcriptome, modular toxin

Database linking: The protein sequences of spiderines (OtTx) reported in this paper have

been submitted to the UniProt Knowledgebase (UniProtKB) under the accession numbers

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This article is protected by copyright. All rights reserved.

P86716–P86719. The nucleotide sequences encoding OtTx have been submitted to the

GenBank under the accession numbers JX134894–JX134897.

Abstract

Apart from the conventional neurotoxins and cytotoxins, venom of the lynx spider

Oxyopes takobius was found to contain two-domain modular toxins named spiderines

OtTx 1a, 1b, 2a and 2b. These toxins show both insecticidal activity (median lethal dose

against flesh fly larvae of 75 µg/g) and potent antimicrobial effects (minimal inhibitory

concentrations in the range of 0.1–10 µM). Full sequences of the purified spiderines were

established by a combination of Edman degradation, mass spectrometry and cDNA cloning.

They are relatively large molecules (~110 residues; 12.0–12.5 kDa) and consist of two

distinct modules separated by a short linker. The N-terminal part (~40 residues) contains no

cysteine residues, is highly cationic, forms amphipathic α-helical structures in membrane-

mimicking environment, and shows potent cytolytic effects on cells of different origin. The

C-terminal part is disulfide-rich (~60 residues; 5 S-S bonds) and contains the inhibitor cystine

knot (ICK/knottin) signature. The N-terminal part of spiderines is much alike linear cytotoxic

peptides found in different organisms, whereas the C-terminal part corresponds to the usual

spider neurotoxins. We synthesized the modules of OtTx 1a and compared their activity to

that of the full-length mature toxin produced recombinantly, highlighting the importance of

the N-terminal part that in both insecticidal and antimicrobial assays retained full-length toxin

activity. The unique structure of spiderines fills up the missing part of the two-domain spider

toxin ensemble.

Introduction

Spiders are widely acknowledged to produce potent and selective toxins [1, 2]. For

instance, the black widows (genus Latrodectus, one of the few that is dangerous to humans)

produce an array of latrotoxins, exerting specific lethal effects to mammals (α-latrotoxin),

insects (latroinsectotoxins), and crustaceans (α-latrocrustatoxin) at doses in the order of 10–

100 μg/kg [3]. Moreover, it turns out that spider venoms are complex mixtures of sometimes

very different compounds, and each compound may have its own specific biological activity.

For instance, Agelenopsis aperta studied by the Adams laboratory produces an array of α-

agatoxins (low-molecular-weight blockers of postsynaptic glutamate receptors), μ-agatoxins

(peptide activators of sodium channels) and ω-agatoxins (peptide blockers of diverse calcium

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channels) [4]. With the recent description of compounds that affect “unconventional” targets,

such as pain receptors in humans [5, 6], the pharmacological potential of spider venom seems

vast but largely unexplored. Only some dozen different species have been investigated into

detail, representing a minor faction of the known biodiversity. It seems apparent that the toxin

repertoire available in spider venoms will grow with the progress of studies.

At present the known spider venom compounds are conventionally split into three

categories: (i) low-molecular-mass chemicals of diverse nature (from simple compounds like

salts to elaborate molecules like acylpolyamines [7, 8]), (ii) peptides (up to 10 kDa), and

(iii) proteins [9]. The peptidic component is the most abundant and functionally important in

most investigated species, and peptides may come in two flavors: (a) neurotoxins, most

usually rich in disulfide bridges and conforming to the inhibitor cystine knot (ICK/knottin)

fold [10], and (b) cytotoxins, typically linear peptides adopting helical conformation upon

interaction with lipid membranes [11]. The latter group is also referred to as membrane-active

peptides (MAPs) or antimicrobial peptides (AMPs) due to the activities of those molecules.

There is growing evidence that “spoils” this simple and clear-cut classification. For

example, the Liang group has discovered in spider venom disulfide-rich peptides presenting

non-ICK types of fold, such as the disulfide-directed hairpin (DDH) [12] and the Kunitz fold

[13]. The colipase fold was assumed for a polypeptide isolated from Hadronyche versuta

venom [14]. It was found that cytotoxic peptides may contain S-S bridges [15]. Our group

has been particularly intrigued by the variety of so-called “modular” toxins that comprise two

modules or domains, each corresponding to a “usual” spider toxin. To date, we have

described the following combinations: (i) ICK-ICK, two ICK domains in one toxin [16],

(ii) ICK-AMP, an ICK domain decorated with a long linear C-terminal extension conferring

membrane activity [17], and (iii) AMP-AMP, two short linear MAPs joined into one long

structure [18]. Importantly, other research groups have also described modular toxins of the

ICK-ICK [19] and ICK-AMP types [20].

In this communication, we describe spider toxins with the fourth (and final) possible

combination of modules, AMP-ICK, which has long remained elusive. The lynx spider

Oxyopes takobius (Oxyopidae) is one of the fairly well investigated species. The Corzo

laboratory has published data on the major neurotoxin oxytoxin 1 (OxyTx 1), and two

families of cytotoxins, oxyopinins 1 and 2a–2d [21], and we have more recently presented

oxyopinin 4a [15]. We find that four new toxins from O. takobius, OtTx 1a, 1b, 2a and 2b,

are exceptionally long (~110 residues) and consist of a long N-terminal linear part (~40

residues), a short linker (a potential cryptic maturation motif), and a C-terminal disulfide-rich

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domain with the ICK signature (~60 residues; 5 S-S bonds). We show our data on

purification, sequencing, and activity tests of OtTx. In addition, we synthesized the modules

of OtTx 1a and compared their activity to that of the full-length mature toxin. Our results

suggest that spiders evolved to produce all possible kinds of two-domain modular toxins.

Results

Toxin purification

To isolate insecticidal toxins from the crude venom of O. takobius, we followed our

usual strategy [22] that combines size-exclusion chromatography (SEC) and reversed-phase

(RP) HPLC (Fig. 1). The major peptide-containing SEC fraction was found to contain

numerous components, including those previously described by the Corzo group [21] and by

us [15] (labeled in Fig. 1). Most of the molecular mass species detected in this fraction by

MALDI MS had standard m/z values, characteristic of spider toxins (2–8 kDa). Interestingly,

we also found comparatively high-molecular-mass components (12–12.5 kDa) that eluted at

~35-45% acetonitrile from the C4–C5 reverse phase (Fig. 1 B,C). These components were

insecticidal against flesh fly larvae and also showed cytolytic effects against cells of different

origin (see below). Two components were purified by rechromatography to achieve ~95%

homogeneity (assessed by RP-HPLC) and named OtTx 1 (O. takobius toxin 1; measured

molecular mass 12030 Da) and OtTx 2 (12415 Da).

OtTx sequencing

N-Terminal Edman degradation of the purified toxins yielded the following partial

sequences: KFKWGKLFSTAKKLYKKGKKLSKNKNFKKALKFGK (OtTx 1) and

KFKLPKINWGKLASKAKDVYKKGQKLAKNKNVKKALK (OtTx 2).

cDNA database was created for O. takobius venom glands in collaboration with

DuPont Agriculture and Nutrition. We then performed library analysis in accordance with an

earlier elaborated strategy that included searches for structural elements and motifs specific

for secreted polypeptide sequences and spider toxins [24, 29, 30]. As a result, putative spider

toxin sequences were deduced. OtTx N-terminal sequences were used to query the library,

and clones coding for the full-length toxins were identified. In addition to sequences

matching those established by Edman degradation exactly, we also found close homologues

to both OtTx 1 and OtTx 2. The isoforms identified in the venom were therefore named

OtTx 1a and OtTx 2a, whereas the homologous putative toxins received the names OtTx 1b

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and OtTx 2b (Fig. 2A). OtTx 1b differs from OtTx 1a at position 10: alanine substitutes for

threonine. Phenylalanine is found at position 4 in OtTx 2b instead of leucine in OtTx 2a. The

calculated molecular masses of OtTx 1a (12031.0 Da) and OtTx 2a (12416.4 Da) are in

agreement with the measured values (12030 Da and 12415 Da, see above).

10 cysteine residues are found in all toxins. Attempt to alkylate native OtTx with

vinylpyridine fails, whereas alkylation of the toxins with disulfide bonds reduced by

dithiothreitol increases the measured molecular masses to 13090 Da (OtTx 1a) and 13475 Da

(OtTx 2a), indicating that all 10 cysteine residues participate in formation of 5 intramolecular

S-S bridges.

OtTx sequence analysis

OtTx 1 (108 residues) and OtTx 2 (113 residues) are highly homologous, they share

~80% identity (see alignment in Fig. 2A); the major difference is the N-terminal five-residue

extension in OtTx 2.

Close inspection of the new toxin sequences results in delineation of two parts

(modules), or domains. The N-terminal part (residues 1–44 in OtTx 1 and 1–49 in OtTx 2)

contains no cysteine residues, whereas the C-terminal domain (residues 50–108 in OtTx 1

and 55–113 in OtTx 2) is cysteine-rich: 10 Cys residues account for 17% of the sequence.

The short linker that joins the two modules (EEHEP, residues 45–50 in OtTx 1; and

EVHEP, residues 50–55 in OtTx 2) resembles closely the processing quadruplet motif

(PQM). This motif is known to indicate the processing cleavage site in precursors of spider

toxins and demarcate the prosequence from the mature chain [24]. It can be represented as

X1X2X3R, where any Xn = E, and R is the functionally important arginine residue: cleavage

occurs at its carbonyl group (PQM = EEAR in OtTx precursors, see below); in some cases,

glutamic acid residues may be found further upstream. In the OtTx linker, we find a mutated

version of PQM: the crucial arginine is changed to proline.

Homology searches using the UniProt database identifies two toxins sharing

considerable similarity with the C-terminal part of OtTx. Both are the neurotoxins

antagonizing voltage-gated calcium channels and described previously by Corzo et al.:

oxytoxin 1 (OxyTx 1) from the same O. takobius venom [21] (this toxin was also later found

in Oxyopes lineatus), and oxytoxin 2 (OxyTx 2) from O. lineatus [31]. There are ~40% and

~45% identical residues in the C-terminal domain of OtTx 1 and OxyTx 1 and OxyTx 2,

respectively (see alignment in Fig. 3). Moreover, these toxins present the same cysteine

signature, pointing to a common folding pattern:

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C1X6C2X3C

3XC4C5X4C6XC7XmC8XC9XnC

10, m = 11 in OtTx and OxyTx 2, 24 in OxyTx 1, n

= 9 in OxyTx 2, 10 in OtTx, and 11 in OxyTx 1. This signature can be reduced to the more

general principal structural motif (PSM) CX6CXnCC (C1X6C2X5C

4C5 in our case), and extra

structural motif (ESM) CXCXnCXC (C6XC7XmC8XC9 in our case) characteristic of spider

neurotoxins [24]. PSM and ESM in turn conform to the more general ICK motif CX2–7CX3–

11CX0–7CX1–17CX1–19C (C1X6C2X5C

4C5X4C6Xm+4C

9 in our case) found in numerous peptides

[32, 33]. We conclude that in OtTx, as well as in OxyTx, the three core ICK disulfides (C1–

C5, C2–C6, C4–C9) are supplemented by two additional S-S bonds (C3–C10, C7–C8, the latter is

in fact part of the ESM and is found in most spider toxins containing 8 cysteine residues).

Lower similarity (~38% identical residues in OtTx 1) is noted with the Tx3-6 [34] and

PRTx23C2 [35] toxins from the Brazilian spiders Phoneutria nigriventer and Phoneutria

reidyi (Ctenidae) that are thought to target calcium channels. Interestingly, a further sixth

disulfide bridge is found in these Phoneutria toxins. Another feature of the C-terminal

domain of OtTx is the high proline residue content (~10%).

To the contrary, no significantly similar sequences were found in public databases for

the N-terminal linear domain of OtTx. This domain is highly cationic (charge of +16 and +13

at pH 7 in OtTx 1 and OtTx 2, respectively; pI ~11), and has a high α-helix-forming

propensity as predicted by different algorithms such as [36]. The positively charged and

hydrophobic residues are arranged over the sequence so that they get segregated in α-helical

conformation (see below). The mentioned properties are common to MAPs/AMPs found in

several spider venoms, including O. takobius [15, 21]. It is interesting to note the differential

abundance of certain residues in the N-terminal linear domain of OtTx. For example, in

OtTx 1a: Lys (~36%) vs. other charged residues Asp, Arg, and Glu (0%); Leu (~14%) and

Phe (~9%) vs. Ile, Met, and Val (0%).

We conclude that OtTx form a new and unique family of spider toxins, encompassing

an N-terminal linear and C-terminal ICK domains (Fig. 2B).

OtTx precursor structure

As described above, O. takobius venom glands cDNA database analysis permitted

identification of clones coding for OtTx. Fig. 4 presents sequence of one cDNA clone

encoding OtTx 1a. Just like most of the known spider toxins [1], OtTx are synthesized in the

form of conventional prepropeptides (OtTx 1 precursors, pOtTx 1, contain 166 residues) that

consist of three parts: (i) N-terminal signal (pre-) peptide (18 residues, identified by the

SignalP 4.0 Server [37]), (ii) prosequence (40 residues), and (iii) C-terminal mature toxin.

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The prepro- part of the precursors is well conserved (only one substitution between OtTx 1a

and OtTx 2a). The prosequence is highly acidic (charge −14 at pH 7, pI ~3), effectively

cancelling out the cationicity of the mature chain (the charge of the full-length OtTx 1

propeptide is −3 at pH 7). One of the functions of the acidic prosequences in precursors of

cytolytic peptides is believed to be inhibition of the mature chain activity during biosynthesis

[38]. It ends with the PQM, signature of the posttranslational processing cleavage (see

explanation above): 37EEAR40.

Production of the full-length OtTx 1a and its modules

To provide enough material for functional studies, we produced recombinant OtTx 1a.

Since the toxin contains multiple disulfide bonds, we chose to produce it in the form of a

cleavable fusion with the helper protein thioredoxin (Trx) [39], which is known to assist

correct folding in disulfide-rich partners. Mature OtTx 1a contains no methionine residues;

this property permitted usage of cyanogen bromide as the cleavage agent to process the

fusion protein: a Met residue was introduced in between Trx and OtTx 1a. To mask the

membrane-damaging activity of the N-terminal domain of the toxin, we used the natural

prosequence from pOtTx 1a. Such approach has been shown effective in case of a number of

MAPs/AMPs, and enabled recombinant production of otherwise highly cytotoxic compounds

[38, 40]. The fusion protein Trx-OtTx 1a also contained an oligohistidine sequence for

affinity purification by metal-chelate chromatography (see Fig. 5A for the fusion protein

organization scheme). Following cleavage of the chimeric protein by CNBr, the target

polypeptide was purified by RP-HPLC (Fig. 5B). Recombinant OtTx 1a was compared with

the native toxin, and found to have the same molecular mass, insecticidal activity against

flesh fly larvae, antimicrobial activity against Escherichia coli (see below), and

chromatographic retention time. It was concluded that OtTx 1a was successfully produced

recombinantly; the final yield was ~0.5 mg/L of bacterial culture. Further in-depth

investigations were performed using the recombinant OtTx 1a due to the drastic shortage of

biological material for native toxin purification: only as much as 0.5 μL of venom could be

milked from single specimen.

To compare their activity with the full-length toxin, we produced the separate

domains of OtTx 1a. We should note here that not only the linker sequence resembles the

PQM, but also we find a mutated version of the inverted PQM (iPQM), another processing

motif found in so-called complex precursors containing multiple mature chains and also

precursors of two-chain toxins [30, 41]. This motif delineates the C-terminus of the mature

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chain, just like the regular PQM delineates the N-terminus. iPQM can be represented as

RX1X2X3, where any Xn = E, and R is the functionally important arginine residue: cleavage

occurs at its carbonyl group. In our case, Gln-42 seems to substitute the crucial arginine. With

this reasoning, we decided to synthesize the “unmutated processed” version of the N-terminal

domain of OtTx 1a (residues 1–41) as well as the C-terminal domain (residues 50–108). Due

to the predicted membrane activity we named the N-terminal domain OtTx 1a-AMP, whereas

structural peculiarities urged us to name the C-terminal domain OtTx 1a-ICK.

OtTx 1a-AMP was synthesized by conventional peptide chemistry, whereas OtTx 1a-

ICK was produced recombinantly. In the latter case, we did not include the prosequence into

the expression construct, since the potentially harmful N-terminal cytolytic domain was

removed from the toxin. A Met codon was introduced immediately upstream of OtTx 1a-

ICK. After affinity purification the fusion protein was subjected to air oxidation, and the

folded C-terminal domain of OtTx 1a was recovered after CNBr cleavage of the chimera and

RP-HPLC separation of the products. Fraction corresponding to OtTx 1a-ICK gave a

symmetric peak on the chromatogram testifying in favour of unequivocal formation of the S-

S bonds. The yield was ~5 mg/L of bacterial culture, exceeding considerably that of the full-

length toxin and pointing to its cytolytic effects. Purity of both OtTx 1a-AMP and OtTx 1a-

ICK was checked by MALDI MS and analytical chromatography and amounted to >98%. We

should note here that since the cysteine connectivity was not determined, we cannot totally

rule out the possible incorrect folding of the recombinant polypeptides.

OtTx 1a secondary structure

Similarly to primary structure analysis performed above, secondary structure

prediction algorithms [36] result in segregation of OtTx 1a modules. The first ~45 residues

(OtTx 1a-AMP) are predicted to form an α-helix, whereas the C-terminal part (OtTx 1a-ICK)

shows high β-turn propensity. Residues in the linker region (~46–55) have the highest

random coil propensity.

CD measurements corroborate the predictions. They indicate a high content of β-sheet

and β-turn conformation in OtTx 1a (Table 2), consistent with the assumption that this toxin

adopts the ICK fold. The same is true for the isolated toxin ICK domain, OtTx 1a-ICK.

Moreover, OtTx 1a-ICK conformation shows little if any alterations upon peptide transfer

from phosphate buffer to membrane-mimicking environment. To the contrary, the

conformation of the linear domain, OtTx 1a-AMP, showed marked dependency on the

environment: <10% α-helix content in phosphate buffer changed to >70% upon addition of

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membrane-mimicking TFE or SDS (Fig. 6A, Table 2). Changes of corresponding magnitude

were detected in the full-length toxin.

Biological activity of OtTx 1a and its modules

The full-length toxin OtTx 1a possessed equally pronounced insecticidal and wide-

range cytolytic activity: the median lethal dose (LD50) against flesh fly larvae and the median

paralyzing dose (PD50) against tobacco hornworm larvae were 75 μg/g, and minimal

inhibitory concentration (MIC) values against different Gram-positive and Gram-negative

bacterial strains were in the order of 0.1–10 μM (Table 3). To localize the functionally

important part of the toxin, we tested the activity of both OtTx 1a domains. It turned out that

OtTx 1a-AMP retained all of the full-length toxin activity: LD50 against flesh fly larvae and

PD50 against tobacco hornworm larvae were 50 μg/g, and MIC values against different

bacteria were in the low micromolar range (Table 3). To the contrary, OtTx 1a-ICK was

ineffective in both types of test: at doses up to 200 μg/g in insect larvae and concentrations up

to 25 μM in bacterial assays.

OtTx 1a-AMP was further found to induce lysis of human erythrocytes, its effective

concentration that provides 50% hemoglobin release (EC50) is equal to 8 μM. The peptide

presented cytotoxic activity: 50% death of A549 and HeLa cells occurred at concentration of

19±2 μM and 13±2 μM, respectively.

Mechanism of antibacterial activity of OtTx 1a-AMP

Both full-length OtTx 1a and OtTx 1a-AMP are MAPs and get effectively depleted

from solution in the pull-down assay. OtTx 1a-AMP inhibits bacterial growth and possesses

bactericidal activity. For Staphylococcus aureus and Enterococcus faecalis, the minimal

bactericidal concentrations are equal to the MIC values (Table 3). The bactericidal effect of

OtTx 1a-AMP develops quickly. Thus, a 15-min exposure to the peptide at 2×MBC results in

total death of S. aureus. Not more than 2–3 colonies per agar dish are formed after 15–30 min

pre-incubation of S. aureus with the peptide at 1.25 μM, and longer pre-incubation (>1 h)

eliminates viable bacteria totally.

Confocal laser scanning microscopy (CLSM) analysis indicates that the bactericidal

effect of OtTx 1a-AMP is related to permeabilization of the bacterial membrane. A short

exposure of S. aureus to OtTx 1a-AMP results in simultaneous leakage of 2′,7′-bis(2-

carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF) from the cytoplasm

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and intracellular penetration of propidium iodide (PI) accompanied with its binding to DNA

and fluorescence enhancement (Fig. 7C, D). Intact bacterial membrane retains BCECF in the

cytoplasm and excludes PI (Fig. 7A, B).

Discussion

Two-domain modular structure of OtTx

The newly described toxins possess unique modular architecture (see Fig. 2A for

module locations). The N-terminal part of OtTx (OtTx-AMP) corresponds to a more usual

single-domain cytotoxin devoid of cysteine residues. Although no homology is found

between OtTx-AMP modules and other known protein sequences, the former possess the

characteristic traits of linear AMPs. Thus, OtTx 1a-AMP is a highly cationic peptide (39%

lysine residues; charge of +16 at pH 7.0) with a high α-helix-forming propensity (either

predicted by conventional methods or observed experimentally, see Fig. 6A, Table 2). In

membrane-mimicking environment it adopts an amphiphilic α-helical structure with clear

separation of hydrophilic vs. hydrophobic clusters (Fig. 6B). It also shows membrane-binding

activity as probed by the pull-down assay. Most importantly, OtTx 1a-AMP possesses strong

cytolytic activity associated with its membrane-damaging properties (see discussion below).

To the contrary, the C-terminal part of OtTx (OtTx-ICK) contains the archetypical

ICK motif signature most common for spider neurotoxins. Moreover, considerable level of

homology is noted between OtTx-ICK modules and single-domain ICK toxins from the same

or a closely related spider (Fig. 3). Thus, the expectation value of the alignment with

oxytoxin-2 is ~10−7 pointing to a very high probability of common ancestry for the toxins.

Most notably, the cysteine signature (with two additional disulfides grafted onto the ICK

motif) is exactly preserved in OtTx and oxytoxins. We may speculate therefore that OtTx

evolved from oxytoxin-like single-domain peptide by incorporation of the N-terminal AMP

module.

An intriguing observation, to our mind, is the similarity between OtTx and modular

scorpion toxins named scorpines [42, 43]. They share common architecture design: in

scorpines, an N-terminal AMP module is also followed by a C-terminal neurotoxin module.

The C-terminal module in this case corresponds to a typical single-domain scorpion toxin

with the cysteine-stabilized alpha/beta (CSαβ) motif. It turns out that venomous creatures

with non-homologous venom glands have followed common evolutionary pathways to

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produce modular toxins with strikingly similar organization. To emphasize the noted

likelihood, we propose the term “spiderines” for OtTx molecules.

The N-terminal module of OtTx 1a is a powerful cytolytic toxin

The structural features of OtTx 1a-AMP noted above led us to propose that it is a

cytolytic MAP. And indeed the peptide was shown to present wide-spectrum cytolytic

activity. It effectively kills bacterial (Table 3) and eukaryotic cells at micromolar

concentrations and its non-selective potency may be compared to that of the classic cytotoxin

melittin from the honeybee venom.

We conducted experiments to unravel the molecular mechanism of OtTx 1a-AMP

cytolytic activity. The peptide was directly shown to interact with membranes by the pull-

down assay. It was further shown to possess bactericidal as opposed to bacteriostatic effect,

favoring the presumed membrane-damaging activity. The latter was eventually observed by

CLSM (Fig. 7): PI penetrated into the cells treated by the toxin, while BCECF simultaneously

leaked out. Both observations are considered solid arguments in favor of the plasma

membrane breakdown. Full-length OtTx 1a also presents potent antimicrobial activity

(Table 3), and its mode of action most probably corresponds to that of OtTx 1a-AMP.

However, additional studies are needed to find out whether disruption of the capsule and cell

wall occurs, or spiderines are able to diffuse through and target the cytoplasmic membrane.

It is the AMP module that accounts for the toxicity of the full-length molecule

observed in our tests. OtTx 1a-AMP renders OtTx 1a with membrane activity including its

cytolytic effects. It seems that the N-terminal AMP domain is the functional core of OtTx.

We should note, however, that the observed lack of activity in the ICK domain OtTx 1a-ICK

may be due to incorrect disulfide formation. It is also possible that spiderines have evolved to

target certain insects that differ significantly from those tested by us and express a specific

receptor to OtTx-ICK.

Venom composition of lynx spiders

The lynx spider O. takobius is one of the two species from the family Oxyopidae

(lynx spiders) that has been investigated in terms of molecular composition of the venom (the

other is O. lineatus). The Corzo group has described two particular groups of toxins in

Oxyopes. Oxytoxins, including OxyTx 1 from O. takobius [21] and an identical toxin from

O. lineatus, and OxyTx 2 from O. lineatus [31], are typical ICK neurotoxins (see Fig. 3 for

sequences). To the contrary, oxyopinins, including oxyopinin 1 and the family of four closely

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related oxyopinins 2a–2d, are typical linear cytolytic peptides [21]. Our recent study

described the cytolytic peptide oxyopinin 4a that contains one disulfide bond in the N-

terminal part [15]. The present paper further widens the known variability of toxins in lynx

spiders. Spiderines (OtTx 1a and OtTx 2a) discovered in the venom of O. takobius and their

close homologs (OtTx 1b and OtTx 2b) found in the cDNA database contain two distinct

domains, each bearing significant similarity to the usual spider toxins.

Diversity of modular toxins in spiders

The present work adds a fourth class to the known modular toxin variability in spider

venoms (see Fig. 2B, Table 4). The observed combination of modules in OtTx can be

presented as “AMP+ICK”, referring to an N-terminal linear amphiphilic domain and a C-

terminal knottin. The “reversed” combination of modules, ICK+AMP, was previously

described in several toxins from spiders of the Lycosoidea superfamily: LtTx from

Lachesana tarabaevi (Zodariidae) [17], and CSTX from Cupiennius salei (Ctenidae) [20].

Moreover, these “hetero” combinations are joined by both possible “homo” variants. The first

example of modular spider toxin was cyto-insectotoxins (CITs) in the venom of L. tarabaevi

containing two linear modules (AMP+AMP) [18]. Later two-domain spider toxins containing

two knottin modules (ICK+ICK) were discovered in the venom of Cheiracanthium

punctorium (Miturgidae) and Haplopelma schmidti (Theraphosidae) [16, 19]. It is seen that

modular toxins are quite widespread in spiders, and further studies will probably reveal even

greater diversity.

The C-terminal AMP parts of LtTx and CSTX were shown to present membrane-

active properties. It was proposed for the ICK+AMP toxins that the AMP module acts like a

membrane anchor, which may either assist target receptor binding through “membrane

access” or stabilize the complex by preventing the dissociation [17]. It was also found that the

AMP module endows the toxins with membrane-damaging activity. Since the same module

types, albeit in a reversed manner, are found in OtTx, one may assume similar functional

roles of the modules. We note, however, that the AMP modules of the ICK+AMP toxins

show much weaker cytotoxicity compared to OtTx 1a-AMP. Indeed, the latter peptide is one

of the most powerful cytotoxins of its kind (see above). Moreover, the AMP part seems to

account for the most of the full-length OtTx biological properties, in contrast to LtTx and

CSTX, where the modules seem equally important.

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Experimental Procedures

Our general guidelines regarding spider venom separation, peptide sequencing and

antimicrobial/insecticidal activity tests are published elsewhere [22].

OtTx isolation

The venom of Oxyopes takobius (Oxyopidae)1 was purchased from Fauna

Laboratories, Ltd. (Almaty, Republic of Kazakhstan). 25 µl of liquid venom was lyophilized

and redissolved in 100 µl of buffer used in SEC: 150 mM NaCl, 20 mM NaH2PO4.

Fractionation was performed on a TSK 2000SW column (7.5×600 mm, 12.5 nm pore size, 10

µm particle size; Toyo Soda Manufacturing Co., Tokyo, Japan) at a flow rate of 0.5 mL/min.

The dominant fraction was then separated by RP-HPLC on a Delta-Pak C4 column

(3.9×150 mm; Waters, Milford, MA, USA) in a linear gradient of acetonitrile concentration

(0–10% in 10 min, 10–50% in 80 min, and 50–70% in 20 min) in 0.1% trifluoroacetic acid

(TFA) at a flow rate of 1 mL/min. One of the three late-eluting highly active antimicrobial

fractions was further separated on a Jupiter C5 column (2×150 mm; Phenomenex, Torrance,

CA, USA) in a linear gradient of acetonitrile concentration (20–60% in 60 min) and 0.1%

TFA at a flow rate of 0.3 mL/min. Final purification was performed by rechromatography on

the same column, and >95% purity was achieved. At each stage, eluate absorbance was

monitored at 210 nm and 280 nm.

Analytical methods

Reduction of disulfide bonds by 1,4-dithiothreitol and alkylation of thiol groups with

4-vinylpyridine were performed in accordance with the published guidelines [22].

Samples were analyzed by MALDI MS with M@LDI-LR (Micromass, Manchester,

UK) and Ultraflex TOF-TOF (Bruker Daltonik GmbH, Bremen, Germany) spectrometers as

reported [18].

N-terminal sequencing was carried out by automated stepwise Edman degradation

using a Procise Model 492 protein sequencer (Applied Biosystems, Foster City, CA, USA)

according to the manufacturer’s protocol.

Absorption spectra were recorded on a Hitachi U-3210 spectrophotometer (Tokyo,

Japan). OtTx 1a, OtTx 1a-ICK and OtTx 1a-AMP concentration was determined using the

molar extinction coefficients at 280 nm (ε280) of 18,950 M−1cm−1, 11,980 M−1cm−1 and 6,970

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M−1cm−1 respectively, calculated by the GPMAW program (Lighthouse Data, Odense,

Denmark).

Peptide synthesis

The N-terminal fragment of OtTx 1a (OtTx 1a-AMP) was synthesized on a peptide

synthesizer Syro I (MultiSynTech, Witten, Germany) using the Fmoc/t-butyl chemistry

following a described procedure [23]. The purity of the synthetic peptide was verified by RP-

HPLC and MALDI MS.

Venom gland cDNA library

mRNA was isolated from O. takobius venom glands. All steps were carried out in

collaboration with DuPont Agriculture and Nutrition (Newark, DE, USA) as described in

detail elsewhere [24].

Expression vector construction

DNA sequence encoding OtTx 1a propeptide was constructed from synthetic

oligonucleotides (Table 1) using the PCR technique. A methionine codon was introduced

downstream of the prosequence. The full-length DNA was amplified using a forward primer

containing a KpnI restriction site and a reverse primer containing an XhoI restriction site and

a stop codon. The DNA sequence encoding OtTx 1a-ICK was obtained using a forward

primer containing a BamHI restriction site and a methionine codon. The PCR fragments

encoding target polypeptides were gel purified, digested by suitable restriction enzymes and

cloned into the expression vectors pET-32b (OtTx 1a propeptide) and pET-32a (OtTx 1a-

ICK) (Novagen, Madison, WI, USA). The resulting constructs were checked by sequencing.

Fusion protein production, purification, and refolding

A detailed procedure of transgene expression and fusion protein purification by

affinity chromatography can be found elsewhere [25]. 2 M guanidine hydrochloride was

added in all solutions used to purify Trx-OtTx 1a-ICK. This protein was further subjected to

air oxidation: 0.01-0.02 mM of protein solution was dialyzed against 100 mM Tris-HCl (pH

8.0), 80 mM NaCl, and 50 mM imidazole and then incubated under permanent vigorous

stirring at 4° C for 5 days. The refolding procedure was followed by CNBr cleavage, and

disulfide bond formation in OtTx 1a-ICK was monitored by HPLC and MS.

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Fusion protein cleavage and target product purification

HCl was added to the fusion protein solution to the final concentration of 100 mM and

protein cleavage with cyanogen bromide was performed as recommended in [26].

Recombinant polypeptides were purified using RP-HPLC on a Jupiter C5 column (250×10

mm, 300 Å, 10 μm; Phenomenex) in a linear gradient of acetonitrile concentration (0–80% in

60 min) in 0.1% TFA at a flow rate of 5 mL/min. The purity of the target polypeptides was

checked by MS, N-terminal sequencing, and the concentration was determined by absorption

spectroscopy.

Liposome preparation

Zwitterionic 1,2-dioleoyl-glycero-3-phosphocholine (DOPC) liposomes and anionic

1,2-dioleoyl-glycero-3-phosphoethanolamine/1,2-dioleoyl-glycero-3-phosphoglycerol

(DOPE/DOPG) liposomes were used in the study (all lipids from Avanti Polar Lipids,

Alabaster, AL, USA). Dry DOPC or DOPE/DOPG mixture (3:7) was dissolved in

chloroform-methanol (2:1, v/v), dried on a rotor evaporator to obtain a lipid film. It was

further lyophilized and suspended in 110 mM NaCl, 50 mM phosphate buffer (pH 7.2) to a

final lipid concentration of 10 mM. The suspension was ultrasonicated and extruded (21

times, room temperature) using a Mini-Extruder (Avanti Polar Lipids) through a

polycarbonate filter with 1000 nm-diameter pores (Whatman, Maidstone, UK) to obtain giant

unilamellar vesicles (GUVs). Large unilamellar vesicles (LUVs) were produced by

consequent extrusion of the GUV suspension through a filter with 100 nm-diameter pores

(Whatman).

Pull-down assay

Polypeptides were added to GUV suspension to the final concentration of 5 µM.

Polypeptide solution in phosphate buffer was used as control. After incubation at 37° C for 1

h the mixture was centrifuged for 20 min at top speed using a MiniSpin centrifuge

(Eppendorf, Hamburg, Germany). The supernatant was analyzed by RP-HPLC.

CD

Polypeptides were dissolved in 50 mM phosphate buffer (pH 7.2), 50% (v/v)

trifluoroethanol, and 20 mM SDS to the concentration of 1 mg/mL. CD spectra were obtained

on a J-810 spectropolarimeter (Jasco, Tokyo, Japan) as described previously [18].

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Antimicrobial assays

Determination of MIC values was performed with a two-fold microtiter broth dilution

assay, for details see [22]. Arthrobacter globiformis VKM Ac-1112, Bacillus subtilis VKM

B-501, E. faecalis VKM B-871, E. coli DH5α, Pseudomonas aeruginosa PAO1, and

S. aureus 209P were used.

Bactericidal effect of the peptide OtTx 1a-AMP against E. faecalis and S. aureus was

estimated by seeding bacteria to liquid broth (LB)-agar media after 3 h incubation with the

peptide at MIC, 2×MIC and 4×MIC and examining the appearance of bacterial colonies in 24

h. Kinetics of the bactericidal action was studied by incubating S. aureus with OtTx 1a-AMP

at 1.25 and 2.5 μM for 0.25, 0.5, 1 and 2 h followed by twenty-fold dilution of the culture

with peptide-free medium, inoculation into LB-agar and examination of colony appearance in

24 h.

Hemolysis

Human capillary blood was collected in a tube with heparin (10 units/mL), diluted to

(1.0±0.1)×107 cells/mL with Roswell Park Memorial Institute 1640 (RPMI-1640) medium

(PanEco, Moscow, Russia) containing 10% fetal bovine serum (FBS; HyClone, Logan, UT,

USA) and incubated with the peptide OtTx 1a-AMP (0.16–20 μM, twofold increment) for 3 h

at 37° C and gentle shaking. Hemoglobin release was measured as described previously [27].

Cytotoxicity

Human cervix epitheloid carcinoma HeLa cells and human lung adenocarcinoma

A549 cells were grown (37° C, 5% CO2) in Dulbecco’s minimum essential medium (MEM)

and Eagle’s MEM (both from PanEco), respectively, supplemented with 10% FBS and 2 mM

L-glutamine. For the survival assays cells were seeded into 96-well plates at 1×105 cells/mL,

and in 24 h OtTx 1a-AMP was added into the wells (0.16–20 μM, twofold increment). Cells

were incubated with the peptide for 24 h, and cell viability was estimated using a colorimetric

3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT)-based assay [28].

Microscopy

CLSM measurements were performed with the SP2 confocal inverted microscope

(Leica Microsystems GmbH, Wetzlar, Germany) using the water immersion 63×/1.2 NA

HCX PL APO objective. A typical voxel size was 0.15×0.15×0.9 μm. Excitation wavelengths

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were 561 nm for PI (Sigma-Aldrich, St. Louis, MO, USA) and 488 nm for BCECF (Sigma-

Aldrich). Laser power was 3–10 μW at the sample. Fluorescence emission was collected in

the 650–700 nm range for PI and in the 500–550 nm range for BCECF.

S. aureus cells were pre-incubated with BCECF (10 μM) in Hank’s balanced salt

solution for 30 min at 37° C, exposed to OtTx 1a-AMP (1.25 μM) and PI (15 μM) for 5–10

min in Mueller-Hinton broth (Sigma-Aldrich), centrifuged gently (in order to concentrate the

cells) and examined with CLSM. Control cells were subjected to the same procedures but

without addition of the peptide.

Insecticidal assay

Insect toxicity was assayed against flesh fly Sarcophaga carnaria maggots as

described previously [22] and the tobacco hornworm Manduca sexta larvae. The latter (the

first instar) were received from the Severtsov Institute of Ecology and Evolution, Russian

Academy of Sciences, and injected into the third segment analogously to the fly maggots.

Footnotes: 1Previously classified as Oxyopes kitabensis by Andrey Feodorov (Fauna

Laboratories, Ltd., Almaty, Republic of Kazakhstan) and then corrected by Wolfgang

Nentwig (University of Bern).

Acknowledgements

We thank Drs. Oksana V. Nekrasova and Yuliya V. Korolkova for their advice

concerning recombinant protein production. We are grateful to Andrey A. Zagorinsky

(Laboratory for Soil Zoology and General Entomology, the Severtsov Institute of Ecology

and Evolution) for providing M. sexta larvae.

This work was supported by the Russian Foundation for Basic Research [grant

numbers 11-04-00706 and 12-04-33151], the Program of Molecular and Cell Biology of the

Russian Academy of Sciences, and by the Ministry of Education and Science of the Russian

Federation [agreement number 8794].

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Tables

Table 1. Synthetic oligonucleotides used to construct toxin-encoding DNA.

Restriction sites (KpnI in f-OtTx 1a, XhoI in rev-OtTx 1a, and BamHI in f-OtTx 1a-

ICK) are underlined, the methionine codon is in bold and italics, the stop codon is in bold.

Name Sequence (5’→3’)

f-OtTx 1a CTGCGGTACCACCGGTGACCTGGAAACCGAACTGGAGGCTTCTGAA

2f-OtTx 1a CTGCAGGAACTGCAGGAAGCTCTAGACCTGATCGGTGAAACCTCTCTGGA

3f-OtTx 1a-M ATCTCTCGAAGCTGAGGAGCTCGAAGAAGCCCGTATGAAATTCAAGTGG

4f-OtTx 1a GGCAAGCTTTTCTCCACCGCTAAAAAACTATACAAGAAGGGTAAGAAACT

5f-OtTx 1a GTCCAAAAACAAGAACTTCAAGAAAGCTCTGAAATTCGGCAAACAGCTCG

6f-OtTx 1a CTAAAAACCTGCAGGCTGGTGAAGAGCACGAACCGGGTACTCCAGTTGGT

7f-OtTx 1a AACAACAAATGCTGGGCTATCGGCACCACTTGCTCTGACGACTGCGACTG

8f-OtTx 1a CTGTCCGGAACACCACTGCCACTGCCCGGCTGGTAAATGGCTGCCGGGTC

9f-OtTx 1a TGTTCCGTTGCACCTGCCAGGTTACCGAATCTGACAAAGTTAACAAATGC

1/2rev-OtTx 1a TTCCTGCAGTTCCTGCAGTTCAGAAGCCTCCAGTTC

2/3rev-OtTx 1a TCCTCAGCTTCGAGAGATTCCAGAGAGGTTTCACCG

3/4rev-OtTx 1a-M GGTGGAGAAAAGCTTGCCCCACTTGAATTTCATACGG

4/5rev-OtTx 1a GAAGTTCTTGTTTTTGGACAGTTTCTTACCCTTCTTGT

5/6rev-OtTx 1a CAGCCTGCAGGTTTTTAGCGAGCTGTTTGCCGAATT

6/7rev-OtTx 1a AGCCCAGCATTTGTTGTTACCAACTGGAGTACCCGG

7/8rev-OtTx 1a CAGTGGTGTTCCGGACAGCAGTCGCAGTCGTCAGAG

8/9rev-OtTx 1a GGCAGGTGCAACGGAACAGACCCGGCAGCCATTTAC

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rev-OtTx 1a GCCGCTCGAGCTATTCAGCCGGTGGGCATTTGTTAACTTTGTCAGA

f-OtTx 1a-ICK AAAAAAGGATCCATGGGTACTCCAGTTGGTAACAACAAATGCTGGGCTATCGGCACCACT

Table 2. Circular dichroism data.

Peptide Solution Secondary structure content, %

α-Helix β-Sheet β-Turn Unordered

OtTx 1a

Phosphate buffer 9.0 29.9 24.0 37.0

20 mM SDS 47.8 7.0 15.0 30.1

50% TFE 48.4 17.7 19.9 14.1

OtTx 1a-AMP


20 mM SDS 81.3 2.0 1.0 15.7

50% TFE 73.5 1.7 6.2 18.6

OtTx 1a-ICK


20 mM SDS 21.5 24.6 22.6 31.3

50% TFE 17.8 42.2 16.8 23.1

Table 3. Antimicrobial activity of OtTx 1a and its modules.

Bacterial strain

Peptide

OtTx 1a (full-length) OtTx 1a-AMP OtTx 1a-ICK

MIC, μM

Arthrobacter globiformis VKM

Ac-1112 0.6–1.25 0.6–1.25 >25

Bacillus subtilis VKM B-501 0.12–0.25 0.12–0.25 >25

Enterococcus faecalis VKM B-871 N/T 5–10 N/T

Escherichia coli DH5α 0.12–0.25 0.12–0.25 >25

Pseudomonas aeruginosa PAO1 6–12 0.37–0.75 >25

Staphylococcus aureus 209P N/T 0.6–1.25 N/T

N/T, not tested

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Table 4. Modular toxin diversity in spider venoms.

N-Terminal

module

C-Terminal module

AMP ICK

AMP AMP+AMP

CITs in Lachesana tarabaevi [18]

AMP+ICK

OtTx in Oxyopes takobius, present work

ICK

ICK+AMP

LtTx in Lachesana tarabaevi [17]

CsTx-1 in Cupiennius salei [20]

ICK+ICK

CpTx in Cheiracanthium punctorium [16]

DkTx in Haplopelma schmidti [19]

Supplementary Material

Suppl. Fig. 1. Pull-down assay of OtTx 1a-AMP. 1 nmol of peptide was incubated in

phosphate buffer alone (control, solid line) or with DOPE/DOPG GUV suspension (dashed

line) and analyzed by HPLC on a Jupiter C5 column (250×4.6 mm, 300 Å, 10 μm;

Phenomenex) in a linear gradient of acetonitrile concentration (0–60% in 60 min).

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Fig. 1. OtTx isolation from the venom of Oxyopes takobius. (A) O. takobius crude

venom separation by size-exclusion chromatography. (B) HPLC of the dominant fraction.

(C) Second step HPLC of a late-eluting fraction. In (A, B), the acetonitrile concentration

gradient is shown with solid lines, and the fractions containing OtTx are marked by shading.

Fractions containing the known compounds are labeled.

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Fig. 2. Amino acid sequences of OtTx precursors and the diversity of modular toxins

in spiders. (A) Alignment of OtTx precursors. Gaps are introduced into OtTx 1 to enable

sequence comparison. Negatively charged residues are in red; positively charged, blue;

cysteine residues are shaded in green; different residues are on a blue background. Regions

corresponding to the signal peptide, propeptide and parts of the mature toxins are marked by

arrows above the sequences. Propeptide processing site is marked by a red arrow. Disulfide

bridges contributing to the canonical ICK motif are shown by lines above the sequences,

additional disulfides are drawn below. (B) Schemes showing possible organization of

modular toxins in spiders. Rectangles represent linear modules that form amphiphilic α-

helices. Circles designate ICK domains.

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Fig. 3. Sequence alignment of OtTx 1a domains and similar peptides. Above, the N-

terminal part of OtTx 1a (OtTx 1a-N) is compared to the N-terminal part of

cytoinsectotoxin 1a (CIT 1a-N) and oxyopinin 2a. Below, the C-terminal part of the toxin

(OtTx 1a-C) is compared to oxytoxins 1 and 2. Cysteine residues are on a dark gray

background. Positively charged residues are in bold type. Differences from OtTx 1a sequence

are highlighted in light gray. The right column shows percentage of identical residues to

OtTx 1a.

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Fig. 4. cDNA sequence encoding OtTx 1a precursor. Translated protein sequence is

shown below the DNA sequence. The signal peptide is in italics and the prosequence is

underlined.

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Fig. 5. Structure of Trx-OtTx 1a fusion protein (A) and purification of recombinant

OtTx 1a after fusion protein cleavage with CNBr by HPLC (B). In (A), functional elements

in Trx-OtTx 1a are shown schematically by shaded rectangles. Trx, thioredoxin domain;

His6, oligohistidine sequence; Pro, pOtTx 1a prosequence; M, methionine residue

demarcating the mature OtTx 1a. Numbers above correspond to the fusion protein residue

numbers. In (B), the fraction containing OtTx 1a is marked by an asterisk.

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Fig. 6. CD spectra (A) and helical net projection (B) of OtTx 1a-AMP. In (A), curve 1

represents the spectrum of the peptide in 50 mM phosphate buffer (pH 7.2); 2, 50%

trifluoroethanol; 3, 20 mM SDS. In (B), positively charged residues are shaded in blue;

hydrophobic, black; glycine residues, grey; hydrophilic uncharged, yellow. Hydrophobic

clusters are enclosed with dashed borders.

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Fig. 7. Ability of intact (A, B) and OtTx 1a-AMP-exposed (C, D) Staphylococcus

aureus cells to retain BCECF in cytoplasm and exclude PI as probed with CLSM. (A,

C) Green channel, distribution of BCECF in cytoplasm. (B, D) Red channel, distribution of

PI complexes with DNA. Bar length is 2 µm. In (C, D), bacteria were exposed to 1.25 µM

OtTx 1a-AMP for 10 min.

spider toxins comprising disulfide-rich and linear amphipathic domains: a new class of molecules...

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