An overview of lysine-49 phospholipase A2 myotoxins from

crotalid snake venoms and their structural determinants

of myotoxic action

Bruno Lomonte*, Yamileth Angulo, Leonel Calderon

Facultad de Microbiologıa, Instituto Clodomiro Picado, Universidad de Costa Rica, San Jose, Costa Rica

Abstract

In 1984, the first venom phospholipase A2 (PLA2) with a lysine substituting for the highly conserved aspartate 49 was

discovered, in the North American crotalid snake Agkistrodon p. piscivorus [J. Biol. Chem. 259 (1984) 13839]. Ten years later, the

first mapping of a ‘toxic region’ on a Lys49 PLA2 was reported, in Bothrops asper myotoxin II [J. Biol. Chem. 269 (1994) 29867].

After a further decade of research on the Lys49 PLA2s, a better understanding of their structural determinants of toxicity and mode

of action is rapidly emerging, with myotoxic effector sites identified at the C-terminal region in at least four proteins: B. asper

myotoxin II, A. p. piscivorus K49 PLA2, A. c. laticinctus ACL myotoxin, and B. jararacussu bothropstoxin I. Although important

features still remain to be established, their toxic mode of action has now been understood in its more general concepts, and a

consistent working hypothesis can be experimentally supported. It is proposed that all the toxic activities of Lys49 PLA2s are

related to their ability to destabilize natural (eukaryotic and prokaryotic) and artificial membranes, using a cationic/hydrophobic

effector site located at their C-terminal loop. This review summarizes the general properties of the Lys49 PLA2 myotoxins,

emphasizing the development of current concepts and hypotheses concerning the molecular basis of their toxic activities.

q 2003 Elsevier Ltd. All rights reserved.

Keywords: Myotoxin; Phospholipase A2; Snake venom; Skeletal muscle

1. Snake venom myotoxins and their classification

Myotoxins can be generally defined as natural com-

ponents (usually small proteins and peptides) of venom

secretions, that induce irreversible damage to skeletal

muscle fibers (myonecrosis) upon injection into higher

animals. They are particularly abundant and widespread in

venomous snakes, but can also be found in the venoms of

other organisms. Some myotoxins act locally, damaging

muscle fibers at the site of injection and its surroundings,

whereas others act systemically, causing muscle damage at

distant sites. Myonecrosis is an important medical compli-

cation of snakebites. In severe cases, local myonecrosis can

lead to drastic sequelae such as permanent tissue loss,

disability, or amputation (Milani et al., 1997; Otero et al.,

2002). On the other hand, widespread systemic myotoxicity

(rhabdomyolysis) can lead to myoglobinuria and acute renal

failure (Azevedo-Marques et al., 1985), a frequent cause of

death in snakebite victims.

Myotoxins described in snake venoms can be classified

into three main groups (Harris and Cullen, 1990) that

constitute structurally distinct protein families. These

include (1) the ‘small’ myotoxins (i.e. Crotalus durissus

terrificus crotamine, Crotalus v. viridis myotoxin a), (2)

the cardiotoxins, and (3) the PLA2 myotoxins (Fig. 1).

The PLA2 myotoxins form the largest group, which can be

further categorized into neurotoxic and non-neurotoxic

types (Mebs and Ownby, 1990). Among the latter, a clear

division between ‘Asp49’ and ‘Lys49’ myotoxins exists,

as will be further detailed. A fourth group of myotoxic

proteins has been considered (Gutierrez and Cerdas,

1984), comprising a variety of venom components that

may damage skeletal muscle by indirect mechanisms. As

an example, hemorrhagic toxins that cause local blood

flow impairment, ischemia, and secondary myonecrosis

of slow onset, would be considered as indirect myotoxic

factors.

0041-0101/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.

doi:10.1016/j.toxicon.2003.11.008

Toxicon 42 (2003) 885–901

www.elsevier.com/locate/toxicon

* Corresponding author. Fax: þ506-292-0485.

E-mail address: blomonte@cariari.ucr.ac.cr (B. Lomonte).

The neurotoxic PLA2 myotoxins are commonly found in

the venoms of elapid snakes, where they play a major role in

its overall lethal effect. Their lethal dose 50% (LD50) values

are extremely low, due to potent pre-synaptic effects at the

neuromuscular junction (Rosenberg, 1990). In addition,

these PLA2s cause impressive skeletal muscle necrosis

at very low doses (i.e. 1–2 mg) in rodents. A well-

characterized example of this myotoxin group is notexin,

from Notechis s. scutatus (Harris et al., 1975; Dixon and

Harris, 1996), an Australian elapid. Neurotoxic PLA2

myotoxins can be also present in a number of viperid/

crotalid species, as exemplified by crotoxin, a thoroughly

studied venom component of C. d. terrificus from South

America (Hendon and Fraenkel-Conrat, 1971; Gopalakrish-

nakone et al., 1984; Salvini et al., 2001).

On the other hand, the non-neurotoxic PLA2 myotoxins

are most commonly found in the venoms of viperids and

crotalids, where they are noted as abundant components. In

contrast to their neurotoxic counterparts, these PLA2s

generally display high LD50 values (Gutierrez et al.,

1986b; Homsi-Brandeburgo et al., 1988; Rosenberg, 1990;

Angulo et al., 1997; Andriao-Escarso et al., 2000; Soares

et al., 2000a,b), being of little relevance for the overall lethal

effect of their corresponding venoms (Lomonte et al., 1985).

Their myotoxic potencies are also weaker when compared

to the neurotoxic-type PLA2s (i.e. doses of 25–100 mg are

commonly utilized in mouse models of myonecrosis).

However, due to their abundance in the venom of

viperids/crotalids, and to the large amounts of venom

frequently injected in such accidents, these myotoxins are

undoubtedly central to the development of myonecrosis.

Their predominant role in the myotoxicity of the corre-

sponding crude venoms has been demonstrated by using

specific neutralizers, such as antibodies or other inhibitory

molecules. When the PLA2 myotoxins are selectively

neutralized, most of the muscle-damaging effect of whole

venoms is prevented (Lomonte et al., 1987, 1990a, 1992;

Moura-da-Silva et al., 1991b; Melo and Ownby, 1999;

Trento et al., 2001). Moreover, venoms that contain these

PLA2 myotoxins induce a significantly higher muscle

damage than venoms that lack them (Moura-da-Silva et al.,

1990).

Among the non-neurotoxic PLA2 myotoxins, two

different types can be recognized: ‘classical’ Asp49

PLA2s, which catalyze the hydrolysis of the ester bond in

the sn-2 position of glycerophospholipids; and ‘variant’

Lys49 PLA2s, or ‘PLA2-like’ proteins, which are devoid of

enzymatic activity (Fig. 1). In addition, at least two variants

with serine occupying position 49 (Ser49 PLA2s) have been

described (Krizaj et al., 1991; Polgar et al., 1996).

In the general structural classification of PLA2 (EC

3.1.1.4) enzymes (Six and Dennis, 2000; Kudo and

Murakami, 2002), the non-neurotoxic PLA2 myotoxins of

viperids/crotalids belong to group IIA. Irrespectively of

their ability to catalyze phospholipid hydrolysis (Asp49-

type), or not (Lys49-type), all of the non-neurotoxic PLA2s

myotoxins, as implied by their name, induce skeletal muscle

damage. Thus, the Lys49 myotoxins have attracted attention

Fig. 1. Classification and general characteristics of snake venom myotoxins.

B. Lomonte et al. / Toxicon 42 (2003) 885–901886

as models for the induction of myonecrosis by a catalytically

independent mechanism of action.

2. The Lys49 PLA2 myotoxins

Maraganore et al. (1984) described a new type of PLA2,

present in the venoms of Agkistrodon p. piscivorus and

Bothrops atrox, which showed significant substitutions in

amino acid residues previously considered to be invariant

among PLA2s. The most notable variation was the

replacement of Asp49, a key residue for binding the

essential Ca2þ-cofactor, by lysine. Therefore, they named

these proteins as ‘Lys49 PLA2s’, and proposed a catalytic

mechanism operating with a reversed order of addition of

calcium and substrate in the formation of the ternary

catalytic complex (Maraganore et al., 1984; Maraganore and

Heinrikson, 1986). However, this proposal was sub-

sequently challenged by the studies of van den Bergh et al.

(1988) and Scott et al. (1992), who suggested that the low

level of enzymatic activity originally detected in the A. p.

piscivorus Lys49 PLA2 was probably due to contamination

with basic Asp49 PLA2 isoforms present in the same venom,

and that the Lys49 proteins were catalytically inert.

Thereafter, the controversy on the inactivity/activity of

Lys49 PLA2s as enzymes has been continuously present in

the literature, and authors have frequently referred to them

as ‘PLA2s with no (or very low) catalytic activity’. This

issue will be further discussed in detail.

Reports on the toxic activities exerted by the Lys49

PLA2s appeared few years after their discovery, since initial

studies only focused on their structure, and its implications

for the catalytic mechanism of PLA2s. Dhillon et al. (1987)

first showed that the A. p. piscivorus Lys49 protein was

cardiotoxic, and affected the directly evoked contractions of

nerve-diaphragm preparations. They observed that the

potency of these effects did not correlate with the low level

of PLA2 activity of the protein. On the other hand, Gutierrez

et al. (1986a), Homsi-Brandeburgo et al. (1988), and

Lomonte and Gutierrez (1989), reported the isolation of

three basic proteins with myotoxic activity from different

Bothrops spp. venoms, which had structural features of

PLA2s, but without a detectable catalytic activity. In

retrospective, it is interesting to note that no relationship

was initially established between these three myotoxins, and

the novel type of Lys49 PLA2 reported earlier by Maraganore

et al. (1984). However, this relationship soon became

evident, probably starting with the isolation and sequencing

of ‘basic proteins I and II’ from Trimeresurus flavoviridis

(Yoshizumi et al., 1990; Liu et al., 1990). Since then, the

Lys49 PLA2 myotoxin family has been gradually expanded,

with proteins newly isolated or cloned from a variety of

crotalid/viperid species. A recent review on this subject listed

nine Lys49 myotoxins (Ownby et al., 1999), while this

number has quadrupled in the last 4 years (Table 1).

3. Are the Lys49 PLA2 myotoxins active

as phospholipolytic enzymes?

An essential step for understanding the toxic effects of the

Lys49 PLA2s is to define if they possess enzymatic activity or

not, due its potential implications for their mechanism of

action. Following the site-directed mutagenesis studies with

Asp49 ! Lys recombinants of porcine (van den Bergh et al.,

1988) and bovine (Li et al., 1994) pancreatic PLA2s, and the

extensive purification strategies of natural Lys49 proteins

utilized by van den Bergh et al. (1988) and Scott et al. (1992),

a strong argument against the originally described catalytic

activity of Lys49 PLA2s was made. Due to the difficulty of

achieving an absolute chromatographic separation of Lys49

and Asp49 isoforms, which often coexist in a given venom

(Lomonte and Carmona, 1992), contaminant traces of the

latter have been assumed to be responsible for the low levels

of enzymatic activity frequently observed in Lys49 PLA2

preparations, depending on the sensitivity of the assays.

However, some studies have opposed this interpretation,

claiming that this low enzymatic activity is inherent to the

Lys49 proteins, and that it becomes even more significant on

certain types or forms of substrates (Yoshizumi et al., 1990;

Liu et al., 1990; Shimohigashi et al., 1995; Rodrigues-Si-

mioni et al., 1995; Yamaguchi et al., 1997; Mancin et al.,

1997; Soares et al., 2002). Until recently, these analyses had

always been conducted with proteins isolated from their

natural sources, and therefore, with the possibility of trace

contamination remaining as a confounding factor. However,

the recent availability of recombinant Lys49 PLA2s (Ward

et al., 2001; Giuliani et al., 2001) has provided new tools to

approach this problem. In particular, a study using recombi-

nant bothropstoxin I significantly contributed to clarify this

essential issue (Ward et al., 2002), by demonstrating that the

protein was enzymatically inactive in vitro, in contrast to an

extensively purified natural sample, which displayed a very

low, but detectable activity. Moreover, the Lys49 ! Asp

mutant of bothropstoxin I was still catalytically inactive,

demonstrating that not only the single Asp49 replacement

with lysine, but other structural changes as well, are

important for the lack of enzymatic activity of this protein

(Ward et al., 2002). These findings clearly favor the concept

that Lys49 PLA2s are enzymatically inert in vitro, and it will

be of interest to learn of future studies re-examining the

enzymatic activity of more Lys49 proteins, when obtained in

recombinant form.

A possible explanation for the lack of enzymatic activity

of Lys49 proteins was suggested in a crystallographic study

of piratoxin II (Lee et al., 2001). These authors proposed

that the Lys49 PLA2s may possess structural features that

preclude the release of the free fatty acid produced after an

initial phospholipid hydrolysis, thereby interrupting the

catalytic cycle, and turning these proteins, in practice, into

inactive enzymes.

On the other hand, an argument has been presented that

Lys49 PLA2s, despite being catalytically inactive on

B. Lomonte et al. / Toxicon 42 (2003) 885–901 887

artificial substrates in vitro, are able to hydrolyze specific

forms or types of natural substrates present only in

biological systems (Fletcher et al., 1997; Fletcher and

Jiang, 1998). This hypothesis was proposed on the basis of

assays that utilize radiolabeled cell cultures as a natural

substrate, in which an extensive release of fatty acids is

observed after exposure to Lys49 PLA2s (Fletcher et al.,

1996, 1997; Soares et al., 2002). However, as admitted in

such studies, these assays cannot discern if fatty acid release

originates from a direct catalytic action of the Lys49 PLA2

on some substrate, or is due to an indirect activation of

endogenous cellular lipases. The latter would seem highly

probable, given the fact that all Lys49 PLA2 myotoxins

evaluated to date cause cell membrane disturbances in a

broad variety of cell types in vitro, ultimately leading to

their cytolysis (Lomonte et al., 1994b, 1999a). It is

conceivable, for example, that an influx of Ca2þ(Gutierrez

et al., 1989; Incerpi et al., 1995; Rufini et al., 1996) or Naþ

(Johnson and Ownby, 1994) ions, caused even by

subcytolytic concentrations of a Lys49 PLA2 (or other

membrane-active factors, for that matter) could initiate a

variety of intracellular processes, including the activation of

Table 1

Lys49 phospholipases A2 isolated from snake venoms

Snake species Protein common name Entry codea References

Agkistrodon piscivorus piscivorus AppK49 P04361 Maraganore et al. (1984) and Maraganore and

Heinrikson (1986)

Agkistrodon bilineatus PLA2-II –b Nikai et al. (1994)

Agkistrodon contortrix laticinctus ACL myotoxin P49121 Johnson and Ownby (1993) and Selistre

de Araujo et al. (1996a)

Atropoides (Bothrops) nummifer Myotoxin I –b Gutierrez et al. (1986a)

Atropoides (Bothrops) nummifer Myotoxin Ih –b Rojas et al. (2001)

Atropoides (Bothrops) nummifer Myotoxin II P82950 Angulo et al. (2000, 2002)

Bothrops asper Myotoxin II P24605 Lomonte and Gutierrez (1989) and

Francis et al. (1991)

Bothrops asper Myotoxin IV –b Dıaz et al. (1995)

Bothrops asper Myotoxin IVa –c Lizano et al. (2001)

Bothrops atrox Ba-K49 –b Maraganore et al. (1984)

Bothrops atrox BaPLA2-I –b Kanashiro et al. (2002)

Bothrops jararacussu Bothropstoxin I Q90249 Homsi-Brandeburgo et al. (1988) and

Cintra et al. (1993)

Bothrops moojeni Myotoxin I P82114 Lomonte et al. (1990b) and Soares et al. (2000a)

Bothrops moojeni Myotoxin II Q9I834 Lomonte et al. (1990b) and Soares et al. (1998)

Bothrops neuwiedi Myotoxin I –b Geoghegan et al. (1999)

Bothrops neuwiedi pauloensis BnSP-7 Q9IAT9 Rodrigues et al. (1998) and Soares et al. (2000b)

Bothrops pirajai Piratoxin I P58399 Toyama et al. (1995, 1998)

Bothrops pirajai Piratoxin II P82287 Toyama et al. (1995, 2000)

Bothrops pradoi PRA-1 –b Moura-da-Silva et al. (1991a)

Bothriechis (Bothrops) schlegelii Myotoxin I P80963 Angulo et al. (1997) and Tsai et al. (2001)

Calloselasma rhodostoma CRV-K49 Q9PVF3 Tsai et al. (2000)

Cerrophidion (Bothrops) godmani Myotoxin II P81165 Dıaz et al. (1992) and de Sousa et al. (1998)

Cerrophidion (Bothrops) godmani PgoK49 Q8UVU7 Tsai et al. (2001)

Crotalus atrox Cax-K49 Q8UVZ7 Tsai et al. (2001)

Crotalus molossus molossus Cmm-K49 –b Tsai et al. (2001)

Deinagkistrodon (Agkistrodon) acutus Dac-K49 O57385 Wang et al. (1996) and Fan et al. (1999)

Deinagkistrodon (Agkistrodon) acutus Dac-K49b –c Tsai et al. (2001)

Trimeresurus albolabris Tal-K49 –b Tsai et al. (2001)

Trimeresurus flavoviridis BP-I P20381 Yoshizumi et al. (1990)

Trimeresurus flavoviridis BP-II E48188 Liu et al. (1990)

Trimeresurus gramineus PLA2-V P70090 Nakai et al. (1995)

Trimeresurus gramineus PLA2-VII P70089 Nakashima et al. (1995)

Trimeresurus mucrosquamatus TMV-K49 P22640 Wang et al. (1996) and Liu et al. (1991)

Trimeresurus okinavensis To3 Q92152 Nobuhisa et al. (1996)

Trimeresurus puniceus Tpu-K49 –b Tsai et al. (2001)

Names in parentheses indicate former genera designations.a Identification codes in the SwissProt database.b Partial or unavailable amino acid sequences.c Sequence not submitted.

B. Lomonte et al. / Toxicon 42 (2003) 885–901888

endogenous lipases. Indeed, membrane-active peptides such

as cardiotoxins or bee venom mellitin (of synthetic origin,

free of PLA2 contamination), which are not catalytic, induce

breakdown of phospholipids and production of free fatty

acids in skeletal muscle cell cultures, compatible with an

activation of endogenous phospholipase C enzymes

(Fletcher et al., 1991). On these grounds, the hypothesis of

a highly selective catalytic action of Lys49 PLA2s, being

exerted exclusively on biological substrates, is difficult to

sustain, at least until novel unambiguous evidence is

provided.

In summary, there is currently no unambiguous proof

that the Lys49 PLA2 myotoxins are enzymatically active

phospholipolytic proteins per se, and thus, their mechanism

of action should be explained via a catalytic-independent

initial event on their target cells.

4. Activities of the Lys49 PLA2 myotoxins and their

in vivo and in vitro study models

In vivo, the main action of the Lys49 PLA2s, when

injected intramuscularly (in resemblance of a snakebite), is

local myotoxicity. This effect has been generally studied in

mice, using histological and ultrastructural techniques

(Gutierrez et al., 1986a, 1989; Homsi-Brandeburgo et al.,

1988; Lomonte and Gutierrez, 1989; Lomonte et al., 1990b;

Soares et al., 1998; Toyama et al., 1998; Melo and Ownby,

1999), intravital microscopy (Lomonte et al., 1994d), and

the plasma creatine kinase release assay (Gutierrez et al.,

1986a; Lomonte and Gutierrez, 1989; Lomonte et al.,

1990a,b; Kihara et al., 1992; Angulo et al., 1997, 2000;

Soares et al., 2002). Ex vivo assays for myotoxicity have

also been described, based on creatine kinase release rates

from isolated muscle preparations (Gutierrez et al., 1986b;

Melo et al., 1993; Melo and Ownby, 1999). In vivo, local

myotoxicity is accompanied by other toxic effects, including

a moderate edema, studied by footpad swelling techniques

(Lomonte and Gutierrez, 1989; Liu et al., 1991; Soares et al.,

1998, 2002; Chaves et al., 1998; Andriao-Escarso et al.,

2000; Angulo et al., 2000; Landucci et al., 2000),

hyperalgesia (Chacur et al., 2003), and release of pro-

inflammatory cytokines such as interleukin-6 (Lomonte

et al., 1993). Another in vivo toxic effect, although studied

in assays utilizing ‘artificial’ routes of administration, is the

general lethal activity via intravenous or intraperitoneal

injections in mice (Gutierrez et al., 1986b; Homsi-Brande-

burgo et al., 1988; Angulo et al., 1997; Andriao-Escarso

et al., 2000; Soares et al., 2000a,b). As mentioned, the lethal

potency of the non-neurotoxic PLA2 myotoxins in LD50

assays is generally very low, and intramuscular injections of

high toxin doses do not result in lethality. The ability of

some Lys49 myotoxins to recruit leukocytes in a pleural

cavity in vivo model has also been described (de Castro

et al., 2000).

In vitro, toxic effects on isolated neuromuscular

preparations, such as the mouse phrenic-diaphragm and

chick biventer cervicis, have been characterized for several

Lys49 myotoxins (Dhillon et al., 1987; Heluany et al., 1992;

Oshima-Franco et al., 2001; Soares et al., 2000b). In

addition, in vitro assays using different cell targets have

demonstrated a broad specificity in their cytolytic action

(Bruses et al., 1993; Lomonte et al., 1994b,c, 1999a; Incerpi

et al., 1995; Rufini et al., 1996; Andriao-Escarso et al., 2000;

Angulo et al., 2000; Soares et al., 2002), and the use of cell

cultures, such as rodent lines of skeletal muscle myoblasts/

myotubes, appears to correlate well with their in vivo

myotoxicity (Incerpi et al., 1995; Lomonte et al., 1999a).

Liposome disruption, particularly if containing negatively

charged phospholipids, is another in vitro effect common to

Lys49 PLA2 myotoxins explored to date (Dıaz et al., 1991;

Rufini et al., 1992; Pedersen et al., 1994, 1995; Shen and

Cho, 1995; Soares et al., 2000a,b). In vitro mast cell

degranulation by some Lys49 myotoxins has been

demonstrated, and related to their edematigenous activity

(Landucci et al., 1998). Also, their ability to induce

neutrophil migration in chemotaxis chambers (Gambero

et al., 2002), and to exert renal damage in isolated perfused

kidneys (Barbosa et al., 2002) have been reported. Lastly, a

wide-spectrum bactericidal activity of Lys49 myotoxins was

described as a novel, catalytic-independent effect for PLA2

molecules, using B. asper myotoxin II and its synthetic

C-terminal peptide 115–129 (Paramo et al., 1998). This

activity has been recently observed for other Lys49

myotoxins (Soares et al., 2000b, 2001), suggesting that it

may be a general property of this protein family.

Given this range of in vivo and in vitro effects of the

Lys49 PLA2 myotoxins, another essential question opens:

are the different activities due to different molecular regions,

or is there a single ‘toxic’ site that exerts a common

mechanism with different manifestations, depending on the

target? In our view, current evidence suggests the latter to be

the case, as discussed below, and we propose that all the

toxic activities of Lys49 PLA2s are related to their ability to

interact with, and destabilize biological membranes, using

the same general effector site.

5. The search for a toxic site in Lys49 PLA2 myotoxins:

approaches and strategies

The lack of enzymatic activity observed in myotoxic

Lys49 PLA2s prompted the search for a protein region that

would be responsible for their toxic effects, in order to

explain their mechanism of action. Different strategies have

been utilized to this end, including: (a) chemical modifi-

cation of the toxins, (b) sequence comparison analyses, (c)

interaction with neutralizing molecules, (d) synthetic

peptide studies, and (e) site-directed mutagenesis analyses.

All these approaches have been greatly enhanced by

B. Lomonte et al. / Toxicon 42 (2003) 885–901 889

the elucidation of the three-dimensional crystal structure of

a number of Lys49 PLA2s (Scott et al., 1986, 1992; Holland

et al., 1990; Arni and Gutierrez, 1993; Arni and Ward, 1996;

Arni et al., 1995, 1999; Treharne et al., 1997; Canduri et al.,

1998; Soares et al., 1998; de Azevedo et al., 1998, 1999;

Fontes et al., 1999; Lee et al., 2001; Liu et al., 2003), which

has allowed fundamental insights for the current under-

standing of their structure–function relationship (reviewed

by Murakami and Arni, this issue).

Chemical modification and site-directed mutagenesis

analyses are reviewed by Soares and Giglio, and Chioato

and Ward, respectively (present issue). They will be briefly

mentioned here, only in relation to other experimental

strategies discussed in detail: the neutralizing interaction

between heparin and Lys49 PLA2 myotoxins, and the

identification of their toxic region by the use of short

synthetic peptides.

In the 1990s, a number of complete sequences for Lys49

PLA2 myotoxins gradually accumulated, allowing to search

for amino acid residues that could be theoretically

associated to their toxic actions, on the basis of comparisons

(Selistre de Araujo et al., 1996a,b; Ward et al., 1998).

However, before these sequence analyses were performed, a

different approach had provided an essential clue towards

identifying the toxic region of these proteins, initially using

B. asper myotoxin II (Lomonte et al., 1994a). In this

approach, it was reasoned that by mapping the interaction

site of the toxin with neutralizing molecules, information on

the molecular region relevant for toxicity could be obtained.

Initially, neutralizing monoclonal antibodies to B. asper

myotoxins were investigated (Lomonte and Kahan, 1988;

Lomonte et al., 1992), but none was able to recognize

denatured myotoxin, implying their binding to confor-

mation-dependent or discontinuous epitopes, which pre-

cluded an easy mapping. However, after reports describing

that heparin could inhibit the myotoxic activity of B.

jararacussu venom (Melo and Suarez-Kurtz, 1988), and its

bohtropstoxin (Melo et al., 1993), a study was undertaken to

identify the heparin-binding site on B. asper myotoxin II.

Candidate segments of its sequence were selected, and by

utilizing their corresponding synthetic peptides, it was

demonstrated that a heparin-binding site mapped within the

sequence 115–129 (numbering of Renetseder et al., 1985),

in the C-terminal loop (Lomonte et al., 1994a). Sub-

sequently, this region was also identified as a heparin-

binding site in the rat class IIA secreted PLA2 (Murakami

et al., 1996).

Since heparin preparations normally consist of poly-

saccharide chains that are heterogeneous in length, it was

informative to determine the minimal structure able to

interact with, and to inhibit, the activity of myotoxin II.

Experiments with heparin fragments identified hexasacchar-

ides as this minimal structure (Fig. 2a; Lomonte et al.,

1994a). As represented in Fig. 2b, the molecular dimensions

of segment 115–129 are roughly comparable to those of

heparin hexasaccharides. This implied that hexasaccharides

were interacting with a narrow protein segment, which

might comprise, or at least be very close to, the putative

toxic site (as opposed to a mechanism in which long heparin

chains could sterically hinder a toxic site distant from their

binding site). Remarkably, when the free synthetic peptide

115–129 was tested by an in vitro cytotoxicity assay,

a weaker, but nonetheless specific cytolytic activity was

observed (Lomonte et al., 1994a), resembling that of the

whole protein. The toxic action of this peptide, like that of

myotoxin II, was also inhibited by heparin. Thus, it was

evident that heparin was neutralizing B. asper myotoxin II

by binding to its C-terminal region 115–129, which was

directly involved in the cytotoxic mechanism of this protein

(Lomonte et al., 1994a).

At the same time, it was observed that cyanogen

bromide (CNBr) treatment of B. asper myotoxin II, by

cleaving its N-terminal octapeptide, significantly

decreased its membrane-damaging activities, including

both liposome disruption and myotoxicity (Dıaz et al.,

1994). This suggested the participation of the N-terminal

region in its mechanism of action, although the

possibility of distant structural alterations caused by the

cleavage of segment 1–8 could not be excluded (Dıaz

et al., 1994). CNBr treatment of other Lys49 myotoxins

such as BnSP-7 (Soares et al., 2000b) and piratoxin I

(Soares et al., 2001) resulted in similar findings, in

agreement with the possible functional relevance of the

N-terminal helix of these proteins in toxicity.

Subsequent studies on B. asper myotoxin II utilized an

immunochemical approach to confirm that the region 115–

129, rich in cationic and hydrophobic residues, was involved

not only in its cytotoxic activity, but also in its myotoxic

effect in vivo. Rabbit polyclonal antibodies raised against

peptide 115–129, were capable of binding to the native

toxin, inhibiting both its cytotoxic and myotoxic actions

(Calderon and Lomonte, 1998, 1999). Unexpectedly, when

the role of the N-terminal a-helix of B. asper myotoxin II

was evaluated by the same approach (using antibodies

directed towards the sequence 1–15), no evidence of

inhibition of toxic activities was obtained (Angulo et al.,

2001). This puzzling result opened doubts as to the exact

participation of the N-terminal region in the toxic

mechanism of Lys49 myotoxins, originally supported by

CNBr-treatment experiments. Thus, more work needs to be

focused on this protein region to settle these contrasting

findings.

The information gathered on B. asper myotoxin II

allowed to propose an initial model for its mechanism of

action (Gutierrez and Lomonte, 1995, 1997), further

extended by the findings of Calderon and Lomonte (1998,

1999). In this model, the toxin would approach the cell

membrane with both the C- and N-terminal regions exposed

on the face of interaction (Fig. 2a). Considering that the

N-terminal region participates in interface recognition by

PLA2s (Scott et al., 1990; Kato et al., 1994), and that

residues 115–129 had been shown to form an amphiphilic,

B. Lomonte et al. / Toxicon 42 (2003) 885–901890

cationic–hydrophobic structure capable of destabilizing

different types of membranes (Lomonte et al., 1994a;

Paramo et al., 1998), the model proposed that the role of the

former site would be related to toxin binding to an

unidentified acceptor on the cell membrane, while the role

of the latter site would involve both a contribution to

binding, and a subsequent perturbation of membrane

integrity, leading to the myotoxic effect of this protein

(Calderon and Lomonte, 1998).

Parallel studies on the structure of bothropstoxin I from

B. jararacussu, using X-ray crystallography and fluor-

escence spectroscopy, added a novel feature to the model of

action of Lys49 myotoxins originally developed with B.

asper myotoxin II. The bothropstoxin homodimer appeared

in two conformations, ‘open’ and ‘closed’ forms, which

varied in the angle formed by the monomers (da Silva Giotto

et al., 1998). Thus, the dimer interface might act as a hinge,

allowing the relative displacement of the C-terminal region

Fig. 2. The interaction between heparin and B. asper myotoxin II. (A) The minimal heparin fragments interacting with (gray bars), and

neutralizing (empty bars) myotoxin II, are hexasaccharides (adapted from Lomonte et al., 1994a); (B) Comparative molecular dimensions of the

heparin-binding C-terminal region 115–129 of myotoxin II and a heparin hexasaccharide. The carboxy (C) and amino (N) termini are indicated

on the dimeric protein, represented as an a-carbon backbone cartoon (PDB entry 1CLP), approaching a membrane at the bottom (adapted from

Calderon and Lomonte, 1998).

B. Lomonte et al. / Toxicon 42 (2003) 885–901 891

by as much as 13 A, which, if occurring upon membrane

binding, could contribute to its insertion and to disorgan-

ization of phospholipid bilayers (da Silva Giotto et al.,

1998). Therefore, this concept of a molecular hinge in

bothropstoxin I, fitted well into the framework of the

proposed model of action of B. asper myotoxin II, which

implicated the C-terminal segment 115–129 as the main

effector of membrane destabilization (Lomonte et al.,

1994a; Paramo et al., 1998; Calderon and Lomonte,

1998). It will be of interest to determine if such quaternary

conformation changes also occur in other dimeric Lys49

PLA2s.

After recognizing the relevance of the C-terminal region

in the toxic effects exerted by B. asper myotoxin II, further

work has explored the structural basis of the membrane-

damaging effects induced by this cationic–hydrophobic

combination of amino acids, in the Lys49 myotoxins. Two

main questions that followed were: (a) is the region 115–

129 involved in the toxic effects of other Lys49 PLA2s? and,

(b) which amino acids within this region play a major role in

the toxic mechanism? These questions have been

approached mostly by two strategies. Our group has utilized

synthetic peptides as tools to investigate mainly the first

point, as summarized below. On the other hand, studies by

Ward and co-workers have tackled the second question,

using an in-depth mutagenesis approach with one Lys49

myotoxin, bothropstoxin I (reviewed by Chioato and Ward,

this issue). The data gradually emerging from these two

strategies are in general good agreement, and together with

other relevant lines of information, have contributed to

construct a better picture of how the Lys49 myotoxins may

function.

6. Experiments with synthetic peptides of Lys49

myotoxins: what has been learned?

The originally described toxic action of synthetic peptide

115–129 of B. asper myotoxin II (KKYRYYLKPLCKK),

was a cytotoxic effect towards cultured endothelial cells—

utilized as a target model at the time—that mimicked the

effect of the whole toxin, although with a lower efficiency

(Lomonte et al., 1994a). This 13-mer peptide alone,

however, was unable to reproduce the myotoxic action of

the protein in vivo, and therefore, the segment 115–129 was

cautiously referred to as a ‘cytolytic region’ of this toxin

(Lomonte et al., 1994a), until further evidence of its role in

myotoxicity could be obtained (Calderon and Lomonte,

1998, 1999). Since the action of peptide 115–129 on

membranes was proposed to depend on its amphiphilic

nature (with a prominent cluster of three tyrosines

surrounded by several cationic residues), it was hypoth-

esized that a modification of its hydrophobic character,

without changing its charge pattern, might influence its toxic

effects. Thus, the activities of a triple Tyr ! Trp substituted

synthetic peptide 115–129, named ‘p115-W3’, were

evaluated. Remarkably, p115-W3 displayed not only an

enhanced membrane-damaging action, but reproduced all

the toxic activities of myotoxin II, including its cytotoxic,

bactericidal, edema-forming, and, most importantly, its

myotoxic action in vivo (Lomonte et al., 1999b). This

provided the first example of a short, PLA2-based peptide,

with the ability to reproduce the myotoxic mechanism of its

parent protein, in support of the proposed relevance of

region 115–129 for the myotoxic mechanism of B. asper

myotoxin II.

Subsequent work led to the identification of the myotoxic

site of A. p. piscivorus Lys49 PLA2 also within its sequence

115–129, by demonstrating that the corresponding peptide

(KKYKAYFKLKCKK) induced myonecrosis in vivo

(Nunez et al., 2001; Lomonte et al., 2003). In particular,

the study of Nunez et al. (2001) constituted the first report of

an unmodified, PLA2-derived synthetic peptide reproducing

the myotoxic effect of its parent molecule, which coin-

cidentally was the first Lys49 PLA2 described (Maraganore

et al., 1984). Interestingly, two C-terminal peptides

corresponding to the Asp49 PLA2 myotoxin counterparts

from B. asper and A. p. piscivorus venoms, respectively,

tested negative for toxic activities. This suggested that the

enzymatically active Asp49 myotoxins may not utilize their

C-terminal region as an effector of membrane damage, in

contrast to the Lys49 isoforms (Nunez et al., 2001).

However, more recent evidence opens alternative interpret-

ations for these findings (Lomonte et al., 2003), which

require further analysis.

The observation of direct toxic actions being exerted

by two peptides 115–129 of Lys49 myotoxins (Lomonte

et al., 1994b; Nunez et al., 2001), prompted a

comparative study, to determine if this would represent

a common feature of this protein family. When a series

of peptides 115–129 were investigated, they varied

widely in their activities, ranging from fully toxic to

innocuous (Lomonte et al., 2003). Notably, the C-

terminal peptide from A. contortrix laticinctus Lys49

PLA2 induced myonecrosis in vivo, thus identifying the

myotoxic site in another protein of this family (Lomonte

et al., 2003). However, this study also showed that the

toxic actions of Lys49 PLA2s cannot always be

reproduced by their free segments 115–129, as several

of them tested negative in the bioassays.

The interpretation of the wide variability in the effects

of the free, synthetic C-terminal peptides 115–129 of

different Lys49 myotoxins, is complex and mostly

speculative at this moment. On one hand, considering

the significant sequence variability of this region in Lys49

proteins (Fig. 3), the possibility of subtle differences in the

mechanism of action between individual toxins cannot be

excluded. However, from an evolutionary perspective, it

would seem unlikely that the Lys49 myotoxins, forming a

closely related phylogenetic group (Ogawa et al., 1995;

Tsai et al., 2001; Angulo et al., 2002), would have

B. Lomonte et al. / Toxicon 42 (2003) 885–901892

developed markedly different membrane-damaging regions

(and mechanisms) in different snake species. In this

regard, the direct evidence of toxicity exerted by C-

terminal peptides of at least three Lys49 PLA2s, namely

from B. asper (Lomonte et al., 1994a, 2003; Chacur et al.,

2003), A. p. piscivorus (Nunez et al., 2001), and A. c.

laticinctus (Lomonte et al., 2003), together with the site-

directed mutagenesis evidence obtained for bothropstoxin

I (Chioato et al., 2002), suggests that this region might

also play a central role in the toxic mechanism of other

members of this family. It is noteworthy that region 115–

129 has also been shown to be relevant for the activity of

ammodytoxin A, a pre-synaptically neurotoxic, class II

Asp49 PLA2 from Vipera ammodytes (Ivanovski et al.,

2000). Also of interest is the observation that a C-terminal

sequence related to snake class IIA PLA2s, when

introduced into the non-toxic porcine pancreatic PLA2,

drastically changed its interfacial kinetics and binding

properties (Janssen et al., 1999).

Sequence variability in the C-terminal extension

among the different Lys49 PLA2s (Fig. 3) would initially

seem contradictory with the proposal of its key role in

toxicity, since function would be expected to associate

with structural conservation. But it is also possible to

conceive the C-terminal loop of this protein family as a

region that allowed mutational ‘experiments’ during

evolution, ultimately leading to the acquisition of a

membrane-perturbing function that is not strictly depen-

dent on a fixed, invariant sequence. Lys115 and Lys122

are so far the only two invariant residues in region 115–

129 of Lys49 myotoxins (Fig. 3). Kini and Chan (1999)

demonstrated that the exposed residues of PLA2s have

evolved at a faster rate than buried amino acids, probably

in relation to the acquisition of variable specificities and

toxic effects of these proteins. The mechanism of

membrane damage exerted by synthetic peptides of

Lys49 myotoxins relies on their amphiphilic nature,

provided by combinations of cationic and hydrophobic

Fig. 3. Sequence variability in Lys49 phospholipase A2 myotoxins. The complete amino acid sequences of 24 Lys49 PLA2s (Table 1) were

aligned using CLUSTAL W (Higgins et al., 1996). In (A), percentage substitution was calculated according to the method of Kini and Chan

(1999), and represents the replacement frequency for a given amino acid position, in comparison to a consensus sequence of the 24 proteins

(SLFELGKMIL QETGKNPAKS YGLYGCNCGV GGRGKPKDAT DRCCYVHKCC YKKLTDCDPK KDRYSYSWKN KTIVCGENNP

_CLKELCECD KAVAICLREN LDTYNKKYKI _YLKPFCKKA _DAC). Dashes indicate gaps introduced to optimize alignment, resulting in

a total of 124 positions. A zero value represents invariant residues. In (B), variability refers to the number of different amino acids found at a

given position. A value of one represents invariant residues. Both calculations were performed with a program written for the Matlab software

(MathWorks, Inc.). The horizontal black bar indicates the location of region 115–129 (numbering of Renetseder et al., 1985), corresponding to

positions 106–119 in this scheme. In both analyses, the C-terminal region 115–129 shows higher variability than other regions, with 7 out of the

13 positions presenting $45% substitution frequency (A), or 8 out of the 13 positions having variability scores $4 (B). Black triangles point to

Lys115 and Lys122, the only invariant residues within region 115–129. Black-filled vertical bars in (B) identify the conserved Cys residues of

class II PLA2s.

B. Lomonte et al. / Toxicon 42 (2003) 885–901 893

amino acids that are somewhat variable. It is noteworthy

that such myotoxin peptides have a functional resem-

blance to the widely distributed cationic antimicrobial

peptides (Kini and Evans, 1989; Paramo et al., 1998;

Santamarıa et al., in preparation), which show high

sequence variability. A great deal of work on antimicro-

bial peptides has demonstrated that membrane-damaging

effects may be achieved through a variety of amino acid

combinations involving cationic and hydrophobic residues

(Hancock and Lehrer, 1998; Hancock and Chapple, 1999;

Ganz and Lehrer, 1999). This might explain the apparent

paradox between the observed sequence variability in the

C-terminal region of the Lys49 PLA2 myotoxins, and its

proposed central role for their toxic functions.

In conclusion, the use of synthetic peptides has provided

solid evidence to identify the C-terminal region as the

effector of toxic activities in Lys49 PLA2 myotoxins, in at

least three examples. The fact that single peptides, as short as

13-mers, can reproduce all the main toxic activities of their

parent molecules, does not support the existence of different

molecular sites responsible for the myotoxic, cytotoxic,

edema-forming, liposome-disrupting, hyperalgesic, or

microbicidal mechanisms. Rather, this evidence strongly

favors the notion that the Lys49 PLA2s utilize a single site to

exert membrane damage, which manifests differently in

various targets and assay models. Our proposal of a single

membrane-damaging site does not imply that other molecu-

lar motifs do not participate in enhancing or complementing

the action of this effector toxic site, as evidenced by the lower

potency of synthetic C-terminal peptides, compared to their

corresponding proteins. In particular, regions involved in

myotoxin binding to membrane acceptor(s), would be

candidates for such enhancing role, for example by

increasing their overall affinity, thus facilitating the mem-

brane-damaging mechanism.

7. Membrane acceptor(s) for Lys49 PLA2 myotoxins:

novel clues from an ‘all-D’ peptide

Knowing that heparin interacted with the C-terminal

region of B. asper myotoxin II and its peptide 115–129

(Lomonte et al., 1994a), prompted an investigation to

explore if heparan sulfate, a common component of cell

surfaces, would act as an acceptor for this Lys49 PLA2.

However, this possibility was ruled out, on the basis of the

unaffected ability of myotoxin II to lyse cells in which

heparan sulfate was eliminated by either enzymatic treat-

ment, synthesis inhibition, or via a specific cellular mutation

(Lomonte et al., 1994c). Sialic acid and N-linked oligosac-

charides neither appear to be involved in recognition by this

Lys49 myotoxin, as shown by neuraminidase and tunica-

mycin cell treatments, respectively (Lomonte et al., 1994b).

On the other hand, the discovery of membrane protein

receptors for endogenous PLA2s on many mammalian cell

types, utilized in some cases as high affinity acceptors by

exogenous venom PLA2s (Lambeau and Lazdunski, 1999),

suggests the possibility that Lys49 myotoxins may utilize

such proteins in their mechanism. However, no evidence has

yet been reported in this regard. On the contrary, despite the

nature of the membrane acceptor for these toxins being

unknown, indirect lines of evidence suggest that it may not

be a protein: (a) Lys49 myotoxins disrupt liposomes

prepared exclusively of phospholipids, with a preference

for negatively charged types (Dıaz et al., 1991; Rufini et al.,

1992; Pedersen et al., 1994, 1995); (b) Lys49 myotoxins

lyse a variety of cell types in culture, showing little

selectivity in their cytotoxic action (Bruses et al., 1993;

Lomonte et al., 1994b,c, 1999a; Incerpi et al., 1995; Rufini

et al., 1996); (c) Lys49 myotoxins also lyse prokaryotic cells

(Paramo et al., 1998; Soares et al., 2000b, 2001) via a

membrane-permeabilization action; (d) the enrichment of

erythrocyte membranes with negatively charged phospho-

lipids increases their susceptibility to lysis by Lys49

myotoxins (Dıaz et al., 2001); and (e) most suggestively, a

synthetic enantiomer of peptide 115–129 of A. p. pisci-

vorus, containing only D-amino acids, expressed the same

cytotoxic and myotoxic activity as its L-counterpart

(Lomonte et al., 2003). This observation, if extrapolable to

the whole parent molecule, provides an important clue to

indicate that the action of myotoxic Lys49 PLA2s may not

involve a configuration-dependent interaction (such as

recognition of a PLA2 receptor protein), but rather would

be compatible with binding to phospholipids. Taken

together, these findings suggest that negatively charged

membrane phospholipids might be utilized as acceptors for

the Lys49 myotoxins. But more work is clearly needed to

properly address this hypothesis.

8. The toxic mechanism of Lys49 PLA2 myotoxins:

current evidence integrated

Two decades after their discovery, the Lys49 PLA2

myotoxins now constitute a well characterized group of

proteins (Table 1), with not less than 24 complete amino

acid sequences, and 11 three-dimensional crystal structures

determined. Their main effect in vivo is myonecrosis,

rapidly induced at the site of injection. Myotoxicity, as well

as other activities, may all be attributed to their ability to

alter membrane integrity in a variety of natural (including

both eukaryotic and prokaryotic cells) and artificial (i.e.

liposome) targets. But mature skeletal muscle fibers would

probably be most susceptible to their action (Lomonte et al.,

1994a, 1999a). Current evidence indicates that their

membrane-damaging mechanism is not dependent on an

intrinsic phospholipolytic activity, if this debated activity

exists at all, but is rather a function of their physical

interaction with some anionic acceptor(s), which might

consist of negatively charged phospholipids. This inter-

action utilizes amino acid residues located on the PLA2

interfacial recognition face of Lys49 myotoxins (Fig. 4),

B. Lomonte et al. / Toxicon 42 (2003) 885–901894

displaying a dense positive charge distribution (Falconi

et al., 2000), which exposes both their C-terminal loop and

N-terminal a-helix towards the membrane. Cationic amino

acids within the C-terminal region (Lomonte et al., 1994a,

1999b, 2003; Calderon and Lomonte, 1998, 1999; Nunez

et al., 2001; Chioato et al., 2002), and possibly also within

the N-terminal region (Dıaz et al., 1994), would establish

initial, weak electrostatic interactions with the anionic

acceptor sites, as suggested by wash-out experiments

(Lomonte et al., 1994b), and in analogy with the principles

of interfacial binding of other PLA2s (Zhou and Schulten,

1996; Stahelin and Cho, 2001). Within the C-terminal

region, the conserved Lys122 might play a central role in the

toxic mechanism, as demonstrated by the Lys122 ! Ala

mutant of bohtropstoxin I, together with significant

contributions by Tyr117 and Arg118 (Chioato et al.,

2002). A dimeric state might be essential in some Lys49

myotoxins, as demonstrated with bothropstoxin I (de

Oliveira et al., 2001). The initially weak toxin-membrane

interaction would be further strengthened by the contri-

bution of hydrophobic and aromatic residues of the effector

C-terminal loop (Lomonte et al., 1994a, 1999, 2003; Nunez

et al., 2001; Chioato et al., 2002), which may partially

penetrate and disorganize the bilayer (Fig. 4). Membrane

fluidity might be required in this step, since in vitro cytolysis

does not occur at 4 8C (Lomonte et al., 1994b). As first noted

by Kini and Evans (1989), this membrane-damaging

mechanism might be analogous to that exerted by numerous

cationic peptides of the innate antimicrobial defenses (Hill

et al., 1991; Hancock and Chapple, 1999). Finally, two

additional factors that might enhance membrane pertur-

bation would be the quaternary conformation changes

through a molecular hinge, in the case of dimeric Lys49

myotoxins (da Silva Giotto et al., 1998; de Oliveira et al.,

2001), and the potential acylation of these toxins, either via

autocatalysis (Pedersen et al., 1995), or via an interrupted

catalytic cycle that fails to release a free fatty acid (Lee et al.,

2001). Membrane perturbation would be the key toxic event,

allowing an uncontrolled influx of ions (i.e. Ca2þand Naþ),

and eventually triggering irreversible intracellular altera-

tions and cell death.

9. Future directions

Although still many important details of the mechan-

ism of action of the Lys49 PLA2 myotoxins remain to be

established, significant advances have been made towards

understanding the structural basis of their function. Their

mode of action has now been understood in its more

general concepts, and a consistent working hypothesis can

be experimentally supported. But further molecular

refinement of these general concepts will certainly come

next, from detailed experiments using mutant and

chimeric proteins, for example. Moreover, relevant clues

Fig. 4. An integrated model to explain the mechanism of action of

Lys49 phospholipase A2 myotoxins. (A) A dimeric myotoxin,

represented by B. asper myotoxin II, is shown with gray a-carbon

backbone, and black C-terminal segments 115–129 (C) and N-

terminal segments 1–8 (N), approaching a membrane (simplified, not

drawn to scale). Gray and black circles represent head groups of

zwitterionic and anionic phospholipids, respectively. (B) The toxin

establishes initial, weak electrostatic interactions with anionic

phospholipids (or other yet unidentified negatively charged accep-

tors), using its basic amino acids within the C-terminal 115–129

region, with the possible (see text) contribution of residues of the N-

terminal a-helix. (C) The binding interaction is strenghtened by

subsequent involvement of hydrophobic and aromatic amino acids

whithin the effector segment 115–129, which partially penetrate and

disorganize the bilayer, altering its permeability to ions (dashed

arrows). Relative motion of the angle formed between the two

monomers (thick empty arrows) may play a role in the membrane

perturbation mechanism of the dimeric Lys49 myotoxins. In addition,

the possible acylation of Lys49 myotoxins (not represented) may have

an enhancing role in their membrane-perturbing mechanism.

B. Lomonte et al. / Toxicon 42 (2003) 885–901 895

may originate from the study of novel myotoxin

inhibitors, increasingly being obtained from a variety of

sources (reviewed by Lizano et al., this issue), particularly

when co-crystallization attempts succeed. Reciprocally, a

better understanding of the structural basis for toxicity in

the Lys49 myotoxins will hopefully open new strategies

for the search of efficient, clinically useful inhibitors, to

aid snakebite victims.

Acknowledgements

We specially thank Jose Marıa Gutierrez, Cecilia Dıaz,

Alberto Alape, Sergio Lizano, Georgina Gurrola, Lourival

Possani, Timoteo Olamendi, Fernando Zamudio, Edgardo

Moreno, Ernesto Moreno, Marco Maccarana, Andrej

Tarkowski, Lars-Ake Hanson, Ulf Bagge, Andreimar

Soares, Rhaguvir Arni, Stefano Rufini, Wonwha Cho, Jose

R. Giglio, Motonori Ohno, Javier Pizarro, Carlos E. Nunez,

and Carlos Santamarıa, for contributions to different aspects

of these studies, as well as Rodrigo Mora for invaluable help

with the Matlab programming. Financial support by

International Foundation for Science (F/2766-2), CONICIT

(FV-058-02), the Embassy of Japan in Costa Rica, and

Universidad de Costa Rica (VI-741-99-269), is gratefully

acknowledged.

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