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UNIVERSIDADE ESTADUAL PAULISTA
INSTITUTO DE BIOCIÊNCIAS
CAMPUS DE BOTUCATU
Estudos estruturais com a BthTX-II, uma Asp49-Fosfolipase A
2
miotóxica e com baixa atividade catalítica do veneno de
Bothrops jararacussu
LUIZ CLÁUDIO CORRÊA
Botucatu
2007
Dissertação apresentada ao Instituto de
Biociências, Ca
mpus de Botucatu,
UNESP, para obtenção do título de
Mestre no Programa de PG em
Biologia Geral e Aplicada.
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UNIVERSIDADE ESTADUAL PAULISTA
INSTITUTO DE BIOCIÊNCIAS
CAMPUS DE BOTUCATU
Estudos estruturais com a BthTX-II, uma Asp49-Fosfolipase A
2
miotóxica e com baixa atividade catalítica do veneno de
Bothrops jararacussu
Luiz Cláudio Corrêa
Orientador: Prof. Adjunto Marcos Roberto de Mattos Fontes
Botucatu
2007
Dissertação apresentada ao Instituto
de Biociências, Campus de Botucatu,
UNESP, para obtenção do títu
lo de
Mestre no Programa de PG em
Biologia Geral e Aplicada.
ads:
2
Dedico este trabalho aos meus pais, Antônio e Nahir, que com tanta
abnegação, se dedicaram à formação e à dignidade dos filhos.
3
Agradecimentos
- Ao meu orientador, o Professor Adjunto Marcos Roberto de Mattos Fontes, pela
oportunidade e pela amizade que me propiciou nesse período tão enriquecedor.
- À Fundação de Auxílio à Pesquisa do Estado de São Paulo (FAPESP) e ao Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq) que financiaram este
projeto de pesquisa.
- Ao Laboratório Nacional de Luz Síncrotron (LNLS), que disponibilizou uso de
equipamento multi-usuário para a realização deste trabalho.
- Aos colegas de curso, pela amizade, companheirismo e disposição para dividir
comigo, o conhecimento necessário para iniciar e concluir os estudos.
- Sobretudo, a Deus, pois sem Ele, nada se realiza.
4
LISTA DE ABREVIATURAS
Acidic-Ag Asp49-PLA
2
ácida de Agkistrodon halys pallas
Asp49-PLA
2
Fosfolipase A
2
com Aspartato na posição 49
Basic-Ag Asp49-PLA
2
básica de Agkistrodon halys pallas
BnSP-6 e BnSP-7 Miotoxina I e Miotoxina II de Bothrops neuwiedi pauloensis
BPB Brometo de p-bromofenacila
BthA-I Asp49-PLA
2
ácida de Bothrops jararacussu
BthTX-I Miotoxina I de Bothrops jararacussu
BthTX-II Bothropstoxina I de Bothrops jararacussu
Lys49-PLA
2
Fosfolipase A
2
com Lisina na posição 49 (homóloga)
MjTX-I Miotoxina I de Bothrops moojeni
NBSF Fluoreto de 2-nitrobenzenosulfonil
NPSC Cloreto de o-nitrofenilsulfenil
PLA
2
Fosfolipase A
2
(E. C. 3. 1. 1. 4.)
PrTX-II Miotoxina II de Bothrops pirajai
PrTX-III Piratoxina III de Bothrops pirajai
5
RESUMO
Fosfolipases A
2
(PLA
2
s) são os componentes responsáveis, nos venenos botrópicos,
pela destruição da membrana celular, através de um mecanismo de hidrólise de
fosfolipídios. Uma classe de PLA
2
s homólogas, que sofreu uma mutação natural no resíduo
Asp49 para Lys49, não apresenta atividade catalítica, porém, estas enzimas podem exercer
diversas atividades farmacológicas, como por exemplo, a miotoxicidade. Várias PLA
2
s têm
sido purificadas de venenos botrópicos. Três enzimas foram purificadas do veneno de
Botrrops jararacussu, uma Lys49-PLA
2
(BthTX-I), com atividade miotóxica, e duas
Asp49-PLA
2
s, sendo uma ácida (BthA-I) com alta atividade catalítica, mas não miotóxica,
e outra básica (BthTX-II), que, embora seja uma Asp49-PLA
2
, possui baixa atividade
catalítica, mostrando-se porém, miotóxica. Isso coloca BthTX-II numa posição
intermediária entre BthTX-I e BthA-I. Este trabalho apresenta a determinação da estrutura
tridimensional da botropstoxina II (BthTX-II), realizada pelo método da cristalografia de
proteínas, assim como um estudo comparativo dessa estrutura com as de outras PLA
2
s
(Asp49 e Lys49) purificadas de venenos de serpentes dos gêneros Bothrops e Agkistrodon.
Os estudos revelam uma grande modificação na posição do loop de ligação do cálcio,
região de resíduos que coordenam este íon, fundamental para a atividade catalítica, em
BthTX-II, quando comparada com outras Asp49-PLA
2
s que apresentam essa atividade. A
cadeia lateral do resíduo Tyr28, presente nessa região, encontra-se em posição oposta à
mesma nas cataliticamente ativas Asp49-PLA
2
s, o que a leva a se ligar ao átomo O2 do
resíduo Asp49, que deveria fazer parte da coordenação do íon cálcio. Estas modificações
impedem a ligação do íon cálcio e assim, podem ser responsáveis pela baixa atividade
catalítica encontrada em BthTX-II, a qual também não apresenta a Lys122, que é associada
com a atividade miotóxica nas Lys49-PLA
2
. Em contrapartida, esta possui um resíduo
ácido (Asp) nesta posição, o que indica um processo alternativo para esta atividade na
BthTX-II.
Palavras chave:
Cristalografia de Raio-X; Asp49-fosfolipase A
2
; Veneno de Bothrops
jararacussu; Miotoxicidade; Baixa atividade catalítica.
6
ABSTRACT
Phospholipases A
2
are components of Bothrops venoms responsible for disruption
of cell membrane integrity via hydrolysis of its phospholipids. A class of homologous
PLA
2,
that underwent a natural mutation in the residue Asp49 for a Lys49, doesn’t show
catalytic activity, however, these enzymes can perform different pharmacology activities,
such as myotoxicity. Several PLA
2
s have been purified from bothropic venoms. Three
enzymes were purified from Bothrops jararacussu venom, a Lys49-PLA
2
(BthTX-I), with
myotoxic activity, and two Asp49-PLA2s; an acidic (BthA-I) with high catalytic activity,
but no myotoxic, and a basic (BthTX-II), that, although being an Asp49-PLA
2
, presents
low catalytic activity, however displaing myotoxicity. This put BthTX-II in an
intermediate position between BthTX-I and BthA-I. This work presents the three-
dimensional structure determination of bothropstoxin II (BthTX-II) performed by protein
crystallography method and, a comparative study of this protein with other PLA
2
(Asp49
and Lys49) purified from venom of Bothrops and Agkistrodon genus. The studies showed
a severe distortion of calcium binding loop - fundamental region for the catalytic activity -
in the BthTX-II when it is compared with other Asp49-PLA
2
s that show this activity. The
side chain of the residue Tyr28, present in this region, is in an opposite position in relation
to the same residue in the catalytic activity Asp49-PLA
2
s, making bond with the atom O2
of the residue Asp49, which should coordinate the calcium. BthTX-II does not present
Lys122, responsible for the polarization of the residue Cys29, which causes the direction
of the amine group (NH) for the Gly30 for the solvent in the hydrophobic channel. These
modifications prevent the binding of the calcium ion, so, they are responsible for the low
catalytic activity found in BthTX-II. Additionally, BthTX-II does not present a Lys residue
in the position 122, which is associated with myotoxic activity. In contrast, it has an acidic
residue in this position (Asp), indicating an alternative process for this activity in the
BthTX-II.
Keyboards: X-ray crystallography; Asp49-phospholipase A
2
; Bothrops jararacussu
venom; Myotoxicity; low catalytic activity.
7
SUMÁRIO
Lista de abreviaturas
Resumo
Abstract
1. Introdução 8
2. Artigo referente à dissertação 20
3. Conclusões 56
4. Referências bibliográficas 59
Apêndice 69
1. INTRODUÇÃO
9
No Brasil, existem 24 espécies de serpentes pertencentes ao gênero Bothrops
(Melgarejo, 2003), que habitam, preferencialmente, ambientes úmidos como matas, áreas
cultivadas e locais de proliferação de roedores (zonas rurais e periferia das grandes
cidades). Estas serpentes são responsáveis por cerca de 85-90% do total de acidentes
ofídicos que ocorrem no país (Rosenfeld, 1971; Ferreira et al., 1992 e Ribeiro et al., 1993),
o que as tornam, assunto de grande interesse científico, médico e social. Venenos de
serpentes são compostos por uma complexa mistura de proteínas, enzimas, peptídeos e
compostos inorgânicos que podem apresentar diversas atividades farmacológicas e/ou
biológicas, sendo seu estudo, uma área bastante promissora na busca e desenvolvimento de
novos medicamentos.
A natureza e as propriedades biológicas dos componentes dos venenos de serpentes
são peculiares para cada espécie animal e a concentração destes componentes pode variar
intra-especificamente, devido a diversos fatores como variações climáticas, geográficas,
sexuais, etárias, alimentares, tempo decorrido entre as extrações de venenos, heranças
genéticas e outros (Lomonte & Carmona, 1992; Valiente et al., 1992; Monteiro et al., 1998
e Soares et al., 2004).
Dentre as diversas toxinas que compõem os venenos botrópicos, estão as
fosfolipases (PLA
2
- E.C.3.1.1.4), enzimas largamente distribuídas na natureza e
extensivamente estudadas. (Dennis, 1983; Waite, 1987 e Dennis, 1994). A primeira vez
que se estudou a atividade enzimática, hoje denominada atividade PLA
2
, foi no século
XIX, utilizando-se venenos de serpentes (Stephens et al., 1898).
PLA
2
s são proteínas multifuncionais capazes de participar como mediadoras de
vários processos inflamatórios, podendo ser aplicadas em diversas áreas da medicina, por
exemplo, na detecção de pré-eclampsia severa, ações gerais de anestésicos, tratamento de
artrites reumatóides, atuando como agentes bacteriológicos em glândulas lacrimais e outros
tecidos, bloqueando a entrada do vírus dentro da célula hospedeira e destacando-se como
um potencial agente antimalárica (Uhl et al., 1997; Moreira et al., 2002 e Soares et al.,
2004).
Diversas PLA
2
(s) têm sido descobertas em secreções (sPLA
2
ou extracelulares) e
no citosol (cPLA
2
ou intracelulares). As PLA
2
s intracelulares apresentam alta massa
molecular (85 kDa), geralmente estão associadas a membranas, são cálcio-dependentes e
estão envolvidas no metabolismo de fosfolipídios (perturbações das membranas) na
sinalização celular e no remodelamento de fosfolipídios (Mukherjee et al., 1994; Arni &
Ward, 1996 e Six & Dennis, 2000). Já as extracelulares são as encontradas em fluidos
10
biológicos, particularmente em secreções pancreáticas, exudados inflamatórios e em
venenos de répteis (serpentes e lagartos) e insetos (Rosenberg, 1990; Arni & Ward, 1996 e
Ownby, 1999). Trata-se de enzimas pequenas (119 a 143 aminoácidos), de baixa massa
molecular (12 a 15 kDa), estáveis e cálcio-dependentes e que atuam sobre substratos
lipídicos (principalmente fosfolipídios) hidrolisando a ligação éster sn-2 (“sequentially
numbered”) (Figura 01).
Figura 01: Representação esquemática de um fosfolipídeo e os locais de atuação das
diferentes fosfolipases (Lehninger et al., 1993).
Os produtos liberados na atividade catalítica das PLA
2
s são lisofosfolipídios,
importantes na sinalização celular e perturbação de membranas (Moolenaar et al.,1997), e
ácidos graxos (Waite, 1987 e Arni & Ward, 1996) tais como o ácido oléico (reserva
energética) e o ácido aracdônico (precursor de eicosanóides, os quais atuam como
mediadores de inflamações).Inicialmente, as PLA
2
(s) foram divididas em dois grupos,
baseado nas posições das pontes dissulfeto (Heinrikson et al., 1977; Dufton & Hider,1983),
mas, com o avanço das técnicas de seqüenciamento e caracterização estrutural, tem havido
um constante aumento dos seus grupos e subgrupos. Atualmente, a superfamília das PLA
2
s
é composta por um conjunto de quinze grupos (Schaloske et al., 2006) que apresentam um
alto grau de homologia seqüencial e estrutural, diferenciando-se somente pela localização e
quantidade de pontes dissulfeto e pelos comprimentos de seus loops (Dennis, 1994). PLA
2
s
11
dos grupos I e II são os componentes principais dos venenos de serpentes (Rosenberg,
1990), sendo que aquelas do segundo grupo são os compostos predominantes dos venenos
de serpentes da família Viperidae, à qual pertencem às espécies do gênero Bothrops.
Apesar dessa pequena variação seqüencial e estrutural, as PLA
2
s destacam-se por
seu grande espectro de atividades biológicas, como: neurotoxicidade pré e/ou pós-
sináptica, miotoxicidade, cardiotoxicidade, mionecrose, anticoagulante (inibição de
agregação plaquetária), efeitos convulsivos, indução de edema, atividades hipotensora,
hemolítica, hemorrágica, entre outros efeitos (Kini & Evans, 1989; Rosenberg, 1990;
Evans & Kini, 1997; Kini, 1997; Gutiérrez & Lomonte, 1997; Gerrard et al., 1993; Ownby,
1999; Braud et al., 2000; Valentin & Lambeau, 2000; Condrea et al., 1981; Lloret &
Moreno, 1993; Andrião-Escarso et al., 2000; Gutiérrez et al., 1980 e Andrião-Escarso et
al., 2002).
Os principais elementos da estrutura secundária das PLA
2
s (Figura 02) são a região
N-terminal, constituída por uma -hélice I, denominada “h1” (resíduos 1-12, os quais
formam o canal hidrofóbico das PLA
2
s; região altamente conservada envolvida na ligação
e orientação de substratos à proteína), em seguida tem-se a hélice curta (“short helix”)
constituída pelos resíduos 18-23. Entre os resíduos 25-34, está localizada a região de
ligação do íon Ca
+2
, que é coordenado por duas ligações com átomos de oxigênio
carboxílicos do Asp49, três ligações com átomos de oxigênio dos resíduos Tyr28, Gly 30,
Gly32, além de duas moléculas de água. Na seqüência, vem a -hélice II (“h2”) constituída
pelos resíduos 40-55. Esta se liga a uma curta folha β antiparalela (“β-wing”) (resíduos 75-
77 e 82-84). Estes dois últimos seguimentos, “h2” e “β-wing”, alocam resíduos que
constituem o sítio catalítico das PLA
2
s. Após a região de “β-wing”, os resíduos 90-107
formam -hélice III (“h3”) que se liga a uma região bastante flexível, denominada C-
terminal (resíduos 108-133), (Arni & Ward, 1996; Ward et al.,1998; de Azevedo, 1999 e
Magro et al, 2003). Este modelo segue o sistema de numeração proposto por Renetseder et
al., 1985.
12
Figura 02: Representação esquemática do enovelamento de uma PLA
2
. Figura feita pelo
programa “Pymol” (Delano, 2002).
Para uma melhor compreensão dos mecanismos de ação das PLA
2
s, muitos estudos
têm sido realizados, tanto com proteínas em sua forma nativa como utilizando complexos
destas com diversos ligantes, que podem inibir de forma parcial ou completa as
propriedades farmacológicas das proteínas, ou simplesmente revelar as interações que
determinam a ligação destas substâncias aos seus sítios protéicos específicos, além da
análise de variedades mutantes, uma vez que a mutação de resíduos específicos tem como
objetivo estabelecer a importância de determinados aminoácidos e/ou segmentos protéicos
para a atividade da proteína como um todo.
Estudos com modificações químicas têm mostrado efeitos importantes nas
atividades farmacológicas das PLA
2
s. A alquilação da His48, resíduo altamente
conservado do sítio catalítico das PLA
2
s, pelo brometo de p-bromofenacil (BPB) induz à
perda da atividade hidrolítica de fosfolipídios e à diminuição dos efeitos tóxicos e
farmacológicos destas proteínas (Rodrigues et al., 1998; Zhao et al., 1998; Soares et al.,
13
2000 a,b e Soares et al., 2001a). Outras modificações químicas como a sulfonação de
resíduos de Tyr pelo fluoreto de p-nitrobenzenosulfonil (NBSF) e a sulfonação de resíduos
Trp pelo cloreto de o-nitrofenilsulfenil (NPSC) também levam à redução de efeitos tóxicos
dessas enzimas (Yang et al., 1985; Soares et al., 2000 a,b; Soares et al., 2001b; Soares &
Giglio, 2003).
Outro ponto interessante no estudo das relações estruturais e funcionais das PLA
2
s
diz respeito à ação de inibidores naturais, que atuam de forma a minimizar os efeitos destas
enzimas durante o processo inflamatório. Entre as substâncias naturais que
comprovadamente apresentam capacidade de inibir o processo inflamatório está a vitamina
E (-tocoferol). Esta substância atua inibindo tanto a atividade das PLA
2
s quanto o ciclo da
cicloxigenase (Pentland et al., 1992; Traber & Packer, 1995) e por isso, tem sido utilizada
nos tratamentos das doenças de Alzheimer e Parkinson e especula-se que bloqueie a
progressão da doença de Alzheimer inibindo a atividade PLA
2
e estabilizando as
membranas neurais (Ebadi et al., 1996; Sano et al., 1997).
Em 2002, Chandra et al. co-cristalizaram uma PLA
2
dimérica do veneno de Daboia
russeli pulchella complexada com -tocoferol e observaram a presença de somente uma
molécula de -tocoferol ligada a um dos monômeros. Os testes bioquímicos mostraram
uma diminuição de 50% em sua atividade catalítica ao serem tratadas com -tocoferol,
mesmo em alta concentração do inibidor.
Uma mutação natural do resíduo Asp49, fundamental para a atividade catalítica das
PLA
2
s, por uma Lys deu origem a um outro grupo de enzimas denominadas Lys49-PLA
2
ou
PLA
2
s homólogas (Francis et al., 1991; Homsi-Brandenburgo et al., 1988; Ward et al.,
1995). Estas não apresentam atividade catalítica, embora mantenham diversas outras
funções farmacológicas (Lomonte et al., 1994a). A mutação em questão ocorre numa
conhecida região de ligação do íon Ca
+2
das PLA
2
s cataliticamente ativas (Asp49-PLA
2
s)
(Figura 03). A presença da Lys no lugar do Asp na posição 49 torna impossível a
coordenação do íon Ca
+2
(Scott et al. 1990). O N da cadeia lateral do resíduo Lys49 ocupa
a posição do íon, o que inviabiliza a ocorrência da reação catalítica (Lee et al., 2001), uma
vez que este íon, coordenado pelo Asp, é responsável, conjuntamente com outros
aminoácidos, pela fixação do substrato ao sítio enzimático das PLA
2
s.
14
Figura 03: a. Região de ligação do íon Ca
+2
(“calcium binding loop”) de uma PLA
2
cataliticamente ativa de Naja naja naja (Asp49). b. Região análoga à região de ligação do
Ca
+2
de uma Miotoxina II (PLA
2
homóloga) cataliticamente inativa de Bothrops asper
(Lys49) (Arni & Ward, 1996 e Ward et al., 1998).
Estudos de mutagênese sítio-dirigida com a substituição do resíduo Lys49 por um
Asp em BthTX-I (Lys49-PLA
2
– cataliticamente inativa) (Ward et al., 2002)
demonstraram que esta proteína continuou não apresentando atividade catalítica, sugerindo
que a perda dessa atividade não está somente relacionada à mutação de Asp49 por Lys49,
mas a um conjunto de outros fatores estruturais, como a interação da região do C-terminal
(Lys122) com a Gly30. O N da cadeia lateral do resíduo Lys122 forma ponte de
hidrogênio com o resíduo Cys29, direcionando o grupo amina (NH) da Gly30 para a
exposição do solvente no canal hidrofóbico (Ward et al., 2002) e o N da cadeia lateral do
resíduo Lys49 forma pontes de hidrogênio com átomos de oxigênio das cadeias principais
dos resíduos Asn28 e Gly30. Este arranjo de pontes de hidrogênio está conservado na
maioria das estruturas das Lys49-PLA
2
s. A posição e orientação dos resíduos His48,
Tyr52, Asp99 e demais resíduos que participam da atividade catalítica estão altamente
conservadas tanto nas PLA
2
s, como nas PLA
2
s homólogas.
Apesar da incapacidade de promover a hidrólise de substratos lipídicos, as Lys49-
PLA
2
s apresentam um pronunciado efeito miotóxico tanto in vivo quanto in vitro
(Gutierrez & Lomonte, 1997). Esta atividade pode ocorrer independentemente da
substituição da Asp49 por Lys, sugerindo a independência da ligação do íon Ca
2+
ao sítio
ativo, ao contrário do verificado no processo normal de catálise de lipídios (Rufini et al.,
1992; Díaz et al., 1991; Díaz et al., 1992 e Ward et al., 1998).
Lomonte et al. (1994b) e Gutiérrez & Lomonte (1995) propõem que a região C-
terminal seja responsável pela miotoxicidade, por ser rica em resíduos hidrofóbicos e
15
básicos. A região de C-terminal, por ser bastante flexível, se insinuaria através das
membranas (celulares ou artificiais), promovendo a desestabilização destas, ou atuaria
como “âncora”, possibilitando o contato de sítios protéicos reativos desconhecidos.
Experimentos de mutação sítio-dirigida comprovaram o envolvimento da região C-
terminal nas atividades miotóxica e de rompimento de membranas das Lys49-PLA
2
s
(Chioato et al., 2002 e Ward et al., 2002). A substituição dos resíduos Lys115, 116 e 122
por alaninas levou à redução da atividade de dano à membrana, porém, não houve
alteração da atividade miotóxica. Substituições dos resíduos Tyr117 Trp117, Arg118
Ala118, Tyr119 Trp119 e Lys122 Ala122 eliminaram as cargas positivas de suas
cadeias laterais, reduzindo significante a atividade miotóxica, porém a atividade de dano à
membrana não foi alterada. Por fim, quando apenas Lys122 foi substituída por Ala, foram
reduzidas, tanto a atividade miotóxica (40%) como a de dano à membrana, além de não
ocorrer nenhuma atividade hidrolítica, indicando que a lisina da posição 122 é um suporte
evidente para a ativação da interrupção do ciclo catalítico. Assim, os experimentos
sugerem que estes resíduos da região C-terminal estão envolvidos no motif estrutural que
determina as atividades de miotoxicidade e dano à membrana e ainda indicam que estas
são independentes.
A análise de PLA
2
s de venenos de serpentes mostra que as mutações ocorrem mais
freqüentemente na região dos éxons, o que sugere a duplicação genética e uma evolução
acelerada dos éxons como responsáveis pela multiplicidade de isoformas encontradas em
um único veneno.
Acredita-se que substituições naturais ocorridas principalmente na superfície da
molécula destas proteínas possam ser produtos de mudanças de especificidade frente a
tecidos específicos (Soares et al., 2004).
Características regionais também podem influenciar na ampla variedade da
composição das toxinas do veneno de serpentes. O veneno da espécie Bothrops neuwiedi
pauloensis pode apresentar, dependendo do local, somente a toxina BnSP-7; somente sua
isoforma BnSP-6, além de haver um terceiro grupo que abriga as duas toxinas (BnSP-7 e
BnSP-6) (Rodrigues et al., 1998, Soares et al., 1998 e Soares et al., 2000b).
Estudos comparativos entre as Lys49-PLA
2
s e as Asp49-PLA
2
s mostram que os
resíduos que compõem o sítio catalítico (His48, Tyr52 e Asp99) e a região de ligação do
lipídio (Leu5, Val102 e Leu106) destacam-se como os mais relevantes e altamente
conservados.
16
O grande leque de ações farmacológicas e bioquímicas, a despeito da significativa
uniformidade estrutural, é a base do amplo interesse despertado por estes dois grupos
protéicos na comunidade científica. A identificação de prováveis relações estruturais e
funcionais torna-se mais fácil devido às pequenas variações existentes entre estas
moléculas.
O conhecimento de estruturas tridimensionais e respectivos estudos das relações
estrutura/função, tanto das PLA
2
s e PLA
2
s homólogas nas formas nativas como
complexadas e co-cristalizadas são de extrema importância devido ao interesse médico,
social e econômico, sendo uma informação indispensável para o desenvolvimento racional
de medicamentos e/ou derivados de toxinas menos agressivos a serem utilizados na
produção de soros antiofídicos.
A cristalografia de raios-X é o método mais utilizado para a determinação
tridimensional de estruturas macromoleculares. Determinar estruturas de cristais de
proteína requer monocristais, os quais são analisados por experimentos de difração de
raios-X. A formação de monocristais de proteínas só ocorre em condições limitadas
determinadas empiricamente (Kobe et al., 1999). Dentre os principais parâmetros que
afetam a solubilidade e conseqüentemente o processo de cristalização podemos destacar:
Parâmetros físico-químicos intrínsecos:
• Supersaturação (volume e concentração de proteína e precipitante);
• Variação da temperatura;
• pH da solução de cristalização (uso de soluções tampão, junto à solução protéica);
• Força iônica;
•
Pureza das substâncias químicas utilizadas;
• Efeitos de densidade, pressão, viscosidade e gravidade;
• Partículas sólidas (solubilização incompleta da proteína ou do precipitante).
Parâmetros bioquímicos e biofísicos:
• Presença de ligantes (ligação de substratos, co-fatores e íons metálicos);
• Aditivos específicos (agentes redutores, detergentes não-iônicos e poliaminas);
• Degradação e/ou desnaturação da amostra.
Amostra protéica:
• Contaminantes macromoleculares (falhas durante o processo de purificação;
poeira);
• Heterogeneidade conformacional ou da seqüência dos aminoácidos;
17
• Concentração da amostra.
Assim, a obtenção de cristais com qualidade cristalográfica não é uma atividade
trivial, podendo levar meses para se alcançar indícios da formação de cristais e, ainda
assim, pode ser necessário mais um tempo significativo para melhorar a qualidade dos
mesmos. Há ainda casos em que cristais, simplesmente não são obtidos.
Grande esforço tem sido empreendido e, nos últimos anos, diversas Asp49-PLA
2
s e
Lys49-PLA
2
s de veneno de serpentes têm sido cristalizadas e elucidadas estruturalmente
com o intuito de obterem-se tais correlações (Holland et al., 1990; Arni et al., 1995; de
Azevedo Jr. et al., 1997; da Silva-Giotto et al., 1998; de Azevedo Jr. et al, 1999; Arni et
al., 1999; Lee et al, 2001; Magro et al., 2003; Magro et al., 2004).
Três frações identificadas como PLA
2
s foram purificadas do veneno de B.
jararacussu (Fig. 04). Esta serpente, de nome popular jararacussu, se distribui desde o sul
da Bahia até o noroeste do Rio Grande do Sul, pode atingir 1,80 m de comprimento, sendo
talvez, a mais imponente do gênero. É a espécie que maior quantidade de veneno produz
(Melgarejo, 2003).
A primeira enzima é uma Lys49-PLA
2
básica denominada BthTX-I (Homsi-
Brandeburgo et al., 1988). Trata-se de uma enzima básica (pI= 8.2) com 121 resíduos de
aminoácidos e peso molecular de 13,667 KDa (Cintra et al, 1993; Ward et al., 1995) .
Apesar de não possuir atividade catalítica, estudos mostram que essa enzima apresenta alto
potencial miotóxico (Cintra et al., 1993). A segunda e a terceira foram caracterizadas como
Asp49-PLA
2
, sendo uma ácida (BthA-I) e outra básica (BthTX-II).
BthA-I é uma enzima que apresenta 122 resíduos de aminoácidos, peso molecular
de aproximadamente 14 KDa e pI= 4,5. Estudos de caracterização mostraram que esta
apresenta uma alta atividade catalítica e também, efeitos hipotensivo, de indução de edema
e inibição de agregação plaquetária, porém, não foram detectadas, atividades miotóxica,
neurotóxica, citotóxica (Andrião-Escarso et al., 2002).
BthTX-II foi purificada por Homsi-Brandeburgo et al (1988) e, desde então, tem
sido alvo de vários estudos. Pereira et al. (1998) realizaram seu sequenciamento pelo
método de Edman (Edman & Begg, 1967) e encontraram 120 resíduos de aminoácidos,
sendo apenas uma metionina. A enzima apresentou atividade PLA
2
residual. Um outro
estudo de sequenciamento, dessa vez por cDNA, foi realizado por Kashima et al. (2004),
que encontraram 122 resíduos, sendo 2 metioninas. Outras diferenças foram encontradas
nas seqüências, como a presença, no primeiro sequenciamento, de um Trp e uma Arg nas
18
posições 5 e 34, respectivamente, enquanto no segundo, tratava-se de uma Phe na posição
5 e uma Gln na posição 34.
Um estudo de purificação e caracterização enzimática de uma nova PLA
2
básica do
veneno de B. jararacussu utilizando-se três passos de HPLC (troca iônica, fase reversa e
exclusão molecular) separou oito frações, sendo que a fração sete (Bj VII) correspondeu a
BthTX-II (Bonfim et al., 2001). Esta apresentou alta atividade miotóxica e significativa
atividade neuromuscular em nervo frênico de camundongos; no entanto, não apresentou
atividade PLA
2
.
Figura 04: Espécime adulto de Bothrops jararacussu. (Foto: Leonardo, 2006).
Essa variação funcional encontrada em três enzimas extraídas do veneno da mesma
espécie, com estruturas e seqüências de aminoácidos tão similares, faz com que o estudo
estrutural destas se torne objeto de grande interesse para os ramos das ciências da saúde,
uma vez que pequenas modificações são responsáveis pela redução, ou até mesmo perda
da(s) atividade(s) tóxica(s). As estruturas de BthTX-I e BThA-I foram elucidadas
recentemente e estudos bioquímicos estruturais destas com ligantes e/ou inibidores têm
sido realizados ( da Silva-Giotto et al., 1998; Soares & Giglio, 2003; Magro et al., 2004;
Takeda et al., 2004; Murakami et al., 2006). Dessa forma, o objetivo deste trabalho foi
19
determinar a estrutura tridimensional de BthTX-II e assim, realizar um estudo comparativo
com PLA
2
s extraídas do veneno de B. jararacussu, bem como de outras espécies da família
Viperidae, a fim de tentar elucidar as bases moleculares da ampla gama de efeitos
farmacológicos apresentados por essas enzimas.
20
2. ARTIGOS REFERENTES À DISSERTAÇÃO
21
2.1. O artigo: “Preliminary X-ray crystallographic studies of BthTX-II, a myotoxic Asp49-
phospholipase A
2
with low catalytic activity from Bothrops jararacussu venom”, referente
aos estudos de cristalização de BthTX-II, foi publicado pelo periódico Acta
Crystallographica section F- Structural Biology and Crystallization communications (ver
apêndice).
2.2. O artigo: “Crystal structure of BthTX-II, a myotoxic Asp49-phospholipase A
2
with
low catalytic activity from Bothrops jararacussu venom”, referente aos estudos estruturais
de BthTX-II, será submetido para publicação.
22
Preliminary X-ray crystallographic studies of BthTX-II, a
myotoxic Asp49-phospholipase A2 with low catalytic activity
from Bothrops jararacussu venom
L. C. Corrêa,
a
D. P. Marchi- Salvador,
a
A. C. O. Cintra,
b
A. M. Soares
b
and
M. R. M. Fontes
a
*
a
Departamento de Física e Biofísica, Instituto de Biociências, UNESP, CP 510, CEP
18618-000, Botucatu-SP, Brazil, and
b
Departamento de Análises Clínicas, Toxicológicas e
Bromatoloógicas, FCFRP, USP, Ribeirão Preto/SP, Brazil
*Correspondence e-mail: fontes@ibb.unesp.br
Abstract
For the first time, a complete X-ray diffraction data set has been collected from a myotoxic
Asp49-phospholipase A
2
(Asp49-PLA
2
) with low catalytic activity (BthTX-II from
Bothrops jararacussu venom) and a molecular-replacement solution has been obtained
with a dimer in the asymmetric unit. The quaternary structure of BthTX-II resembles the
myotoxin Asp49-PLA
2
PrTX-III (piratoxin III from B. pirajai venom) and all non-catalytic
and myotoxic dimeric Lys49- PLA
2
s. In contrast, the oligomeric structure of BthTX-II is
different from the highly catalytic and non-myotoxic BthA-I (acidic PLA
2
from B.
jararacussu). Thus, comparison between these structures should add insight into the
catalytic and myotoxic activities of bothropic PLA
2
s.
1. Introduction
Phospholipases A
2
(PLA
2
s; EC 3.1.1.4) belong to a superfamily of proteins which
hydrolyze the sn-2 acyl groups of membrane phospholipids to release fatty acids,
arachidonic acid and lysophospholipids (van Deenen & de Haas, 1963). The coordination
of the Ca
2+
ion in the PLA
2
calcium-binding loop includes an Asp at position 49 which
plays a crucial role in the stabilization of the tetrahedral transition-state intermediate in
catalytically active PLA
2
s (Scott et al., 1992). In the genus Bothrops, PLA
2
s are the main
components of the venoms produced by species classified into this animal group. In
23
addition to their primary catalytic role, snake-venom PLA
2
s show other important
toxic/pharmacological effects, including myonecrosis, neurotoxicity, cardiotoxicity and
haemolytic, haemorrhagic, hypotensive, anticoagulant, platelet-aggregation inhibition and
oedemainducing activities (Gutiérrez & Lomonte, 1997; Ownby, 1998; Andrião-Escarso et
al., 2002). Some of these activities are correlated with the enzymatic activity, but others
are completely independent (Kini & Evans, 1989; Soares & Giglio, 2004). It has been
suggested that some specific sites of these molecules have biochemical properties that are
responsible for the pharmacological and toxic actions, including the anticoagulant and
platelet-inhibition activities (Kini & Evans, 1989). PLA
2
s are also one of the enzymes
involved in the production of eicosanoids. These molecules have physiological effects at
very low concentrations; however, increases in their concentration can lead to
inflammation (Needleman et al., 1986). Thus, the study of specific PLA
2
inhibitors is
important in the production of structure based anti-inflammatory agents. Many non-
catalytic homologous PLA
2
s (Lys49-PLA
2
s) have been purified from Bothrops snake
venoms and have been structurally and functionally characterized (Marchi-Salvador et al.,
2005, 2006; Watanabe et al., 2005; Soares et al., 2004; Magro et al., 2003; Lee et al.,
2001; Arni et al., 1995, 1999; da Silva-Giotto et al., 1998). However, little is known about
the bothropic catalytic Asp49-PLA
2
(Magro et al., 2004, 2005; Rigden et al., 2003;
Serrano et al., 1999; Pereira et al., 1998; Daniele et al., 1995; Homsi-Brandeburgo et al.,
1988). Despite the structures of a large number of PLA
2
s having been solved by
crystallography to date, many questions still need to be clarified. For example, there are
PLA
2
s with high, moderate and no catalytic activity (Magro et al., 2004; Rigden et al.,
2003; da Silva- Giotto et al., 1998). However, for all these ‘classes’ of PLA
2
s the majority
of residues of the catalytic machinery are conserved. Similarly, toxic (e.g. myotoxicity,
cytotoxicity) and pharmacological effects (e.g. anticoagulant, hypotensive and platelet-
aggregation activities) are far from being completely understood. An acidic catalytic PLA
2
(BthA-I) has been isolated from B. jararacussu venom and characterized (Andrião-Escarso
et al., 2002; Roberto et al., 2004). BthA-I is three to four times more catalytically active
than BthTX-II (bothropstoxin-II from B. jararacussu) and other basic Asp49-PLA
2
s from
Bothrops venoms, but is not myotoxic, cytotoxic or lethal (Andrião-Escarso et al., 2002).
Other activities demonstrated by this enzyme are time-independent oedema induction,
hypotensive response in rats and platelet-aggregation inhibition (Andrião-Escarso et al.,
2002). The crystal structure of BthA-I has been recently described in two conformational
states: monomeric and dimeric (Magro et al., 2004). Additionally, Magro et al. (2005)
24
solved the structure of BthA-I chemically modified with BPB (p-bromophenacyl bromide)
and showed important tertiary and quaternary structural changes in this enzyme. This novel
oligomeric structure is more energetically and conformationally stable than the native
structure and the abolition of pharmacological activities (including anticoagulant,
hypotensive effect and platelet-aggregation inhibition) by the ligand may be related to the
oligomeric structural changes. The isolation, biochemical/pharmacological characterization
and amino-acid sequence of bothropstoxin II from B. jararacussu (BthTX-II) have been
reported (Homsi-Brandeburgo et al., 1988; Gutiérrez et al., 1991; Pereira et al., 1998).
Protein sequencing indicated that BthTX-II is an Asp49-PLA
2
and consists of 120 amino
acids (MW = 13 976 Da). The protein shows myotoxic, oedematogenic and haemolytic
effects and low phospholipase activity (Homsi- Brandeburgo et al., 1988; Gutiérrez et al.,
1991). Recently, it has been shown that BthTX-II induces platelet aggregation and
secretion through multiple signal transduction pathways (Fuly et al., 2004). Despite
BthTX-II having been crystallized more than ten years ago (Bortoleto et al., 1996), the
structure has not been solved to date, probably owing to the low completeness of the data
set (50–60% completeness). The crystals belonged to the tetragonal crystal system and
preliminary analysis indicated the presence of three molecules in the asymmetric unit
(Bortoleto et al., 1996). However, a careful analysis of the Matthews coefficient indicated
that a tetrameric conformation is also possible (VM = 2.4 Å
3
Da
_1
), which also occurs in
the Lys49-PLA
2
MjTX-I (myotoxin I from B. moojeni venom) structure formed of two
Lys49-PLA
2
dimers (Marchi-Salvador et al., 2005; personal communication). In the
present paper, we describe the crystallization of BthTX-II (bothropstoxin-II) from B.
jararacussu venom in the monoclinic system, the collection of a complete X-ray
diffraction data set and molecular-replacement solution. This study should improve the
understanding of the relation of the myotoxic and low catalytic activity mechanisms to the
structural features of this protein when compared with BthTX-I (Lys49-PLA2 from B.
jararacussu venom) and BthA-I, which possess no and high catalytic activity, respectively.
2. Experimental procedures
2.1. Purification
BthTX-II was isolated from B. jararacussu snake venom by gel filtration and ion-exchange
chromatography as previously described (Homsi-Brandeburgo et al., 1988).
2.2. Crystallization
25
A lyophilized sample of BthTX-II was dissolved in ultrapure water at a concentration of 12
mg ml
_1
. The sparse-matrix method (Jancarik & Kim, 1991) was used to perform initial
screening of the crystallization conditions (Crystal Screens I and II; Hampton Research).
Large crystals of BthTX-II were obtained by the conventional hanging-drop vapour-
diffusion method (MacPherson, 1982), in which 1 µl protein solution and 1 µl reservoir
solution were mixed and equilibrated against 500 µl of the same precipitant solution. The
BthTX-II was crystallized using a solution containing 20% (v/v) 2-propanol, 13% (w/v)
polyethylene glycol 4000 and 0.1 M sodium citrate pH 5.6. The best crystals measured
approximately 0.4 x 0.2 x 0.1 mm after two months at 291 K (Fig. 1).
Figure 1: Crystals of BthTX-II from B. jararacussu venom.
2.3. X-ray data collection and processing
X-ray diffraction data from BthTX-II crystals were collected using a wavelength of 1.427
Å at a synchrotron-radiation source (Laboratório Nacional de Luz Síncrotron, LNLS,
Campinas, Brazil) with a MAR CCD imaging-plate detector (MAR Research). A crystal
was mounted in a nylon loop and flash-cooled in a stream of nitrogen at 100 K using no
26
cryoprotectant. The crystal-to-detector distance was 100 mm and an oscillation range of 1°
was used; 149 images were collected. The data were processed to 2.13 Å resolution using
the HKL program package (Otwinowski & Minor, 1997).
3. Results and discussion
The data-collection statistics are shown in Table 1. The data set is 96.1% complete at 2.13
Å resolution, with R
merge
= 9.1%. The crystals belong to space group C2, with unit-cell
parameters a = 58.9, b = 98.5, c = 46.7 Å and = 125.9°. Packing parameter calculations
based on the protein molecular weight indicate the presence of a dimer in the asymmetric
unit. This corresponds to a Matthews coefficient (Matthews, 1968) of 2.0 Å
3
Da
_1
with a
calculated solvent content of 37.4%, which are within the expected range for typical
protein crystals (assuming a value of 0.74 cm
3
g
_1
for the protein partial specific volume).
The crystal structure was determined by molecular-replacement techniques implemented in
the program AMoRe (Navaza, 1994) using the coordinates of a monomer of PrTX-III
(PDB code 1gmz). The quaternary structure of BthTX-II is very similar to those of PrTX-
III and all dimeric bothropic Lys49-PLA
2
s (Rigden et al., 2003; Soares et al., 2004) and
totally different from those of native dimeric BthA-I and BthA-I–bromophenacyl bromide
and BthA-I-α-tocopherol complexes (Magro et al., 2004, 2005; Takeda et al., 2004). In
conclusion, a complete X-ray diffraction data set has been collected from a low catalytic
activity Asp49-PLA
2
for the first time (to 2.13 Å) and a molecular-replacement structure
solution has been obtained. The quaternary structure of BthTX-II resembles those of the
myotoxin PrTX-III (which does not bind Ca
2+
ions) and all noncatalytic and myotoxic
dimeric Lys49-PLA
2
s (Rigden et al., 2003; Soares et al., 2004). In contrast, the oligomeric
structure of BthTX-II is different from that of the high catalytic activity and non-myotoxic
BthA-I (Magro et al., 2004). Thus, comparison between these structures should add insight
into the catalytic and myotoxic activities of bothropic PLA
2
s.
27
Table 1
X-ray diffraction data-collection and processing statistics.
Values in parentheses are for the highest resolution shell.
!" #$%$%
& '" #
( "")
!
( *
+
)
!," -./0/$12
3( 4 ##
*( ''5',""*
6
$%
5785 #%
!, ,- %#
1.9"'':
1
%
3
$
#
1 ".;
<) %
†R
merge
= Σ
hkl
(Σ
i
(|I
hkl,i
-<I
hkl
>|))/Σ
hkl,i
<I
hkl
>, where I
hkl,i
is the intensity of an individual
measurement of the reflection with Miller indices hkl, and <I
hkl
> is the mean intensity of
that reflection. Calculated for I>-3σ (I). ‡ Data processing used the HKL suite
(Otwinowski & Minor, 1997).
The authors gratefully acknowledge financial support from Fundação de Amparo à
Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq), Fundação para o Desenvolvimento da UNESP
(FUNDUNESP) and Laboratório Nacional de Luz Síncrontron (LNLS, Campinas-SP).
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31
Crystal structure of a myotoxic Asp49-phospholipase
A
2
with low catalytic activity, insights into Ca
2+
independent
catalytic mechanism
Corrêa, L.C.
a
, Marchi-Salvador, D.P.
a
, Cintra, A.C.O.
b
, Soares, A.M.
b
, Fontes, M.R.M.
a
a
Departamento de Física e Biofísica, Instituto de Biociências, UNESP, Botucatu-SP,
Brazil
b
Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, FCFRP, USP,
Ribeirão Preto/SP, Brazil
Abstract
Phospholipases A
2
belong to the superfamily of proteins which hydrolyze the sn-2 acyl
groups of membrane phospholipids to release arachidonic acid and lysophospholipids. A
myotoxic Asp49-phospholipase A
2
(Asp49-PLA
2
) with low catalytic activity (BthTX-II
from Bothrops jararacussu venom) was crystallized and the molecular-replacement
solution has been obtained with a dimer in the asymmetric unit. The quaternary structure of
BthTX-II resembles the myotoxin Asp49-PLA
2
PrTX-III (piratoxin III from B. pirajai
venom) and all non-catalytic and myotoxic dimeric Lys49- PLA
2
s, however it is different
from the highly catalytic and non-myotoxic BthA-I (acidic PLA
2
from B. jararacussu) and
other Asp49-PLA
2
s. BthTX-II structure showed a severe distortion of calcium binding loop
leading to displacement of C-terminal region. Tyr28 side chain, present in this region
(calcium binding loop), is in an opposite position in relation to the same residue in the
catalytic activity Asp49-PLA
2
s, making hydrogen bond with the atom O2 of the catalytic
residue Asp49, which should coordinate the calcium. Additionally, BthTX-II was
crystallized in presence of calcium ions, and the resulting showed Ca
2+
-binding loop
adopts very similar conformation and any ion is present in this region. These facts lead to
the conclusion that the BthTX-II is not able to bind calcium ions, consequently, we suggest
that its low catalytic function is based in an alternative way compared with other
phospholipases A
2
.
Keywords: X-ray crystallography; Asp49-phospholipase A
2
; Bothrops jararacussu venom;
Myotoxicit; low catalytic activity.
32
1. Introduction
Phospholipases A
2
(PLA
2
s; EC 3.1.1.4) belong to a superfamily of proteins which
hydrolyze the sn-2 acyl groups of membrane phospholipids to release fatty acids,
arachidonic acid and lysophospholipids (van Deenen & de Haas, 1963). The coordination
of the Ca
2+
ion in the PLA
2
calcium-binding loop includes an Asp at position 49 which
plays a crucial role in the stabilization of the tetrahedral transition-state intermediate in
catalytically active PLA
2
s (Scott et al., 1992). In the genus Bothrops, PLA
2
s are the main
components of the venoms produced by species classified into this animal group. In
addition to their primary catalytic role, snake-venom PLA
2
s show other important
toxic/pharmacological effects, including myonecrosis, neurotoxicity, cardiotoxicity and
haemolytic, haemorrhagic, hypotensive, anticoagulant, platelet-aggregation inhibition and
oedemainducing activities (Gutiérrez & Lomonte, 1997; Ownby, 1998; Andrião-Escarso et
al., 2002). Some of these activities are correlated with the enzymatic activity, but others
are completely independent (Kini & Evans, 1989; Soares & Giglio, 2003). It has been
suggested that some specific sites of these molecules have biochemical properties that are
responsible for the pharmacological and toxic actions, including the anticoagulant and
platelet-inhibition activities (Kini & Evans, 1989). PLA
2
s are also one of the enzymes
involved in the production of eicosanoids. These molecules have physiological effects at
very low concentrations; however, increases in their concentration can lead to
inflammation (Needleman et al., 1986). Thus, the study of specific PLA
2
inhibitors is
important in the production of structure based anti-inflammatory agents. Many non-
catalytic homologous PLA
2
s (Lys49-PLA
2
s) have been purified from Bothrops snake
venoms and have been structurally and functionally characterized (Marchi-Salvador et al.,
2005, 2006; Watanabe et al., 2005; Soares et al., 2004; Magro et al., 2003; Lee et al.,
2001; Arni et al., 1995, 1999; da Silva-Giotto et al., 1998). However, little is known about
the bothropic catalytic Asp49-PLA
2
s (Magro et al., 2004, 2005; Rigden et al., 2003;
Serrano et al., 1999; Pereira et al., 1998; Daniele et al., 1995; Homsi-Brandeburgo et al.,
1988). Despite the structures of a large number of PLA
2
s having been solved by
crystallography to date, many questions still need to be clarified. For example, there are
PLA
2
s with high, moderate and no catalytic activity (Magro et al., 2004; Rigden et al.,
2003; da Silva- Giotto et al., 1998). However, for all these ‘classes’ of PLA
2
s the majority
of residues of the catalytic machinery are conserved. Similarly, toxic (e.g. myotoxicity,
cytotoxicity) and pharmacological effects (e.g. anticoagulant, hypotensive and platelet-
33
aggregation activities) are far from being completely understood. An acidic catalytic PLA
2
(BthA-I) has been isolated from B. jararacussu venom and characterized (Andrião-Escarso
et al., 2002; Roberto et al., 2004). BthA-I is three to four times more catalytically active
than BthTX-II (bothropstoxin-II from B. jararacussu) and other basic Asp49-PLA
2
s from
Bothrops venoms, but is not myotoxic, cytotoxic or lethal (Andrião-Escarso et al., 2002).
Other activities demonstrated by this enzyme are time-independent oedema induction,
hypotensive response in rats and platelet-aggregation inhibition (Andrião-Escarso et al.,
2002). The crystal structure of BthA-I has been recently described in two conformational
states: monomeric and dimeric (Magro et al., 2004). Additionally, Magro et al. (2005)
solved the structure of BthA-I chemically modified with BPB (p-bromophenacyl bromide)
and showed important tertiary and quaternary structural changes in this enzyme. This novel
oligomeric structure is more energetically and conformationally stable than the native
structure and the abolition of pharmacological activities (including anticoagulant,
hypotensive effect and platelet-aggregation inhibition) by the ligand may be related to the
oligomeric structural changes. The isolation, biochemical/pharmacological characterization
and amino-acid sequence of bothropstoxin II from B. jararacussu (BthTX-II) have been
reported (Homsi-Brandeburgo et al., 1988; Gutiérrez et al., 1991; Kashima et al., 2004).
Protein sequencing indicated that BthTX-II is an Asp49-PLA
2
and consists of 122 amino
acids (MW = 13 976 Da). The protein shows myotoxic, oedematogenic and haemolytic
effects and low phospholipase activity (Homsi- Brandeburgo et al., 1988; Gutiérrez et al.,
1991). Recently, it has been shown that BthTX-II induces platelet aggregation and
secretion through multiple signal transduction pathways (Fuly et al., 2004). BthTX-II has
been crystallized and a complete data set has been collected (Corrêa et al., 2006).
In the present paper, we describe crystal structure of basic Asp49-PLA
2
-BthTX-II
isolated from the venom of Bothrops jararacussu and a comparative analysis with other
Asp49 and Lys49 phospholipases A
2
from snake venoms. This study provides deeper
understanding of structural basis of low catalytic and high myotoxic activity of this protein
and may introduce to a totally new Ca
2+
-independent catalytic mechanism for PLA
2
s.
2. Material and methods
2.1. Purification
BthTX-II was isolated from B. jararacussu snake venom by gel filtration and ion-
exchange chromatography as previously described (Homsi-Brandeburgo et al., 1988).
34
2.2. Crystallization
A lyophilized sample of BthTX-II was dissolved in ultra pure water at a
concentration of 12 mg ml
_1
. Crystals of BthTX-II were obtained by the conventional
hanging-drop vapour-diffusion method (MacPherson, 1982) using Crystal Screens I (by
Hampton Research) with the sparse-matrix method (Jancarik & Kim, 1991). Better crystals
were obtained using a solution containing 20%(v/v) 2-propanol, 13%(w/v) polyethylene
glycol 4000 and 0.1 M sodium citrate pH 5.6 (Corrêa et al., 2006) in which 1 µl protein
solution and 1 µl reservoir solution were mixed and equilibrated against 500 µl of the same
precipitant solution. The crystals measured approximately 0.4 x 0.2 x 0.1 mm after two
months at 291 K.
2.3. X-ray data collection and processing
X-ray diffraction data from BthTX-II crystals were collected using a wavelength of
1.427 Å at a synchrotron-radiation source (Laboratório Nacional de Luz Sincrotron, LNLS,
Campinas, Brazil) with a MAR CCD imaging-plate detector (MAR Research). A crystal
was mounted in a nylon loop and flash-cooled in a stream of nitrogen at 100 K using no
cryoprotectant. The crystal-to-detector distance was 100 mm and an oscillation range of 1
o
was used; 149 images were collected. The data were processed to 2.19 Å of resolution
(Corrêa et al., 2006), using the HKL program package (Otwinowski & Minor, 1997).
2.4. Structure determination and refinement
The crystal structure of BthTX-II was solved by the Molecular Replacement
Method using the program AMoRe (Navaza, 1994) and coordinates of PrTX-III (Rigden et
al., 2003). The model choice was based on the best results of correlation and R-factor from
the AMoRe program. After a cycle of simulated annealing refinement using the CNS
program (Brunger et al., 1998), the electron densities were inspected and the amino acid
sequence as obtained from the cDNA of BthTX-II (Kashima et al., 2004) was inserted. The
modeling process was always performed by manually rebuilding with the program “O”
(Jones et al., 1990). Electron density maps calculated with coefficients 3|Fobs|-2|Fcalc| and
simulated annealing omit maps calculated with analogous coefficients were generally used.
The model was improved, as judged by the free R-factor (Brunger, 1992), through rounds
of crystallographic refinement (positional and restrained isotropic individual B-factor
refinement, with an overall anisotropic temperature factor and bulk solvent correction)
using the CNS program (Brunger et al., 1998), and manual rebuilding with the program
35
“O” (Jones et al., 1990). Solvent molecules were added and refined also with the program
CNS (Brunger et al., 1998). The refinement converged to R
free
and R
cryst
of 22.7% and
20.7% respectively. The final models comprise 1891 protein atoms, 229 water molecules
and 2 sodium ions. The refinement statistics are shown in Table 1. The quality of the
model was checked with the program Procheck (Laskowski et al., 1993). The contacts
were analyzed with the program Dimplot (Wallace et al., 1995) and the buried surface
areas were calculated using the program CNS (Brunger et al., 1998). The coordinates have
been deposited in the RCSB Protein Data Bank with ID codes 2OQD.
2.5. Comparative analyzes
For molecular comparisons of the Asp49-PLA
2
and Lys49-PLA
2
structures, the
program “O” (Jones et al., 1990) was used with only the Cα coordinates. The comparative
analysis of PLA
2
s has been performed with BthTX-II, BthTX-I and BthA-I (from B.
jararacussu), PrTX-II and PrTX-III (from B. pirajai), apart of a basic and an acid Asp49-
PLA
2
s (from A. h. Pallas). An alignment of the class IIA Asp49 and Lys49 PLA
2
s from
different species (Bothrops jararacussu, Bothrops pirajai and Agkistrodon halys pallas)
was produced using only the secondary structure residues using ClustalW program
((Higgins et al., 1994).
3. Results
3.1. Overall structure of the BthTX-II
The crystals of complex BthTX-II diffract to 2.19 Å and are monoclinic, space
group C2 with unit cell constants of a=58.9, b=98.5, c=46.7 Å and =125.9
o
. The
refinement converged to a crystallographic residual of 20.7 % (R
free
=22.7 %) for all data
between 30.0 Å and 2.19 Å (Table 1).
The structure shows excellent overall stereochemistry with no residues found in the
disallowed or generously allowed regions of the Ramachandran plot. The overall Procheck
G-factor is -0.1 (Laskowski et al., 1993).
36
Table 1. X-ray data collection and refinement statistics
Unit cell (Å)
a= 58.912; b= 98.458; c= 46.720
β
= 125.89
o
Space group C2
Resolution (Å) 30.01-2.19 (2.33-2.19)
a
Unique reflections 10,687 (1619)
a
Completeness (%) 95.9 (92.9)
a
R
merge
b
(%) 9.1 (26.4)
a
I/ (I) 10.6 (3.7)
a
Redundancy 3.0 (2.9)
a
R
cryst
c
(%) 20.7 (35.9)
a
R
free
d
(%) 22.7 (40.4)
a
Number of non-hydrogen atoms:
Protein
Water
Na
+
ion
1891
229
2
Mean B factor (Å
2
)
e
Overall
Na
+
ion
35.1
31.6
R.m.s deviations from ideal values
e
Bond lengths (Å)
Bond angles (°)
0.023
2.3
Ramachandram plot
f
(%)
Residues in most favored region
Residues in additional allowed region
88.9
11.1
Coordinate error (Å)
e
Luzzati plot (cross-validated Luzzati plot)
SIGMAA (cross-validated SIGMAA)
0.27 (0.31)
0.34 (0.35)
a
Numbers in parenthesis are for the highest resolution shell.
b
R
merge
= Σ
hkl
(Σ
i
(|I
hkl,i
-<I
hkl
>|))/Σ
hkl,i
<I
hkl
>, where I
hkl,i
is the intensity of an individual measurement
of the reflection with Miller indices h, k and l, and <I
hkl
> is the mean intensity of that reflection.
Calculated for I>-3σ (I).
c
R
cryst
=
hkl
(||Fobs
hkl
|-|Fcalc
hkl
||)/|Fobs
hkl
|, where |Fobs
hkl
| and |Fcalc
hkl
| are the observed and
calculated structure factor amplitudes.
d
R
free
is equivalent to R
cryst
but calculated with reflections (5 %) omitted from the refinement
process.
e
Calculated with the program CNS (Brünger et al., 1998).
f
Calculated with the program PROCHECK (Laskowski et al., 1993).
BthTX-II is a dimeric structure with seven disulfide bridges in each monomer and
like other class II PLA
2
s, has the following structural features: (i) an N-terminal -helix;
37
(ii) a “short” helix, (iii) a Ca
2+
binding loop; (iv) two anti-parallel -helices (2 and 3); (v)
two short strands of anti-parallel -sheet (-wing); and (vi) a C-terminal loop (Figure 1).
The catalytic network for class II PLA
2
s, formed by His48, Tyr52, Tyr73 and Asp99, is
fully conserved.
Figure 1- Dimeric structure of BthTX-II is showed as a ribbon diagram. The figure was
drawn using Pymol program (DeLano, 2002).
The monomers of BthTX-II are related by an approximate two-fold axis
perpendicular to the -wing (Figure 1). Hydrophobic contacts and two intermolecular
hydrogen bonds (Arg77 (mon.A)-Thr13 (mon. B) and Lys16 (mon. A)-Ala78 (mon. B)
contribute to the stabilization of the dimer. The majority of contacts involve residues of h1
α-helix (residues 10-14), -wing (residues 78-80) and Trp110 from C-terminal (Figure 2a).
It has been found an alternative dimeric conformation analyzing the crystal packing of
protein (Figure 2b) (see discussion section).
38
a
39
Figure 2- Contacts in the dimeric interface of BthTX-II. a) Conformation used in the
structure of BthTX-II; b) Alternative dimer. By Dimplot (Wallace et al., 1995).
The monomers are very similar, where the r.m.s. deviation of C
α
atoms is 0.73 Å
after the superposition between them. The average B-factor for both m-BthA-I and
d-BthA-I is 35.1 Å
2
. The two Na
+
ions in the final model, one bound to each subunit, are
well defined by density, with B-factor of 31.6 Å
2
, which is similar of entire structure (35.1
b
40
Å
2
). These ions are sited in the hydrophobic channel of catalytic site bound to the Phe19
residue, for both monomers.
The Figure 3 shows the BthTX-II structure according with B-factor value of each
residue. It can be observed C-terminal regions of both monomers and -wing region of
monomer B have high B-factors values and are the more flexible regions of protein.
Figure 3- Diagram of B-factor values from BthTX-II. Regions with higher B-factor values
are show in yellow and red. By Pymol program (DeLano, 2002).
The B-factors in C-terminal are 41 Å
2
and 46 Å
2
of the monomers A and B,
respectively, while their values are 31 Å
2
and 44 Å
2
in the -wing regions of monomers A
and B, respectively. In contrast, the three main helixes (h1, h2 and h3) in the core region of
protein are very stable. The Ca
2+
-binding loop of both monomers, despite their unusual
conformation compared to other PLA
2
s, have B-factors values comparable to entire
structure (32 Å
2
and 34 Å
2
, respectively for monomers A and B).
41
3.2. Comparison between PLA
2
s
Figure 4 shows the alignment of the class IIA Asp49 and Lys49 PLA
2
s from
different species: Bothrops jararacussu (Kashima et al., 2003; Magro et al., 2004, Cintra et
al., 1993), Bothrops pirajai (Rigden et al., 2003; Lee et al., 2001) and Agkistrodon halys
pallas (Zhao et al., 1997; Chen et al., 1987) produced using only the secondary structure
residues. The sequence identity related to BthTX-II varies from 80.0 % (basic Asp49-
PLA
2
-PrTX-III) to 56% (Lys49-PLA
2
-BthTX-I) and 57 % (acidic Asp49-PLA
2
-BthA-I).
Figure 4- Amino acid sequence alignments of Asp49 and Lys49-PLA
2
s: BthTX-I,
BthTX-II and BthA-I (from B. jararacussu); PrTX-II and PrTX-III (from B. pirajai);
Basic-Ag and Acidic-Ag (Basic and Acidic Asp49-PLA2 from A. h. pallas). Produced
by the ClustalW program (Higgins et al., 1994).
Table 2 catalogs r.m.s. deviations after superposition between monomer A from
BthTX-II and other monomers A of PLA
2
s: BthA-I (Murakami et al., 2006) and BthTX-I
from B. jararacussu, acidic and basic Asp49-PLA
2
form A. h. pallas (Wang et al., 1996;
Zhao et al., 1998), Lys49-PLA
2
PrTX-II (Lee et al., 2001) and low catalytic basic Asp49-
PLA
2
PrTX-III (Rigden et al., 2003). This comparison shows the effect caused mainly by
Ca
2+
binding loop distortion on the tertiary structure of BthTX-II and PrTX-III. The
BthTX-II and PrTX-III monomers are very similar, with an r.m.s. deviation of about 0.5 Å
between C
α
atoms. By contrast, the comparisons of the BthTX-II with other PLA
2
s have
r.m.s. deviations higher than 1.5 Å.
10
20 30 40 50 60 Identity
BthTX-II DLWQFGQMI-LKETGKLPFPYYTTYGCYCGWGGQGQPKDATDRCCFVHDCCYG---KLTNCK-----P 100%
PrTX-III DLWQFGKMI-LKETGKLPFPYYVTYGCYCGVGGRGGPKDATDRCCFVHDCCYG---KLTSCK-----P 80%
Basic-Ag HLLQFRKMI-KKMTGKEPVVSYAFYGCYCGSGGRGKPKDATDRCCFVHDCCYE---KVTGCD-----P 67%
PrTX-II SLFELGKMI-LQETGKNPAKSYGAYGCNCGVLGRGKPKDATDRCCYVHKCCYK---KLTGCN-----P 59%
Acidic-Ag SLIQFETLI-MKVAKKSGMFWYSNYGCYCGWGGQGRPQDATDRCCFVHDCCYG---KVTGCD-----P 58%
BthA-I SLWQFGKMI-NYVMGESGVLQYLSYGCYCGLGGQGQPTDATDRCCFVHDCCYG---KVTGCD-----P 57%
BthTX-I SLFELGKMI-LQETGKNPAKSHGAYGCNCGVLGRGKPKDATDRCCYVHKCCYK---KLTGCD-----P 56%
70 80 90 100 110 120 130
BthTX-II KTDRYSYSRENGVIICG-EGTPCEKQICECDKAAAVCFRENLRTYK-KRYMAYPDVLCKKP-
AEKC 100%
PrTX-III KTDRYSYSRKDGTIVCG-ENDPCRKEICECDKAAAVCFRENLDTYN-KKYMSYLKSLCKKX-ADDC 80%
Basic-Ag KWDDYTYSWKNGTIVCG-GDDPCKKEVCECDKAAAICFRDNLKTYK-KRYMAYPDILCSSK-SEKC 67%
PrTX-II KKDRYSYSWKDKTIVCG-ENNPCLKELCECDKAVAICLRENLGTYN-KKYRYHLKPFCKKA--DKC 59%
Acidic-Ag KMDVYSFSEENGDIVCG-GDDPCKKEICECDRAAAICFRDNLTLYNDKKYWAFGAKNCPQEESEPC 58%
BthA-I KIDSYTYSKKNGDVVCG-GDDPCKKQICECDRVATTCFRDNKDTYD-IKYWFYGAKNCQEK-SEPC 57%
BthTX-I KKDRYSYSWKDKTIVCG--ENNCLKELCECDKAVAICLRENLGTYN-KKYRYHLKPFCKKA--DAC 56%
42
Table 2. Superposition of monomer A from
BthTX-II with monomer A from other
PLA
2
s (r.m.s. deviation of Cα atoms).
Protein r.m.s.d (Å)
PrTX-III (B. pirajai) 0.490
Basic-Ag (A. h. pallas) 1.527
Acidic-Ag (A. h. pallas) 1.835
BthA-I (B. jararacussu) 1.924
PrTX-II (B. pirajai) 1.711
BthTX-I (B. jararacussu)
1.508
Figure 5a shows the superposition between the three main helices (h1, h2, and h3)
of the monomers A of the BthTX-II, BthTX-I, BthA-I, acidic and basic Asp49 from
Agkistrodon halys pallas, PrTX-II and PrTX-III. This comparison indicates that there are
two main regions with significant structural differences of the BthTX-II in comparison
with other PLA
2
s: the Ca
2+
-binding loop and the C-terminal region. Both structural
differences seem to be generate by extreme distortion of Ca
2+
-binding loop (Figure 5b)
which starts in the different configuration adopted by Tyr28 side chain and, mainly by
important diversion taken by the main chain dihedral angles of Cys29. The Gly30, Trp31
and Gly32 backbones are approximately perpendicular to the all other PLA
2
s, with
exception of PrTX-III, which adopts very similar conformation. In the present structure,
the catalytic residue Asp49, which is in a similar position that other PLA
2
s, is hydrogen
bound to the Tyr28, residue from Ca
2+
-binding loop (Figure 6a). The Trp31 side chain
adopts a conformation totally opposite to the Leu31 in BthA-I (Figure 6b) pointing to the
C-terminal region.
43
Superposition between Cα atoms of the BthTX-II monomers resulted in an r.m.s.
deviation of 0.73 Å while the same superposition between native BthA-I monomers
Figure 5- Superposition between C
of BthTX-I, BthTX-I I and BthA-
I
(B. jararacussu); PrTX-
II and
PrTX-III (B. pirajai); Basic Asp49-
PLA
2
and Acidic Asp49-PLA
2
(
A.
h. pallas
). a) monomers A; b)
Calcium binding loop. This figure
was drawn using Pymol
(DeLano,
2002).
b
a
44
resulted in a value of 0.54 Å (Magro et al., 2004). These results are similar to those
obtained for other dimeric PLA
2
s (Magro et al., 2003).
The superposition of Ca
2+
binding loop of these proteins (Figure 5b) resulted in a
large difference between r.m.s deviations of BthTX-II in comparison with other PLA
2
s
(table 3).
Table 3. Superposition between Calcium binding
loop of the monomers A from BthTX-II and
other PLA
2
s (r.m.s. deviation of Cα atoms).
Protein r.m.s.d (Å)
PrTX-III (B. pirajai) 0.208
Basic-Ag (A. h. pallas) 2.074
Acidic-Ag (A. h. pallas) 2.249
BthA-I (B. jararacussu) 2.299
PrTX-II (B. pirajai) 2.111
BthTX-I (B. jararacussu)
2.097
The quaternary structure of BthTX-II resembles the Lys49-PLA
2
s (Soares et al.,
2004) and one of two possible dimers of PrTX-III (Rigden et al., 2003 - these authors
found two possible biological dimers for PrTX-III) structures. Fig 7 shows the
superposition of the monomers A from BthTX-II, BthTX-I and PrTX-III (Cα atoms of α-
helices h1, h2 and h3 were used in the superposition) indicating similar quaternary
structure. However, as previously demonstrated (Magro et al., 2003), the monomers of
these dimeric PLA
2
s may adopt different relative aperture between them.
45
Figure 6- a) Configuration adopted by Tyr28 in BthTX-II in comparison with BthA-I. b)
Perpendicular position between residues 30 and 31 of BthTX-II and BthA-I. Magenta
sphere is an ion Ca
2+
present in the structure of BthA-I.
b
a
46
Figure 7- Superposition between dimers of BthTX-II, PrTX-III and BthTX-I.
4. Discussion
4.1. Quaternary structure
BthTX-II and PrTX-III are unique proteins among class I/II PLA
2
s studied to this
date. They have actions of both PLA
2
s classes Asp49-PLA
2
s (e.g catalytic activity) and
Lys49-PLA
2
s (myotoxic and cytotoxic actions), however its catalytic activity is three to
four times less than BthA-I. BthTX-II also is able to induce platelet aggregation in a
concentration-dependent manner (Fuly et al., 2004), in contrast with BthA-I which caused
a hypotensive response in rats and inhibited platelet aggregation (Andrião-Escarso et al.,
2002).
The oligomeric state is an important and enigmatic issue for many of phospholipase
A
2
structures solved to this date date (Magro et al., 2003; Pan et al., 2001; de Oliveira et
al., 2001; Dekker, 2000; Snijder et al, 1999; Sanchez et al., 2001). It has been shown the
majority of Lys49-PLA
2
s are dimeric is solution using eletrophoretic and spectroscopic
techniques (da Silva-Giotto et al., 1998; Arni et al., 1999 and Soares et al., - BnSP-7-ABB
47
2000). However, GodMT-II crystal structure is a monomer (Arni et al., 1999) while MjTX-
I has a tetrameric conformation (Marchi-Salvador et al., 2005). For GodMT-II, it is likely
its monomeric conformation is due to physicochemical conditions used in the
crystallization experiments (low pH value). It has been shown oligomeric conformation is
essential for myotoxic, cytotoxic and Ca
2+
-independent membrane damaging effects
(Angulo et al., 2005; de Oliveira et al., 2001 and Ruller et al., 2005). These authors also
showed that this oligomeric conformation is pH dependent, which confirms the hypothesis
GodMT-II is monomeric just because it was crystallized at low pH. Recent site-directed
mutagenesis associated with spectroscopic experiments also demonstrated the importance
of dimeric conformation of Lys49-PLA
2
s in its biological function (Ruller et al, 2005).
For the acidic PLA
2
s from B. jararacussu (BthA-I), in contrast with the Lys49-
PLA
2
s, there is no consensus regarding its oligomeric conformation. BthA-I native
structure was solved in monomeric and dimeric conformations (Magro et al., 2004).
Furthermore, it has been solved BthA-I complexed with p-bromophenacyl bromide
inhibitor in an alternative dimeric and more stable conformation compared to native BthA-
I (Magro et al., 2005a). BthA-I was also crystallized in presence of α-tocopherol inhibitor
showing monomeric and dimeric conformations (Magro et al., 2005b).
Figure 7 shows that the BthTX-II structure resembles those of Lys49-PLA
2
s,
however an alternative crystal lattice can be observed for BthTX-II. In this assembly, the
dimeric conformation is kept only by one hydrogen bond and a few hydrophobic
interactions (Fig. 2) from C-terminal and short helix regions. Similar conformations were
found for PrTX-III structure (Rigden et al., 2003).
We suggest dimeric Lys49-PLA
2
assembly is the conformation for the BthTX-II
based on three main reasons. (i) This conformation is stabilized by larger number
hydrophobic contacts (seven interactions) and two intermolecular hydrogen bonds. In
contrast, the alternative conformation is stabilized by six hydrophobic contacts and one
intermolecular hydrogen bond (Fig. 2a, b). (ii) The higher B factors values are found in the
C-terminus which is in the outer part of structure (Fig. 3), while for the alternative
assembly, this region is in the interface of the monomers. In contrast, the -wing B factors,
which are 31 and 44 in subunits A and B, respectively, are close to the overall B factor of
35.1 Å
2
and should stay in the inner part of structure. It has been show for the monomeric
Lys49-PLA
2
(Arni et al., 1999) high values of B factors compared to the structure. (iii) The
Lys49-PLA
2
conformation is found in more than ten crystallographic structures which
48
have at least five different space groups suggesting no influence of crystal packing of
molecules.
Recently, it has been solved the crystal structure of complex Basp-II bound to
suramin (Murakami et al., 2005). This structure revealed that one molecule suramin binds
both monomers of protein; this configuration is only permitted for an alternative dimer of
Lys49-PLA
2
s where the hydrophobic surfaces surrounding the entrance to the active sites
form the dimer interface. This “alternative dimer” found in this structure is not the same
alternative assembly found for BthTX-II structure whose interfacial area is much smaller
than those of Basp-II/suramin complex.
4.2. The distorted Ca
2+
binding loop – possible relation with low catalytic activity
BthTX-II and PrTX-III structures revealed Ca
2+
-binding loops extremely distorted.
The binding of Ca
2+
, required for phospholipase activity in Asp49-PLA
2
s (Scott & Sigler,
1994), involves the main-chain carbonyl groups of residues 28, 30 and 32. In the BthA-I
and the Acidic Asp49-PLA
2
from A. h. pallas, both monomerics, crystallized in the
presence of Ca
2+
, the distance between this ion and the Gly30 O were 4.92 Å and 2.24 Å,
respectively, while the same measurements in PrTX-III was 8.6 Å in subunit A. In BthTX-
II, this distance was 8.8 Å
2
. These values show clearly, a distortion of the Ca
2+
-binding
loop in the present structure beyond that expected from the simple absence of Ca
2+
ion.
Asp49-PLA
2
s have been solved in the native form and in the presence of Na
+
or
Ca
2+
ions. The crystal structure of native BthA-I was recently described in two
conformational states: monomeric (m-BthA-I – in the presence of Na
+
ions) and dimeric
(d-BthA-I) (Magro et al., 2004). Subsequently, high resolution structures of m-BthA-I
have been described, both in the apo form and in the presence of Ca
2+
ions (Murakami et
al., 2006). Interesting, for all these BthA-I structures (five monomers in the total), the
Ca
2+
-binding loops have approximately the same conformation. The Na
+
and Ca
2+
ions or a
water molecule, for native protein, bond in the same position in the Ca
2+
-binding loop for
all structures. Consequently, we can conclude that Ca
2+
-binding loop does not suffer
substantial conformational alterations for the Asp49-PLA
2
s solved in the presence of Ca
2+
when compared to native ones.
BthTX-II was crystallized in the presence of Na
+
ions. These ions are bound to
Phe19 in both monomers, in contrast with native monomeric BthA-I structure whose ions
are bound to the Ca
2+
binding loop. Similarly, other Asp49-PLA
2
s which are able to bind
49
Ca
2+
ions are also able to bind Na
+
ions at approximately the same position in the Ca
2+
-
binding loop (Singh et al., 2001).
Magro et al. (2005) solved the structure of BthA-I chemically modified with BPB
(p-bromophenacyl bromide) and showed important tertiary and quaternary structural
changes in this enzyme. The comparison between the BPB inhibited and native BthA-I
leads to the observation of three main regions with significant structural differences: the
Ca
2+
-binding loop, the C-terminal, and the -wing. The presence of the inhibitor in the
active site displaces the Ca
2+
binding loop which interacts with C-terminal region, also
displacing it. Residues Gly30, Leu31, and Leu32 are displaced by BPB group, yielding a
different conformation in the Ca
2+
binding loop (Magro et al., 2005). Additionally, it has
been suggested that divalent cations like Ca
2+
protect PLA
2
against inactivation by BPB
and that Ca
2+
does not bind to the inactivated PLA
2
(Volwerk et al., 1974). Similarly,
acidic PLA
2
from A. h. Pallas in the presence of Ca
2+
prevents BPB modification (Zhang et
al., 1994). This fact is probable due to the absence of a water molecule that coordinates the
Ca
2+
ion generated by distorted geometry of Ca
2+
binding loop (Renetseder et al., 1988;
Zhao et al., 1998). These examples of PLA
2
/ligands complexes shows that distorted Ca
2+
binding loops cannot bind bivalent ions.
Then, these two examples above denotes that PLA
2
s, which binds Ca
2+
ions, do not
show significant changes in the Ca
2+
-binding loops, in contrast, PLA
2
s with distorted Ca
2+
-
binding loops cannot bind Ca
2+
ions, consequently we hypothesize BthTX-II cannot bind
Ca
2+
and its low catalytic function is based in a alternative way compared with other
Asp49-PLA
2
s.
In order to test this hypothesis, we crystallized BthTX-II in the same conditions,
however we add CaCl
2.
An X-ray data collection has been performed and processed at 2,35
Å resolution. The
initial refinement of the structure did not show the presence of this ion in
the calcium binding loop, The position of residues of this region are in the same position as
in the native protein.
In conclusion, we showed BthTX-II is not able to bind Ca
2+
, consequently, we
suggest that its low catalytic function is based in an alternative way compared with other
phospholipases. Further studies are in progress in order to establish the details of catalytic
and myotoxic activities of this class of protein. These studies include co-crystallization
with inhibitors, functional studies and site-directed mutagenesis.
50
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3. CONCLUSÕES
57
O entendimento dos sistemas biológicos ao nível molecular tem apresentado
avanços importantes, em grande parte, devido aos conhecimentos gerais e detalhados de
estruturas tridimensionais obtidos com o avanço da área cristalográfica, a ponto de.
Companhias químicas e farmacêuticas se tornarem mais ativas nesta área estrutural devido
a interesses no desenvolvimento de novos produtos industriais e no desenho racional de
novas drogas (Drenth, 1994, Kubinyi, 1998 e Souza et al., 2000).
Nos estudos de cristalização de BthTX-II, foi necessário um mês para serem
encontrados os primeiros cristais, os quais foram levados para a coleta de dados de
difração de raios-X, no entanto, só após mais três meses, foram obtidos monocristais com
qualidade suficiente para a coleta dos dados. Nesse período, foram realizados inúmeros
testes com variações das concentrações dos componentes do sistema, bem como na
temperatura de incubação e nos métodos utilizados (sitting-drop e hanging-drop). Após a
coleta do conjunto de dados de difração de raios-X, outros problemas foram encontrados.
Com a resolução da estrutura cristalina, passou-se ao refinamento da estrutura
tridimensional e para isso, foi utilizada a seqüência de aminoácidos de BthTX-II com 120
resíduos de aminoácidos, proposta por Pereira et al. (1998). Após vários ciclos de
refinamento sem muito êxito, passou-se a utilizar o sequenciamento realizado por Kashima
et al. (2004), que compreende 122 resíduos, uma vez que não se tinha conhecimento deste,
até então. Uma varredura foi realizada e constatou-se que espaços no mapa de densidade
eletrônica, até então não preenchidos pela primeira seqüência, foram ocupados
satisfatoriamente pela segunda, uma vez que esta favorecia a existência de mais dois
resíduos, além do fato de que cadeias laterais mal preenchidas pelos resíduos até então
utilizados, passaram a ser bem ocupadas após a modificação pelos propostos na segunda
seqüência. Após essa modificação, foi possível dar seguimento ao refinamento até a
conclusão da estrutura tridimensional de BthTX-II.
A determinação da estrutura tridimensional de uma proteína é apenas um passo na
elucidação dos mecanismos envolvidos nas relações estrutura/função de uma proteína.
BthTX-II é uma Asp49-PLA
2
que no entanto, apresenta baixa atividade catalítica, diferente
do observado em BthA-I, extraída do veneno da mesma espécie. Assim, um estudo
comparativo com outras fosfolipases A
2,
tanto Asp49 como Lys49, foi fundamental para
aumentar a compreensão das bases estruturais dessa variação na atividade catalítica,
levando-nos a propor que a posição significativamente deslocada da região do cálcio
binding loop em BthTX-II impede a coordenação do íon cálcio, sendo responsável pela
baixa atividade catalítica dessa enzima. Uma nova fase de estudos, já em andamento, que
58
tem por objetivo, confirmar essa proposta, envolve a cristalização de BthTX-II na presença
de íons cálcio, bem como a resolução de sua estrutura cristalina. Estudos preliminares com
um conjunto de dados de difração de raios-X de cristais obtidos nessa condição dão fortes
indícios da inexistência do íon Ca
2+
na região do cálcio binding loop, no entanto, é
necessária a obtenção de um conjunto de dados com melhor estatística, a fim de se concluir
essa análise. Esse estudo deverá ser concluído nos próximos meses, como parte do projeto
de doutorado. A partir destes dados, possivelmente poderemos propor um mecanismo
catalítico independente de cálcio para fosfolipases A
2
, porposta que seria inédita e
impensável até o momento. Para este estudo, serão necessários possíveis estudos de
complexos desta proteína com inibidores, bem como outros estudos bioquímicos,
biofísicos e de mutação sítio dirigida.
59
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APÊNDICE
crystallization communications
Acta Cryst. (2006). F62, 765–767 doi:10.1107/S1744309106025164 765
Acta Crystallographica Section F
Structural Biology
and Crystallization
Communications
ISSN 1744-3091
Preliminary X-ray crystallographic studies of
BthTX-II, a myotoxic Asp49-phospholipase A
2
with
low catalytic activity from Bothrops jararacussu
venom
L. C. Corre
ˆ
a,
a
D. P. Marchi-
Salvador,
a
A. C. O. Cintra,
b
A. M. Soares
b
and
M. R. M. Fontes
a
*
a
Departamento de Fı
´
sica e Biofı
´
sica, Instituto de
Biocie
ˆ
ncias, UNESP, CP 510, CEP 18618-000,
Botucatu-SP, Brazil, and
b
Departamento de
Ana
´
lises Clı
´
nicas, Toxicolo
´
gicas e
Bromatolo
´
gicas, FCFRP, USP, Ribeira
˜
o Preto/SP,
Brazil
Received 17 May 2006
Accepted 29 June 2006
For the first time, a complete X-ray diffra ction data set has been collected from a
myotoxic Asp49-ph ospholipase A
2
(Asp49-PLA
2
) with low catalytic activity
(BthTX-II from Bothrops jararacussu venom) and a molecular-replacement
solution has been obtained with a dimer in the asymmetric unit. The quaternary
structure of BthTX-II resembles the myotoxin Asp49-PLA
2
PrTX-III (piratoxin
III from B. pirajai venom) and all non-catalytic and myotoxic dimeric Lys49-
PLA
2
s. In contrast, the oligomeric structure of BthTX-II is different from the
highly catalytic and non-myotoxic BthA-I (acidic PLA
2
from B. jararacussu).
Thus, comparison between these structures should add insight into the catalytic
and myotoxic activities of bothropic PLA
2
s.
1. Introduction
Phospholipases A
2
(PLA
2
s; EC 3.1.1.4) belong to a superfamily of
proteins which hydrolyze the sn-2 acyl groups of membrane phos-
pholipids to release fatty acids, arachidonic acid and lysophos-
pholipids (van Deenen & de Haas, 1963). The coordination of the
Ca
2+
ion in the PLA
2
calcium-binding loop includes an Asp at posi-
tion 49 which plays a crucial role in the stabilization of the tetrahedral
transition-state intermediate in catalytically active PLA
2
s (Scott et al.,
1992). In the genus Bothrops, PLA
2
s are the main components of the
venoms produced by species classified into this animal group. In
addition to their primary catalytic role, snake-venom PLA
2
s show
other important toxic/pharmacological effects, including myonecrosis,
neurotoxicity, cardiotoxicity and haemolytic, haemorrhagic, hypo-
tensive, anticoagulant, platelet-aggregation inhibition and oedema-
inducing activities (Gutie
´
rrez & Lomonte, 1997; Ownby, 1998;
Andria
˜
o-Escarso et al., 2002). Some of these activities are correlated
with the enzymatic activity, but others are completely independent
(Kini & Evans, 1989; Soares & Giglio, 2004). It has been suggested
that some specific sites of these molecules have biochemical prop-
erties that are responsible for the pharmacological and toxic actions,
including the anticoagulant and platelet-inhibition activities (Kini &
Evans, 1989). PLA
2
s are also one of the enzymes involved in the
production of eicosanoids. These molecules have physiological effects
at very low concentrations; however, increases in their concentration
can lead to inflammation (Needleman et al., 1986). Thus, the study of
specific PLA
2
inhibitors is important in the production of structure-
based anti-inflammatory agents.
Many non-catalytic homologous PLA
2
s (Lys49-PLA
2
s) have been
purified from Bothrops snake venoms and have been structurally and
functionally characterized (Marchi-Salvador et al., 2005, 2006;
Watanabe et al., 2005; Soares et al., 2004; Magro et al., 2003; Lee et al.,
2001; Arni et al., 1995, 1999; da Silva-Giotto et al., 1998). However,
little is known about the bothropic catalytic PLA
2
s (Asp49-PLA
2
s;
Magro et al., 2004, 2005; Rigden et al., 2003; Serrano et al., 1999;
Pereira et al., 1998; Daniele et al., 1995; Homsi-Brandeburgo et al.,
1988).
Despite the structures of a large number of PLA
2
s having been
solved by crystallography to date, many questions still need to be
clarified. For example, there are PLA
2
s with high, moderate and no
catalytic activity (Magro et al., 2004; Rigden et al., 2003; da Silva-
Giotto et al., 1998). However, for all these ‘classes’ of PLA
2
s the
# 2006 Internat ional Union of Crystallography
All rights reserved
majority of residues of the catalytic machinery are conserved. Simi-
larly, toxic (e.g. myotoxity, cytotoxity) and pharmacological effects
(e.g. anticoagulant, hypotensive and platelet-aggregation activities)
are far from being completely understood.
An acidic catalytic PLA
2
(BthA-I) has been isolated from
B. jararacussu venom and characterized (Andria
˜
o-Escarso et al.,
2002; Roberto et al., 2004). BthA-I is three to four times more cata-
lytically active than BthTX-II (bothropstoxin-II from B. jararacussu)
and other basic Asp49-PLA
2
s from Bothrops venoms, but is not
myotoxic, cytotoxic or lethal (Andria
˜
o-Escarso et al., 2002). Other
activities demonstrated by this enzyme are time-independent oedema
induction, hypotensive response in rats and platelet-aggregation
inhibition (Andria
˜
o-Escarso et al., 2002). The crystal structure of
BthA-I has been recently described in two conformational states:
monomeric and dimeric (Magro et al., 2004). Additionally, Magro et
al. (2005) solved the structure of BthA-I chemically modified with
BPB (p-bromophenacyl bromide) and showed important tertiary and
quaternary structural changes in this enzyme. This novel oligomeric
structure is more energetically and conformationally stable than the
native structure and the abolition of pharmacological activities
(including anticoagulant, hypotensive effect and platelet-aggregation
inhibition) by the ligand may be related to the oligomeric structural
changes.
The isolation, biochemical/pharmacological characterization and
amino-acid sequence of bothropstoxin II from B. jararacussu
(BthTX-II) have been reported (Homsi-Brandeburgo et al., 1988;
Gutie
´
rrez et al., 1991; Pereira et al., 1998). Protein sequencing indi-
cated that BthTX-II is an Asp49-PLA
2
and consists of 120 amino
acids (MW = 13 976 Da). The protein shows myotoxic, oedemato-
genic and haemolytic effects and low phospholipase activity (Homsi-
Brandeburgo et al., 1988; Gutie
´
rrez et al., 1991). Recently, it has been
shown that BthTX-II induces platelet aggregation and secretion
through multiple signal transduction pathways (Fuly et al., 2004).
Despite BthTX-II having been crystallized more than ten years ago
(Bortoleto et al., 1996), the structure has not been solved to date,
probably owing to the low completeness of the data set (50–60%
completeness). The crystals belonged to the tetragonal crystal system
and preliminary analysis indicated the presence of three molecules in
the asymmetric unit (Bortoleto et al., 1996). However, a careful
analysis of the Matthews coefficient indicated that a tetrameric
conformation is also possible (V
M
= 2.4 A
˚
3
Da
À1
), which also occurs
in the Lys49-PLA
2
M
j
TX-I (myotoxin I from B. moojeni venom)
structure formed of two Lys49-PLA
2
dimers (Marchi-Salvador et al.,
2005; personal communication).
In the present paper, we describe the crystallization of BthTX-II
(bothropstoxin-II) from B. jararacussu venom in the monoclinic
system, the collection of a complete X-ray diffraction data set and
molecular-replacement solution. This study should improve the
understanding of the relation of the myotoxic and low catalytic
activity mechanisms to the structural features of this protein when
compared with BthTX-I (Lys49-PLA
2
from B. jararacussu venom)
and BthA-I, which possess no and high catalytic activity, respectively.
2. Experimental procedures
2.1. Purification
BthTX-II was isolated from B. jararacussu snake venom by gel-
filtration and ion-exchange chromatography as previously described
(Homsi-Brandeburgo et al., 1988).
2.2. Crystallization
A lyophilized sample of BthTX-II was dissolved in ultrapure water
at a concentration of 12 mg ml
À1
. The sparse-matrix method
(Jancarik & Kim, 1991) was used to perform initial screening of the
crystallization conditions (Crystal Screens I and II; Hampton
Research). Large crystals of BthTX-II were obtained by the
conventional hanging-drop vapour-diffusion method (MacPherson,
1982), in which 1 ml protein solution and 1 ml reservoir solution were
mixed and equilibrated against 500 ml of the same precipitant solu-
tion. The BthTX-II was crystallized using a solution containing
20%(v/v) 2-propanol, 13%(w/v) polyethylene glycol 4000 and 0.1 M
sodium citrate pH 5.6. The best crystals measured approximately
0.4 Â 0.2 Â 0.1 mm after two months at 291 K (Fig. 1).
2.3. X-ray data collection and processing
X-ray diffraction data from BthTX-II crystals were collected using
a wavelength of 1.427 A
˚
at a synchrotron-radiation source (Labor-
ato
´
rio Nacional de Luz Sı
´
ncrotron, LNLS, Campinas, Brazil) with a
MAR CCD imaging-plate detector (MAR Research). A crystal was
mounted in a nylon loop and flash-cooled in a stream of nitrogen at
100 K using no cryoprotectant. The crystal-to-detector distance was
100 mm and an oscillation range of 1
was used; 149 images were
collected. The data were processed to 2.13 A
˚
resolution using the
HKL program package (Otwinowski & Minor, 1997).
3. Results and discussion
The data-collection statistics are shown in Table 1. The data set is
96.1% complete at 2.13 A
˚
resolution, with R
merge
= 9.1%. The crystals
belong to space group C2, with unit-cell parameters a = 58.9, b = 98.5,
c = 46.7 A
˚
, = 125.9
.
Packing parameter calculations based on the protein molecular
weight indicate the presence of a dimer in the asymmetric unit. This
corresponds to a Matthews coefficient (Matthews, 1968) of
2.0 A
˚
3
Da
À1
with a calculated solvent content of 37.4%, which are
within the expected range for typical protein crystals (assuming a
value of 0.74 cm
3
g
À1
for the protein partial specific volume).
The crystal structure was determined by molecular-replacement
techniques implemented in the program AMoRe (Navaza, 1994)
using the coordinates of a monomer of PrTX-III (PDB code 1gmz).
The quaternary structure of BthTX-II is very similar to those of
PrTX-III and all dimeric bothropic Lys49-PLA
2
s (Rigden et al., 2003;
Soares et al., 2004) and totally different from those of native dimeric
crystallization communications
766 Corre
ˆ
a et al.
BthTX-II Acta Cryst. (2006). F62, 765–767
Figure 1
Crystals of BthTX-II from B. jararacussu venom
BthA-I and BthA-I–bromophenacyl bromide and BthA-I–-toco-
pherol complexes (Magro et al., 2004, 2005; Takeda et al., 2004).
In conclusion, a complete X-ray diffraction data set has been
collected from a low catalytic activity Asp49-PLA
2
for the first time
(to 2.13 A
˚
) and a molecular-replacement structure solution has been
obtained. The quaternary structure of BthTX-II resembles those of
the myotoxin PrTX-III (which does not bind Ca
2+
ions) and all non-
catalytic and myotoxic dimeric Lys49-PLA
2
s (Rigden et al., 2003;
Soares et al., 2004). In contrast, the oligomeric structure of BthTX-II
is different from that of the high catalytic activity and non-myotoxic
BthA-I (Magro et al., 2004). Thus, comparison between these struc-
tures should add insight into the catalytic and myotoxic activities of
bothropic PLA
2
s.
The authors gratefully acknowledge financial support from
Fundac¸a
˜
o de Amparo a
`
Pesquisa do Estado de Sa
˜
o Paulo (FAPESP),
Conselho Nacional de Desenvolvimento Cientı
´
fico e Tecnolo
´
gico
(CNPq), Fundac¸a
˜
o para o Desenvolvimento da UNESP (FUNDU-
NESP) and Laborato
´
rio Nacional de Luz Sı
´
ncrontron (LNLS,
Campinas-SP).
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BthTX-II 767
Table 1
X-ray diffraction data-collection and processing statistics.
Values in parentheses are for the highest resolution shell.
Unit-cell parameters (A
˚
,
) a = 58.9, b = 98.5,
c = 46.7, = 125.9
Space group C2
Resolution (A
˚
) 40–2.13 (2.21–2.13)
Unique reflections 11560 (1127)
R
merge
† (%) 9.1 (26.4)
Completeness (%) 96.1 (94.2)
Radiation source Synchrotron (LNLS-MX1)
Data-collection temperature (K) 100
(I) cutoff for data processing‡ À3
Average I/(I) 10.6 (3.7)
Redundancy 3.0 (2.9)
Matthews coeffici ent V
M
(A
˚
3
Da
À1
)2.0
Molecules in the ASU 2
Solvent content (%) 37.4
† R
merge
=
P
hkl
½
P
i
ðjI
hkl;i
ÀhI
hkl
ijÞ=
P
hkl;i
hI
hkl
i, where I
hkl,i
is the intensity of an
individual measurement of the reflection with Miller indices hkl and hI
hkl
i is the mean
intensity of that reflection. Calculated for I > À3(I). ‡ Data processing used the HKL
suite (Otwinowski & Minor, 1997).
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