Amino acid sequence and crystal structure of BaP1,
a metalloproteinase from Bothrops asper snake venom
that exerts multiple tissue-damaging activities
LEANDRA WATANABE,1JOHN D. SHANNON,2RICHARD H. VALENTE,2,3
ALEXANDRA RUCAVADO,4ALBERTO ALAPE-GIRO´N,4,5AURA S. KAMIGUTI,6
R. DAVID G. THEAKSTON,7JAY W. FOX,2JOSE´MARI´A GUTIE´RREZ,4AND
RAGHUVIR K. ARNI1
1Department of Physics, IBILCE/UNESP, CP 136, Sao José de Rio Preto, CEP 15054-000, Brazil
2Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville, Virginia, USA
3Departamento de Fisiologia e Farmacodinâmica, Fiocruz, Rio de Janeiro, Brazil
4Instituto Clodomiro Picado, Facultad de Microbiología and5Departamento de Bioquímica, Escuela de Medicina,
Universidad de Costa Rica, San José, Costa Rica
6Department of Haematology, University of Liverpool, Royal Liverpool University Hospital, Liverpool, UK
7Venom Research Unit, Liverpool School of Tropical Medicine, Liverpool, UK
(RECEIVED March 26, 2003; FINAL REVISION July 8, 2003; ACCEPTED July 8, 2003)
BaP1 is a 22.7-kD P-I-type zinc-dependent metalloproteinase isolated from the venom of the snake Bothrops
asper, a medically relevant species in Central America. This enzyme exerts multiple tissue-damaging
activities, including hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. BaP1 is a single chain
of 202 amino acids that shows highest sequence identity with metalloproteinases isolated from the venoms
of snakes of the subfamily Crotalinae. It has six Cys residues involved in three disulfide bridges (Cys 117–Cys
197, Cys 159–Cys 181, Cys 157–Cys 164). It has the consensus sequence H142E143XXH146XXGXXH152,
as well as the sequence C164I165M166, which characterize the “metzincin” superfamily of metalloproteinases.
The active-site cleft separates a major subdomain (residues 1–152), comprising four ?-helices and a
five-stranded ?-sheet, from the minor subdomain, which is formed by a single ?-helix and several loops.
The catalytic zinc ion is coordinated by the N?2nitrogen atoms of His 142, His 146, and His 152, in addition
to a solvent water molecule, which in turn is bound to Glu 143. Several conserved residues contribute to the
formation of the hydrophobic pocket, and Met 166 serves as a hydrophobic base for the active-site groups.
Sequence and structural comparisons of hemorrhagic and nonhemorrhagic P-I metalloproteinases from
snake venoms revealed differences in several regions. In particular, the loop comprising residues 153 to 176
has marked structural differences between metalloproteinases with very different hemorrhagic activities.
Because this region lies in close proximity to the active-site microenvironment, it may influence the
interaction of these enzymes with physiologically relevant substrates in the extracellular matrix.
Keywords: Snake venom; zinc-dependent metalloproteinases; metzincins; hemorrhagic toxins; amino acid
sequence; crystal structure
Snake venoms are a rich source of zinc-dependent metallo-
proteinases (Bjarnason and Fox 1994; Hati et al. 1999).
Apart from playing an important role in the digestion of
prey tissues, these enzymes also participate in the patho-
physiology of envenoming by inducing local and systemic
bleeding (Ownby 1990; Kamiguti et al. 1996a), as well as
other tissue-damaging activities and hemostatic alterations
(Kamiguti et al. 1996a; Gutiérrez and Rucavado 2000).
Snake venom metalloproteinases (SVMPs) and the ADAMs
form the “reprolysin” group of zinc-dependent metallopro-
teinases, which, together with astacins, matrix metallopro-
Reprint requests to: José María Gutiérrez, Instituto Clodomiro Picado,
Facultad de Microbiología, Universidad de Costa Rica, San José, Costa
Rica; e-mail: email@example.com; fax: 506-2920485; or Raghuvir K.
Arni, Department of Physics, IBILCE/UNESP, CP 136, Sao José de Rio
Preto, CEP 15054–000, Brazil; e-mail: firstname.lastname@example.org; fax: 55-
Article and publication are at http://www.proteinscience.org/cgi/doi/
Protein Science (2003), 12:2273–2281. Published by Cold Spring Harbor Laboratory Press. Copyright © 2003 The Protein Society
teinases, and serralysins, comprise the “metzinczin” super-
family (Bode et al. 1993; Stöcker et al. 1995). All these
enzymes contain the sequence HEXXHXXGXXH in their
zinc-binding region, as well as a Met-turn that serves as a
base for the three active histidine residues (Bode et al. 1993;
Stöcker et al. 1995). Depending on the domain composition,
SVMPs can be classified as belonging to one of four
classes: (1) P-I, which includes enzymes possessing only the
metalloproteinase domain; (2) P-II, enzymes that possess
both the metalloproteinase and a disintegrin-like domain;
(3) P-III, enzymes that possess these two domains in addi-
tion to a high-cysteine domain; and (4) P-IV, enzymes that
comprise all the aforementioned domains and are linked by
a disulfide bridge to a type-C lectin subunit (Hite et al.
1994; Bjarnason and Fox 1994).
The ability of SVMPs to induce hemorrhage has been
associated with their capacity to hydrolyze extracellular ma-
trix proteins present in the basement membrane, a structural
scaffold that surrounds endothelial cells in the microvascu-
lature (Ohsaka 1979; Bjarnason and Fox 1994; Hati et al.
1999). P-III SVMPs induce a greater hemorrhagic effect
than those belonging to the P-I class (Bjarnason and Fox
1994). It has been proposed that the disintegrin-like and
high-Cys domains contribute to this enhanced activity by
influencing the substrate specificity (Jeon and Kim 1999) by
targeting the enzymes to physiologically relevant sites in
capillaries (Jia et al. 1997; Gutiérrez and Rucavado 2000),
and additionally by inhibiting platelet aggregation (Kami-
guti et al. 1996b; Jia et al. 1997; Moura-da-Silva et al.
Large variations in hemorrhagic potency are observed
even within P-I SVMPs; some are quite active, whereas
others have no hemorrhagic activity (Bjarnason and Fox
1994; Tsai et al. 2000). Comparative sequence studies be-
tween hemorrhagic and nonhemorrhagic P-I venom metal-
loproteinases have led to various hypotheses concerning the
structural determinants involved in the expression of hem-
orrhagic activity (Takeya et al. 1989, 1990; Gasmi et al.
2000; Tsai et al. 2000), although some of these hypotheses
need to be revised in the light of new sequence and struc-
tural information (Gomis-Ruth et al. 1994). Recent propos-
als indicate that variations in a stretch of residues located at
the C terminus (Tsai et al. 2000) or in three regions lying on
the periphery of the active site (Bolger et al. 2001) are
associated with differences in hemorrhagic activity. Only
the crystal structures of six P-I SVMPs have been described:
adamalysin II from Crotalus adamanteus venom (Gomis-
Ruth et al. 1993, 1994), atrolysin C from Crotalus atrox
venom (Zhang et al. 1994), H2-proteinase from Trimeresu-
rus flavoviridis venom (Kumasaka et al. 1996), acutolysins
A and C from Agkistrodon acutus venom (Gong et al. 1998;
Zhu et al. 1999), and TM-3 from Trimeresurus mucrosqua-
matus venom (Huang et al. 2002). Because these enzymes
vary greatly in their ability to induce hemorrhage, the com-
parative structural analysis of these and new enzymes will
undoubtedly contribute to improve our understanding of the
structural determinants of hemorrhagic activity.
BaP1 is a P-I basic SVMP isolated from the venom of the
crotaline snake Bothrops asper (Gutiérrez et al. 1995;
Gutiérrez and Ovadia 1998). This species is responsible for
the vast majority of snakebites in Central America and
southern Mexico (Gutiérrez 1995). Envenoming by B. asper
is characterized, among other clinical features, by severe
local tissue damage, often associated with permanent dis-
ability and sequelae (Gutiérrez 1995). Local pathology in-
duced by this venom involves muscle necrosis, hemorrhage,
edema, and blistering, a complex series of events mediated
by venom phospholipases A2 and metalloproteinases
(Gutiérrez and Lomonte 1995; Gutiérrez and Rucavado
2000). Among various SVMPs isolated from B. asper
venom, BaP1 exerts a wide variety of local pathological
alterations, including hemorrhage (Gutiérrez et al. 1995;
Rucavado et al. 1995), myonecrosis (Rucavado et al. 1995),
dermonecrosis and blistering (Rucavado et al. 1998), and
edema (Gutiérrez et al. 1995). In addition, it induces a
prominent inflammatory response characterized by comple-
ment activation (Farsky et al. 2000) and synthesis of cyto-
kines and matrix metalloproteinases (Rucavado et al. 2002).
Because of this wide toxicological profile, BaP1 is a rel-
evant tissue-damaging component in B. asper venom that
warrants better characterization. The present study therefore
describes its amino acid sequence and crystal structure and
compares them with those of related venom enzymes. This
is the first crystal structure described of a SVMP isolated
from the genus Bothrops; these snakes cause the highest
impact in public health in Latin America (Fan and Cardoso
1995; Gutiérrez 1995). Our observations highlight a mo-
lecular region in P-I SVMPs that could be correlated with
the variation in hemorrhagic activity within this group of
Results and Discussion
Amino acid sequence
BaP1 is a single-chain protein consisting of 202 amino acids
and a molecular weight of 22,735, as determined by
MALDI-TOF-MS. As described for several SVMPs, the
N-terminal residue is blocked, and the determination of the
N-terminal sequence was possible only after treatment with
pyroglutamyl aminopeptidase, with the consequent release
of the pyroGlu residue. Such a residue is conventionally
assigned as residue −1 (Gomis-Ruth et al. 1994). Following
this treatment, the first 26 amino acids were sequenced by
direct Edman degradation. Overlapping peptides generated
by digestion with CNBr, lysyl peptidase, clostripain, endo-
proteinase Asp-N, and asparaginyl peptidase were also se-
quenced. Figure 1 presents the complete sequence of BaP1.
Watanabe et al.
Protein Science, vol. 12
The identity of Asn at position 151 was tentatively assigned
based on Edman degradation of a peptide generated by as-
paraginyl peptidase digestion, and confirmed by examina-
tion of the electron density maps. The protein sequence
data were deposited in the SWISS-PROT and TrEMBL
knowledge base under the accession number P83512. Ma-
ture BaP1 comprises only the metalloproteinase domain,
confirming its classification within the P-I class of SVMPs
(Hite et al. 1994). Owing to its basic pI (≈8.5; Gutiérrez
and Ovadia 1998) and its weak hemorrhagic activity
(Gutiérrez et al. 1995), BaP1 can be placed within the sub-
class P-IB (weakly hemorrhagic enzymes) proposed by
Bjarnason and Fox (1995). It possesses the characteristic
and the sequence C164I165M166, associated with the Met-
turn. These structural determinants are characteristic of the
“metzincin” superfamily of zinc-dependent metalloprotein-
ases (Bode et al. 1993), and are highly conserved in a large
number of SVMPs (Fig. 2). BaP1 has six Cys residues at
positions 117, 157, 159, 164, 181, and 197, thus belonging
to the group of SVMPs with three disulfide bonds (see
structural details below).
The sequence identity of BaP1 with various SVMPs of
snakes of the family Viperidae, including both hemorrhagic
and non-hemorrhagic enzymes, ranged from 70% to 48%
(Fig. 2). BaP1 displays the highest sequence identity with
enzymes placed within the clusters of “basic hemorrhagins”
and “non-hemorrhagic fibrinogenases” in the classification
of P-I SVMPs proposed by Tsai et al. (2000; Fig. 2). The
identity percentage with SVMPs from Naja mossambica
and Atractaspis engaddensis, classified in the families
Elapidae and Atractaspididae, was 50% and 48%, respec-
tively (data not shown).
Overall crystallographic structure
The three-dimensional structure of BaP1 is very similar to
those described for other P-I snake venom metalloprotein-
ases (Gomis-Ruth et al. 1993, 1994; Zhang et al. 1994;
Kumasaka et al. 1996; Gong et al. 1998; Zhu et al. 1999;
Huang et al. 2002). Atomic coordinates and structure factors
were deposited in the Protein Data Bank, Brookhaven Na-
tional Laboratory (reference: 1ND1). BaP1 is an ellipsoidal
molecule with a shallow active-site cleft that separates two
subdomains: a major subdomain (residues 1–152) with sec-
ondary structure characteristic of ?/? proteins, comprising
four ?-helices (A, B, C, and D) and a five-stranded ?-sheet.
Strands I, II, III, and V are parallel, whereas strand IV is
antiparallel (Fig. 3). The minor subdomain (residues 153–
202) is formed by an ?-helix and various loops. The mol-
ecule has three disulfide bridges (Cys 117–Cys 197, Cys
159–Cys 181, Cys 157–Cys 164) that stabilize the structure.
The former bond links the two subdomains, whereas the
latter two occur within the minor subdomain. Thus, BaP1
belongs to the three-disulfide-bridge subgroup of P-I metal-
loproteinases; in this regard, it is therefore similar to acu-
tolysins A and C (Gong et al. 1998; Zhu et al. 1999) and
TM-3 (Huang et al. 2002). In contrast, adamalysin II and
atrolysin C present only two disulfide bridges (Gomis-Ruth
et al. 1994; Zhang et al. 1994). Both N and C termini of
BaP1 are located on the surface of the molecule, as in the
Figure 1. Amino acid sequence of metalloproteinase BaP1. The first 26 residues were determined by direct Edman degradation after the protein was
deblocked with pyroglutamyl aminopeptidase. Overlapping peptides were generated by digestion of the protein with CNBr, lysyl peptidase (K), clostripain
(CL), endoproteinase Asp-N (D), and asparaginyl peptidase (N). Only the sequences of the peptides required to show overlap coverage for the whole
sequence are presented. The cleavage sites are depicted by the symbol /. The sequence of each peptide includes only the residues that were confidently
Structure of a snake venom metalloproteinase
Figure 2. Sequence alignment of metalloproteinase BaP1 with other SVMPs from species of the family Viperidae whose hemorrhagic activity has been characterized. The seven top
sequences correspond to SVMPs that are hemorrhagic, and the eight bottom sequences correspond to enzymes devoid of hemorrhagic activity. The numbers at the top correspond to
those of the BaP1 sequence. Accession numbers in SWISS-PROT (sp) or trEMBL (tr) databases are indicated on the left. Invariant and conserved residues are presented against black
and gray backgrounds, respectively. (P20897) Hemorrhagic metalloproteinase HT-2 (ruberlysin) from Crotalus ruber ruber; (P20164) metalloproteinase HR1B (Trimerelysin I) from
Trimeresurus flavoviridis; (P14530) hemorrhagic metalloproteinase HR2A (Trimerelysin II) from Trimeresurus flavoviridis; (P15167) hemorrhagic toxin d (atrolysin-d) from Crotalus
atrox; (Q9PW35) acutolysin A from Agkistrodon (Deinakistrodon) acutus; (P22796) hemorrhagic factor II (LHF-II) from Lachesis muta; (Q919R4) nonhemorrhagic fibrinolytic
metalloprotease from Bothrops neuwiedi; (Q91401) atroxase from Crotalus atrox; (P28891) fibrolase from Agkistrodon contortrix contortrix; (P34179) adamalysin II from Crotalus
adamanteus; (P20165) H2 metalloproteinase from Trimeresurus flavoviridis; (Q98995) lebetase from Vipera lebetina; (Q98SP2) bothrostatin from Bothrops jararaca; (Q90495) ecarin
from Echis carinatus.
Watanabe et al.
Protein Science, vol. 12
other enzymes studied, and the C-terminal helix (helix E) is
stabilized by the disulfide bridge Cys 157–Cys 164. The
N-terminal residue was identified as pyroglutamate (residue
−1), as observed in the electron density maps, in agreement
with the results described above for sequence analysis.
The active site
As described for the other six P-I SVMPs whose structures
have been studied, the zinc ion is tetrahedrally coordinated
in BaP1 by the N?2nitrogen atoms of His 142, His 146, and
His 152, in addition to a water solvent molecule, which, in
turn, is bound to Glu 143 (Fig. 4). His 142, Glu 143, and His
146 are located on helix D. The Met-turn permits His 152 to
be in close contact with the catalytic zinc, and the side chain
of Met 166 comprises a hydrophobic base for the active-site
groups. The bonds in the active site of BaP1 are detailed in
Table 1. In analogy with the catalytic mechanism proposed
for the bacterial metalloproteinase thermolysin (Matthews
1988), it is assumed that Glu 143 polarizes the catalytic
water molecule, an event that is followed by the nucleo-
philic attack of the polarized water on the carbonyl carbon
of the scissile peptide bond in the substrate. A hydrophobic
pocket is present in the S1? site of SVMPs. Residues Phe
178, Val 138, Ala 141, Tyr 176, and Ile 165 form part of the
hydrophobic pocket (Gomis-Ruth et al. 1994) and are con-
served in many SVMPs (Fig. 2). However, there are varia-
tions in the depth of this pocket among various SVMPs,
depending on the presence of the nonconserved disulfide
bridge Cys 159–Cys 181 and on the particular amino acid
composition lining the pocket (Gong et al. 1998; Huang et
al. 2002). The structural characteristics of this pocket influ-
ence the cleavage specificity and the binding of inhibitors
(Zhang et al. 1994; Botos et al. 1996).
Structural calcium ion
The presence of a calcium ion located on the surface has
been reported for several SVMPs and is considered to be
important for structural stabilization (Gomis-Ruth et al.
1994). The ion is coordinated by the carbonyl oxygen atom
of Glu 9, both carboxylate oxygens of Asp 93, the carbonyl
oxygen of Cys 197, and the carboxiamide oxygen of Asn
200, in addition to a structural water molecule (Gomis-Ruth
et al. 1994; Gong et al. 1998). Although these four residues
are conserved, no calcium ion was found in the crystal
structure of BaP1. This can be attributed to the fact that,
because of the crystal packing, the N?group of Lys 131
from a symmetry equivalent molecule interacts with Asp
93, Cys 197, and the water, thereby precluding the binding
of calcium in that region in the crystal structure. Addition-
Table 1. Bond distances at the active site in BaPl
Bonds Distance (Å)
Figure 4. Details of the zinc-binding site of BaP1, superimposed on the
electron density map (2Fo− Fcmap contoured at 1 ?).
Figure 3. Ribbon diagram of the overall structure of metalloproteinase
BaP1, depicted in standard orientation. The catalytic zinc atom is high-
lighted, together with the three histidines, a glutamic acid, and a water
molecule forming the active-site environment. Secondary structures as well
as N and C termini are also labeled.
Structure of a snake venom metalloproteinase
ally, Asn 200 N?2and Lys 131 N?are bridged via a water
molecule. It is not known if such a Ca2+ion is present when
BaP1 is in solution. Absence of the calcium ion has been
also described for H2-proteinase (Kumasaka et al. 1996)
and acutolysin-C (Zhu et al. 1999). A role has been pro-
posed for this calcium in stabilizing the structure of these
enzymes (Gomis-Ruth et al. 1994), not only of the metal-
loproteinase domain, but also of the additional domains
present in P-II, P-III, and P-IV SVMPs (Gomis-Ruth et al.
1994; Gong et al. 1998), particularly the disintegrin-like
domain that follows the catalytic domain in these enzymes.
Calcium ions are known to stabilize the tertiary structure of
collagenase, a matrix metalloproteinase (Lovejoy et al. 1994).
Structural determinants involved in
SVMPs greatly differ in their ability to induce hemorrhage,
which is one of the main manifestations of envenoming by
vipers (Bjarnason and Fox 1994; Gutiérrez and Rucavado
2000). P-III metalloproteinases, containing disintegrin-like
and high-Cys domains in addition to the metalloproteinase
domain, usually induce more intense hemorrhage than the
P-I metalloproteinases, which consist of only the metallo-
proteinase domain (Bjarnason and Fox 1994). It has been
proposed that these additional domains may be responsible
for this enhanced hemorrhagic activity because of their abil-
ity to direct the enzymes to physiologically relevant targets
in the microvessels (Zhou et al. 1995; Jia et al. 1997; Gutiér-
rez and Rucavado 2000), probably resulting in proteolytic
degradation of key structural proteins at the basement mem-
brane (Baramova et al. 1990, 1991). Moreover, disintegrin-
like and high-Cys domains in P-III SVMPs may influence
substrate specificity (Jeon and Kim 1999); they have been
shown to inhibit collagen-induced platelet aggregation (Ka-
miguti et al. 1996b; Jia et al. 1997, 2000; Moura-da-Silva et
al. 1999), an effect that might potentiate hemorrhagic ac-
tivity. In addition to this well-demonstrated difference in
hemorrhagic potency between large and small SVMPs, a
wide range of hemorrhagic activities is also found in the P-I
group of enzymes, indicating that structural features within
the metalloproteinase domain play a role in this effect. Cata-
lytic activity is an absolute requirement for the development
of hemorrhage, because zinc chelation abrogates this effect
(Bjarnason and Fox 1994). Owing to the fact that the cata-
lytic center is identical in these molecules, the structural
basis for their different biological activity must depend on
structural determinants located in other regions, probably in
the region of the catalytic center.
Comparative sequence analyses have led to various pro-
posals concerning the residues and molecular regions puta-
tively involved in hemorrhagic activity (Takeya et al. 1989,
1990; Gasmi et al. 2000; Tsai et al. 2000). Some of these
proposals have been refuted as new sequences and crystal
structures have become available (Gomis-Ruth et al. 1994).
Comparison of the sequences of hemorrhagic and nonhem-
orrhagic P-I SVMPs demonstrated three regions with high
variation between these enzymes: (1) amino acid residues
63–90, (2) residues between 179 and 193, and (3) residues
between 150 and 176 (Tsai et al. 2000; Bolger et al. 2001).
These regions surround the active-site cleft and are located
on the surface of the molecule; it seems likely that they are
involved in modulating the interaction with substrates. A
phylogenetic analysis of 30 P-I SVMPs permitted their
grouping into three different subtypes, these being the
highly hemorrhagic acidic enzymes, moderately hemor-
rhagic basic proteinases, and nonhemorrhagic enzymes
(Tsai et al. 2000). Analysis of the C-terminal regions of
these SVMPs revealed that both acidic and basic hemor-
rhagic metalloproteinases are rich in residues with carboxyl-
and hydroxyl-containing side chains, as well as hydropho-
bic residues, whereas nonhemorrhagic proteinases possess
in that region predominantly residues with small or cationic
side chains (Tsai et al. 2000). On the basis of its sequence
in that particular region, BaP1 can be placed within the
basic (BH) subgroup of metalloproteinases, in agreement
with its basic pI (Gutiérrez and Ovadia 1998) and its rela-
tively low potency in inducing hemorrhage (Gutiérrez et al.
1995). However, as shown in Figure 2, the comparative
sequence analysis of BaP1 with other SVMPs in which
hemorrhagic activity, or its absence, have been well estab-
lished, did not evidence any clear picture of possible resi-
dues or positions specifically conserved among hemor-
rhagic enzymes. Therefore, comparative analysis of three-
dimensional structures is required to identify the structural
motifs determining hemorrhagic activity in SVMPs.
We superimposed the structures of three SVMPs exhib-
iting an identical pattern of disulfide bonds but displaying
large variations in hemorrhagic potency: acutolysin A
(highly hemorrhagic), BaP1 (weakly hemorrhagic), and H2-
proteinase (nonhemorrhagic). Structural differences among
them do not depend on a different disulfide bond arrange-
ment, as occurs when proteinases from the groups having
two and three disulfide bonds are compared. Superimposi-
tion of these structures indicated a high degree of structural
identity in most regions, with the exception of a stretch in
the region of residues 153 to 176, which corresponds to a
loop located near the catalytic site in the crystal structure
(Fig. 5). This observation supports a previous structural
comparison between acutolysin A, H2-proteinase, and ada-
malysin II, which showed variations in the segments 153–
164 and 173–176 (Gong et al. 1998). Within this region,
there is a highly conserved sequence Cys–Ile–Met involved
in the Met-turn. However, major structural variations in the
stretches located before and after this conserved sequence
are observed. In addition, there are some amino acid sub-
stitutions, insertions, and deletions that also occur around
the disulfide bond Cys 159–Cys 164; these contribute to
Watanabe et al.
Protein Science, vol. 12
structural divergences in this local microenvironment, as
described for H2-proteinase and acutolysin A (Gong et al.
1998). Here we show that BaP1 also differs from these two
enzymes in this region, showing that, despite an identical
disulfide bonding pattern in these three enzymes, there are
conspicuous structural differences in this loop. Interest-
ingly, BaP1 and acutolysin, both of which are hemorrhagic,
have more similarities in this region than H2-proteinase,
which is not hemorrhagic (Fig. 5). Because this region lies
close to the active site, it is implied that this loop may be
involved in the interaction with extracellular matrix sub-
strates. This might influence their ability to bind and hy-
drolyze basement membrane components, a key step in the
weakening and disruption of capillary vessel structure lead-
ing to hemorrhage.
It has been shown that structural motifs determining func-
tions of many proteins are often located in flexible loops.
Amino acid substitutions, deletions, and insertions in such
motifs may significantly modify the function of a protein
without affecting the structural stability of the molecule
(Overall 2002). In addition to hemorrhage, BaP1 induces a
wide variety of local pathological effects (Gutiérrez et al.
1995; Rucavado et al. 1995, 1998, 2002). It is not known if
the various toxicological activities depend on a single struc-
tural motif or if different regions are involved. Such a mul-
tiple functional profile makes BaP1 a valuable model for
investigating structure–function relationships associated
with various toxic effects in a single SVMP.
Materials and methods
Isolation of BaP1
Metalloproteinase BaP1 was isolated from a venom pool obtained
from >40 adult specimens of B. asper collected in the Pacific
region of Costa Rica. The isolation protocol previously described
by Gutiérrez et al. (1995) and Rucavado et al. (1998) included
ion-exchange chromatography on CM-Sephadex C-25, gel filtra-
tion on Sephacryl S-200, and affinity chromatography on Affi-Gel
Blue. Molecular mass was determined by MALDI-TOF mass spec-
trometry on a PE Voyager DE Pro equipment.
Reduction and alkylation
For reduction and alkylation, 20 ?g of protein was dissolved in 50
?L of a 0.64 M Tris-HCl (pH 8.0), 8 M urea, 0.16 M methylamine-
HCl solution. Following the addition of 5 ?L of a 2-mercapto-
ethanol solution (7 ?L/mL), the mixture was incubated at 60°C for
1.5 h. Then, 7 ?L of an N-isopropyliodoacetamide solution (2.3
mg in 20 ?L of methanol and 80 ?L of water) was added to the
protein solution, and the mixture was incubated at room tempera-
ture for 30 min before the addition of 10 ?L of 2-mercaptoethanol
Digestion with cyanogen bromide and various enzymes
For deblocking BaP1, 10 ?g of the metalloproteinase in a volume
of 5 ?L was added to 50 ?L of 0.1 M phosphate, 5% glycerol, 10
mM EDTA (pH 8.0) buffer, followed by the addition of 3 ?L of
0.1 M dithiothreitol. Then 2 ?g of pyroglutamate aminopeptidase
was added, and the mixture was incubated at 4°C overnight, fol-
lowed by incubation at 25°C for 4 h. The protein was then desalted
on a Phenomenex C18 column. For CNBr digestion, 5 ?g of BaP1
was incubated with 50 ?L of a 50 mg/mL CNBr solution. After
incubation at room temperature overnight, the mixture was diluted
with 400 ?L of 0.1% trifluoroacetic acid (TFA) for chromato-
graphic separation of the peptides. Lysyl peptidase digestion was
performed by incubating 5 ?g of reduced and alkylated BaP1 with
0.5 ?g of the enzyme (Wako Biochemicals). The mixture was
incubated at 37°C for 4.5 h with shaking. For clostripain digestion,
10 ?g of reduced and alkylated BaP1 was desalted on a Brownlee
C8 column. After neutralization of TFA with triethylamine and
concentration by drying, 0.2 ?g of clostripain (Sigma), in 50 mM
Tris-HCl, 1 mM CaCl2, and 2.5 mM dithiothreitol (pH 7.5) was
added and incubated at room temperature overnight. Endoprotein-
ase Asp-N digestion was carried out by incubating 10 ?g of re-
duced and alkylated BaP1, previously dialyzed against Tris-HCl
buffer (pH 8.0), with 0.092 ?g of endoproteinase Asp-N. Incuba-
tion was performed at 37°C for 18 h. The reaction was stopped
with 2 ?L of TFA. Asparaginyl peptidase digestion was performed
by diluting 10 ?g of reduced and alkylated BaP1 with 50 mM
acetate buffer (pH 5.0). Then, 0.08 mU of asparaginyl peptidase
(Pan Vera) was added, and the mixture was incubated at room
Amino acid sequence determination and
The N-terminal amino acid sequence of BaP1 was determined by
direct Edman degradation on a Applied Biosystems 494 amino
acid sequencer, operated according to the manufacturer’s instruc-
tions, once the enzyme was deblocked by pyroglutamate amino-
peptidase. Whole protein was adsorbed on PVDF in a ProSorb
cartridge (Applied Byosystems) and analyzed using the manufac-
turer’s instructions for PVDF samples. Peptides resulting from the
various digestions were separated by reverse phase HPLC chro-
matography. All separations used a gradient of 0.1% TFA in water
Figure 5. Stereo figure of the superimposition of the main chains of the
segment 153–176 in the structures of three P-I SVMPs that have an iden-
tical pattern of disulfide bonds but differ in their hemorrhagic potencies.
BaP1, a weakly hemorrhagic SVMP (white); H2-proteinase, a SVMP de-
void of hemorrhagic activity from the venom of Trimeresurus flavoviridis
(blue); and acutolysin A, a SVMP with high hemorrhagic activity from the
venom of Agkistrodon (Deinakgistrodon) acutus (orange).
Structure of a snake venom metalloproteinase
(solvent A) and 0.09% TFA in acetonitrile (solvent B), on an
Applied Biosystems 120A or 140 chromatography system, with
detection at 215 nm. The flow rate was 200 ?L/min. The columns
used were Phenomenex Jupiter C18, Vydac C18, Zorbax CN, and
Zorbax SB-C18. The sequences of the peptides resulting from the
hydrolysis of BaP1 by the CNBr and enzymatic treatments were
determined in an Applied Biosystems 494 sequencer after chro-
matographic separation of the peptides. The peptides were applied
to a Polybrene-coated glass fiber filter and analyzed with the
manufacturer’s pulsed liquid cycle. Similarity searches were per-
formed using FASTA (Pearson and Lipman 1988), and amino acid
sequences were aligned using the program CLUSTAL W (Higgins
et al. 1996).
Crystallization was performed by the hanging-drop vapor diffusion
method, as described earlier (Watanabe et al. 2002). Briefly, single
crystals were obtained when a 3-?L protein droplet, at a protein
concentration of 10 mg/mL, was equilibrated over a reservoir con-
taining Bicine buffer (pH 9.0), 10% PEG 20.000, and 2% (v/v)
X-ray diffraction data collection
Three-dimensional X-ray diffraction data were collected from a
single crystal (maximum dimensions of 0.4 mm) at room tempera-
ture at the crystallographic beamline (Polikarpov et al. 1998a,b) of
the Laboratório Nacional de Luz Síncrotron (LNLS, Campinas,
Brazil). The synchrotron-radiation source was set to a wavelength
of 1.54 Å. The diffraction intensities were measured using an
MAR 345 imaging plate detector (MAR Research) and an oscil-
Å. The X-ray diffraction data were processed, scaled, and integrated
using DENZO/SCALEPACK (Otwinowski and Minor 1997).
Molecular replacement was carried out using the program
AmoRe (Navaza 1994) with a homology-built model for BaP1 that
was based on the crystal structure of acutolysin-C from the venom
of Agkistrodon (Deinakgistrodon) acutus (PDB code 1QUA; Zhu
et al. 1999). The rotation function provided a clear solution with a
correlation coefficient of 17.9 (the next highest correlation was
9.5) in the resolution range of 20.0–2.5 Å. In the translation search,
the correlation increased to 48.1 (R-factor ? 45.5%) for data in
the same resolution range. This solution was subjected to rigid-
body refinement, which resulted in a correlation coefficient of 67.6
and an R-factor of 46.1%. The refinement was carried out using the
program CNS (Brunger et al 1998), and the electron density maps
were examined using the program package O (Jones et al. 1991).
The refinement converged to a crystallographic residual of 19.08%
(Rfree? 21.59%) for all data between 500.0 and 1.9 Å with no
outliers in the Ramachandran Plot (Laskowski et al. 1993) and
with excellent stereochemistry (Table 2). Data collection and re-
finement statistics are presented in Table 2. The figures of 3D
structures were prepared using the PyMOL molecular graphics
system (Delano 2002; http://www.pymol.org).
This study was supported by grants from the Wellcome Trust
(grant no. 062043) to J.M.G. and R.D.G.T., the International Foun-
dation for Science (grant no. F-2707-2), FAPESP (grant 99/09162-
4), CNPq (grant 520081/95-NV), SMOLBNet (grant 01/07537-2),
FUNDUNESP (Brazil), and Vicerrectoría de Investigación, Uni-
versidad de Costa Rica (projects 741-A1-504 and 741-A2-036).
L.W. was the recipient of an FAPESP fellowship. Atomic coordi-
nates and structural factors have been deposited in the Protein Data
Bank, Brookhaven National Laboratory (accession code 1ND1).
The publication costs of this article were defrayed in part by
payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 USC section 1734
solely to indicate this fact.
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Table 2. Diffraction data collection and refinement statistics
Crystal type formed Orthorhombic
Cell dimension (Å)
Maximum resolution (Å)
Percentage solvent (%)
No. of observations
No. of unique reflections
Molecules per asymmetric unit
Refinement resolution (Å)
No. of solvent molecules
Average B valuesd(Å2)
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aRsym? 100 × ∑|I (h) − 〈I (h)〉|/∑I (h), where I (h) is the observed intensity
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dB values are average B values for all nonhydrogen atoms.
eRMSD, root-mean-square deviation.
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Structure of a snake venom metalloproteinase