Snake venom metalloproteinases: Structure, function

Biochimica et Biophysica Acta 1824 (2012) 164–176

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a p a p

Review

Snake venom metalloproteinases: Structure, function and relevance to the mammalian ADAM/ADAMTS family proteins☆ Soichi Takeda a,⁎, Hiroyuki Takeya b, Sadaaki Iwanaga c a b c

Department of Cardiac Physiology, National Cerebral and Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565–8565, Japan Division of Pathological Biochemistry, Department of Life Sciences, Tottori University School of Medicine, 86 Nishi-cho, Yonago-shi, Tottori-ken, 683–8503, Japan The Chemo-Sero-Therapeutic Research Institute, KAKETSUKEN, 1-6-1 Okubo, Kumamoto 812–8581, Japan

a r t i c l e

i n f o

Article history: Received 22 March 2011 Received in revised form 11 April 2011 Accepted 11 April 2011 Available online 20 April 2011 Keywords: Snake venom metalloproteinase Disintegrin MDC protein ADAM ADAMTS

a b s t r a c t Metalloproteinases are among the most abundant toxins in many Viperidae venoms. Snake venom metalloproteinases (SVMPs) are the primary factors responsible for hemorrhage and may also interfere with the hemostatic system, thus facilitating loss of blood from the vasculature of the prey. SVMPs are phylogenetically most closely related to mammalian ADAM (a disintegrin and metalloproteinase) and ADAMTS (ADAM with thrombospondin type-1 motif) family of proteins and, together with them, constitute the M12B clan of metalloendopeptidases. Large SVMPs, referred to as the P-III class of SVMPs, have a modular architecture with multiple non-catalytic domains. The P-III SVMPs are characterized by higher hemorrhagic and more diverse biological activities than the P-I class of SVMPs, which only have a catalytic domain. Recent crystallographic studies of P-III SVMPs and their mammalian counterparts shed new light on structure–function properties of this class of enzymes. The present review will highlight these structures, particularly the non-catalytic ancillary domains of P-III SVMPs and ADAMs that may target the enzymes to specific substrates. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Snake venom is a complex mixture of bioactive proteins and polypeptides that contribute to subdue, kill and/or digest the prey [1,2]. Proteinases are present in the venoms of many snakes and are structurally classified into trypsin-like serine proteinases (SVSPs) and metalloproteinases (SVMPs). The composition of most viper venoms contains at least 30% of SVMPs, suggesting their potentially significant roles in envenomation-related pathogenesis, such as bleeding, intravascular clotting, edema, inflammation and necrosis [1,3–5]. SVMPs are the primary factors responsible for local and systemic hemorrhage [6]. They act essentially by degrading the components of basement membranes underlying capillary endothelial cells, and thus causing the disruption of vessel walls and allowing the escape of the blood contents into the stroma [7]. The hemorrhagic activity of SVMPs is among the major lethal factors in viper snake venom. Certain SVMPs do not possess hemorrhagic activity, but act through different mechanisms such as the disruption of hemostasis mediated by pro- or anti-coagulant effects (e.g., fibrinogenase, fibrolase, prothrombin activating activities),

platelet aggregation inhibitor and apoptotic or pro-inflammatory activities [7,8]. SVMPs are monozinc endopeptidases varying in size from 20 to 100 kDa and are grouped into several subclasses according to their domain organization [4,9,10]. SVMPs are phylogenetically most closely related to the mammalian ADAM (a disintegrin and metalloproteinase) family of proteins and, together with ADAM and the related ADAM with thrombospondin type-1 motif (ADAMTS) family of proteinases, constitute the adamalysin/reprolysin/ADAM family or the M12B clan of zinc metalloproteinases (MEROPS classification, http:// merops.sanger.ac.uk/). The name of the family is derived from their dual origins, as the first family member to be structurally characterized was adamalysin II from reptile venom, whereas others belong to a group initially described in male reproductive tissues [11–13]. Recent crystallographic studies of high-molecular-weight SVMPs and ADAM/ADAMTS family proteins have shed new light on structure– function properties of this class of proteinases. This review will highlight these recent structures with emphasis on the non-catalytic ancillary domains, which may modulate the biological properties of higher molecular weight SVMPs. 2. Classification of SVMPs

☆ This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome. ⁎ Corresponding author at: 5-7-1 Fujishiro-dai, Suita, Osaka 565–8565 Japan. Tel.: +81 6 6833 5012x2381; fax: +81 6 6835 5416. E-mail addresses: [email protected] (S. Takeda), [email protected] (H. Takeya), [email protected] (S. Iwanaga). 1570-9639/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2011.04.009

SVMPs are classified into P-I to P-III classes according to their domain organization (Fig. 1) [4,9]. P-I SVMPs are the simplest class of enzymes that contain only a metalloproteinase (M) domain. P-II SVMPs contain a metalloproteinase domain followed by a disintegrin (D) domain. P-III

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Fig. 1. Schematic diagram of the domain structure of SVMPs and related molecules. Each domain or subdomain is represented by a different color. M, metalloproteinase; D, disintegrin (or disintegrin-like) domain; C, cysteine-rich domain; Cw, the cysteinerich “wrist” subdomain; Ch, the cysteine-rich “hand” subdomain; snaclec, snake venom C-type lectin-like domain; E, epidermal growth factor (EGF)-like domain; T, thrombospondin type-1 (TSP) motif; S, spacer domain; X, domain variable among ADAMTSs. Representatives of each class of SVMPs and ADAMs/ADAMTSs, whose crystal structure have been determined, are indicated in red letters. The P-III classes SVMPs are divided into subclasses (IIIa–IIId) based on their distinct post-translation modifications. Recently, it was found that the D domain of ADAMTS family proteinases does not have a disintegrin-like structure but adopt the Ch-subdomain fold, and thus, is represented as D*. The previously called cysteine-rich domain of ADAMTSs is structurally subdivided into the N-terminal Ch-subdomain-fold domain (CA) and the C-terminal domain (CB). The ADAMTS family proteins commonly possess the N-terminal M, D, T, C, S domains, whereas the C-terminal is variable among ADAMTSs (e.g., ADAMTS13 possess six repeats of TSP domain and two CUB (complement, uEGF, and bone morphogenesis) domains that follow the S domain).

SVMPs contain M, disintegrin-like (D) and cysteine-rich (C) domains. P-III SVMPs are further divided into subclasses based on their distinct post-translational modifications, such as homo-dimerization (P-IIIc) or proteolysis between the M and D domains (P-IIIb). Formally called P-IV, the heterotrimeric class of SVMPs that contain an additional snake C-type lectin-like (snaclec) domain [14] is now included in the P-III group as a subclass (P-IIId), as no P-IV mRNA transcript has been found to date. The P-IIId class of SVMPs is therefore considered as a post-translationally modified derivative of the canonical P-III class (P-IIIa) of SVMPs [9]. SVMPs of different classes are often present in the same venom. The gene structure of all these SVMPs contains a signal (pre) and a pro domain sequence before the M domain, but none of the SVMPs with the pro domain have been isolated from the venom. The pro domain is suggested to maintain the proteinase in a latent state during their maturation by a cysteine-switch mechanism [15]. 3. The ADAM/ADAMTS family of proteins ADAM family proteins, which are also called metalloproteinasedisintegrins or metalloproteinase/disintegrin/cysteine-rich (MDC)

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proteins, belong to a class of membrane-bound glycoproteins whose functions have been implicated in cell–cell and cell–matrix adhesion and signaling [16,17]. The best-characterized in vivo function of ADAMs is their involvement in ectodomain shedding of various cellsurface proteins, including the latent forms of growth factor, cytokines and their receptors and cell adhesion molecules. ADAM17 (TNF-α converting enzyme, or TACE) is one of the best characterized due to its key function as the primary sheddase for tumor necrosis factor-α, as well as other surface proteins [18,19]. The ADAMTS family is a subclass of ADAMs, and constitutes a group of secreted proteinases. ADAMTSs are expressed in a broad spectrum of species ranging from human to worms and have diverse functions including procollagen processing, aggrecan degradation, organogenesis and hemostasis [20,21]. Excluding pseudo genes, human ADAMs and ADAMTSs are encoded by 20 and 19 functional genes, respectively (a list of functional ADAM/ADAMTS genes can be found at: http://degradome.uniovi.es/met.html). ADAMs and ADAMTSs play key roles in normal development and morphogenesis and are associated with a number of disease conditions, including arthritis, Alzheimer's disease, heart disease, cancer, and thrombosis [16,17,21,22]. As such, these proteinases have emerged as potential therapeutic targets for a variety of diseases. Although soluble isoforms of certain ADAMs exist, the typical ADAMs are type-I integral membrane proteins that contain an EGF domain and transmembrane/cytoplasmic domain following the MDC domains that are shared by the P-III SVMPs [23] (Fig. 1). Atypical ADAM10 and 17 lack an EGF domain and thus, the transmembrane segment follows the MDC domain [24]. On the other hand, ADAMTS family proteinases have varying numbers of carboxyl terminal thrombospondin type-1(TSP) repeats and additional domains that differentiate family members. They differ from ADAMs in their lack of an EGF domains, and a transmembrane/cytoplasmic region (Fig. 1). In contrast to SVMPs and ADAMTSs, which are catalytically active enzymes, only ~60% of the membrane-type ADAMs contain a functional catalytic signature sequence (see below). The physiological roles of these proteinase-inactive ADAMs remain largely unknown, although several members of this group play important roles in development [25], which suggests that the adhesive activity of these proteins may be relevant to their function. Hemorrhagic SVMPs degrade endothelial basal membrane proteins, which are usually processed by matrix metalloproteinases (MMPs) in normal biological processes. MMPs share a similar catalytic site architecture with SVMPs and ADAMs/ADAMTSs that is characteristic of the metzincin superfamily [26–28], but they have distinct non-catalytic ancillary domains, and thus belong to a different clan (MEROPS classification M10A) of metalloproteinases. Thus far no MMP-type proteinases have been identified in snake venoms, raising the intriguing question of why only adamalysin/reprolysin/ADAM family proteinases, but not MMPs, have evolved as toxins in the snake venom gland. 4. Biological activities of SVMPs Table 1 shows a brief summary of some of the biological activities associated with some of the SVMPs. Most of the functional activities of SVMPs are associated with hemorrhage or the disruption of the hemostatic system, which are primarily mediated by the proteolytic activity of the M domain. SVMPs cause hemorrhage by disturbing the interactions between endothelial cells and the basement membrane through the degradation of endothelial cell membrane proteins (e.g., integrin, cadhelin) and basement membrane components (e.g., fibronectin, laminin, nidogen, type IV collagen) [29]. Blood coagulation proteins (e.g., fibrinogen, factor X, prothrombin) are also targets of their proteolytic activities. Echis carinatus venom contains the specific prothrombin activators, ecarin [30,31] and carinactivase [32]. Adamalysin II, a non-hemorrhagic P-I SVMP isolated from Crotalus adamantus venom, cleaves and inactivates serum proteinase inhibitors including antithrombin III [33]. Kaouthiagin isolated from the

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venom of Naja kaouthia specifically binds and cleaves von Willebrand factor (VWF), resulting in loss of both the ristocetin-induced platelet aggregation and collagen-binding activity of VWF [34]. Additionally, a large number of the P-III SVMPs can inhibit platelet aggregation, thus enhancing the hemorrhagic state [35]. The hemorrhagic P-III SVMP jararhagin from the venom of Bothrops jararaca has been shown to degrade platelet collagen receptor α2β1 integrin in addition to fibrinogen and VWF, resulting in the inhibition of platelet aggregation [36]. Other platelet receptors, such as GPIbα and GPVI, are also degraded by SVMPs (e.g., kistomin, mocarhagin and crotalin cleave GPIbα [37–39], whereas alborhagin, crotarhagin and kistomin cleave GPVI [40,41]). Hemorrhagic activity has been assigned to the all three classes of SVMPs. In terms of hemorrhagic potency and diversity of biological activities, the P-III SVMPs are the most potent and diverse among the three classes. The P-III SVMPs are capable of inducing not only local but also systemic bleeding, whereas the P-I SVMPs mainly induce local hemorrhage [7]. Of note, their proteolytic activities themselves do not parallel their hemorrhagic activities. These observations suggest that additional, non-catalytic domains of the P-III SVMPs may contribute to their activities, most likely through substrate targeting. Several SVMPs (e.g., VAP1, VAP2, graminelysin) have been reported to induce apoptosis of human umbilical vein endothelial cells (HUVECs) [42–44]. The detachment of endothelial cells and resulting apoptosis could be an additional mechanism of a disruption of normal hemostasis by SVMPs. The peptide bond cleavage specificities of SVMPs were studied using insulin and glucagon as substrates [45–47]. These early studies

revealed that SVMPs generally hydrolyze peptide bonds with hydrophobic residues in the P1′ site. This finding was recently confirmed using proteome-based peptide libraries [48]. Because SVMPs do not hydrolyze commonly used synthetic substrates such as 4-nitroanilide and 4-methylcoumaryl-7-amide, intramolecularly quenched fluorogenic peptide substrates have been developed as a convenient and sensitive method to assess SVMP proteolytic activity [10,49]. Both hemorrhagic and non-hemorrhagic types of SVMPs exhibit rather broad peptide bond specificity, and there is no significant difference in residue preference for the primed and non-primed sites of small peptide substrates between both types, although there are distinct differences in their peptide bond specificities with respect to the hydrolysis of native protein substrates (e.g., HR2a and H2-proteinase, hemorrhagic and non-hemorrhagic P-I SVMPs isolated from Trimeresurus flavoviridis venom specifically cleave positions 517 and 413 in the Aα-chain of fibrinogen, respectively [49]). On the other hand, Russell's viper factor X (FX) activator, RVV-X shows a strict requirement for Arg at the P1 site of peptide substrates [49]. 5. Three-dimensional structures of SVMPs Adamalysin II is the first one of the adamalysin/reprolysin/ADAM family proteinases to have its structure resolved [13]. Vascular apoptosis-inducing protein-1 (VAP1), a P-IIIc dimeric class SVMP isolated from Crotalus atrox venom, was the first P-III SVMPs structure to be solved by X-ray crystallography [50]. To date, the structures of nine P-I SVMPs and seven P-III SVMPs are available in the Protein Data Bank

Table 1 Biological activities of selected SVMPs. SVMP

Source

Activities

Reference

P-I class Adamalysin II Atrolysin C Acutolysin A BaP1 H2-proteinase TM-3 Acutolysin C FII BmooMPα-I HT-2 HR2A Graminelysin-I

Crotalus adamanteus Crotalus atrox Agkistrodon acutus Bothrops asper Trimeresurus flavoviridis Trimeresurus mucrosquamatus A. acutus A. acutus Bothrops. moojeni Crotalus rubber rubber T. flavoviridis Trimeresurus gramineus

Inhibition of serum proteinase inhibitors Hemorrhagic Hemorrhagic Hemorrhagic; Inflammatory; myonecrotic Protelolytic; non-hemorrhagic Fibrinogenolytic Hemorrhagic Fibrin(ogen)olytic Fibrin(ogen)olytic Hemorrhagic Hemorrhagic Apoptotic

[33] [103] [104] [105] [106] [107] [108] [109] [110] [111] [112,113] [43]

P-II class MT-d Jerdonitin Bilitoxin-I

Agkistrodon halys brevicaudus Trimeresurus jedonii I Agkistrodon bilineatus

Proteolytic Inhibition of platelet aggregation Hemorrhagic

[114] [115] [116]

C. atrox Bothrops jararaca A. acutus Naja atra N. atra C. atrox B. jararaha T. flavoviridis T. flavoviridis Naja kaothia Echis carinatus Bothrops erythromelas

Appoptotic; inhibition of pletelet-aggregation Hemorrhagic Hemorrhagic Proteolytic Proteolytic Hemorrhagic; inhibition of pletelet-aggregation Hemorrhagic; inhibition of pletelet-aggregation Hemorrhagic Hemorrhagic Cleavage of VWF; inhibition of pletelet-aggregation Activation of prothrombin Activation of prothrombin

[44,74] [117] [118] [54] [54] [103] [119] [120] [121] [34] [31] [122]

C. atrox T. flavoviridis

Apoptotic Apoptotic

[44,123] [55]

Daboia. russelli Vipera lebetina E. carinatus Echis multisquamatus

Activation Activation Activation Activation

P-III class P-IIIa/b Catrocollastatin/VAP2B Bothropasin AaHIV Atragin K-like Atrolysin A Jararhagin HR1A HR1B Kaouthiagin Ecarin Berythractivase P-IIIc VAP1 HV1 P-IIId RVV-X VLFXA Carinactivase Multactivase

of factor X of factor X of prothrombin of prothrombin

[93,94] [124] [32] [57]

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(PDB). P-II SVMP structures are currently unavailable, although crystal or solution structures of more than 10 disintegrins are deposited in the PDB. Structures of partial fragments of eight members of the mammalian ADAM/ADAMTS family of proteins are currently available in the PDB. Table 2 summarizes the structures of the SVMPs and the related molecules determined by X-ray crystallography to date. 5.1. M domain structure The catalytic M domains of SVMPs range from 200 to 210 residues in length [4]. Fig. 2A depicts the crystal structure of adamalysin II [13] viewed from the standard orientation. All the M domain structures of the P-I and P-III SVMPs and the mammalian counterparts described above are topologically equivalent and can be superposed with each other with variability found only in the loop regions connecting helices and strands (Fig. 2B). The M domain has an oblate ellipsoidal shape with a notch in its flat side that separates a relatively small “lower” domain and an “upper” main molecular body, wherein the active-site cleft extends horizontally across the M domain surface to bind peptide substrates from left to right (Fig. 2A). The catalytic zinc ion is located at the bottom of the cleft, and is tetrahedrally coordinated by the Ne2 atoms of the three histidines (His142, His146 and His152 in adamalysin II) in the catalytic consensus sequence HEXGHXXGXXHD and the putative catalytic water molecule. This water molecule is anchored to the glutamic acid residue (His143) that functions as a catalytic base. The conserved methionine side chain (Met166) downstream of the catalytic consensus sequence provides a hydrophobic base beneath the three zinc-binding histidine residues. These structural features are the hallmarks of the metzincin superfamily of metalloproteinases that also contain MMPs, astacins

167

such as crayfish digestive proteinases and serralysins such as bacterial metalloproteinases [26–28]. The amino-terminal upper domain has a central core consisting of a highly twisted five-stranded β-sheet and four helices. The β-sheet is parallel to a substrate when bound to the cleft except for strand 4 (β4 in Fig. 2A), which is anti-parallel and creates an upper rim for the cleft. The carboxy-terminal lower sub-domain consists of a C-terminal helix preceded by an irregularly folded region. This irregular region is presumably important for substrate recognition because it forms, in part, the wall of the substrate-binding pockets, in particular those of the primed sites. 5.2. Substrate recognition at the catalytic site The structures of SVMPs in complex with peptide-like inhibitors have shed light on the molecular mechanism of substrate recognition at the catalytic site of this class of proteinases. Fig. 2C depicts one of these structures, showing a close-up view of the active-site cleft of the catalytic site of BaP-1 determined in complex with the hydroxyamate inhibitor WR2 [51]. The peptide-like portion of the inhibitor adopts an extended geometry and binds to the notched right-hand side of the catalytic site cleft (the S1′ to S3′ sites), closely mimicking the Cterminal part (P1′ to P3′) of an enzyme-bound substrate from left to right. The hydrogen bonding network between the inhibitor and the adjacent pocket-flanking regions of the enzyme resembles that of an antiparallel β sheet, in essence extending the central β sheet by two strands. Inhibitor-binding causes the pocket-flanking loops to sift inward, narrowing the substrate-binding cleft, while the rest of the proteinase is unperturbed. The flexibility at the binding pocket that is generally found in SVMPs is in agreement with the broad substrate specificity of this family of proteinases. These enzymes generally have

Table 2 Selection of the X-ray structures of SVMPs and related proteins deposited in the PDB. Protein SVMPs P-I class Adamalysin II Atrolysin C Acutolysin A BaP1 H2-proteinase TM-3 Acutolysin C FII BmooMPα-I P-III class Catrocollastatin/VAP2B Bothropasin AaHIV Atragin K-like VAP1 RVV-X Disintegrins Trimestatin Schistatin Disintegrin Mammalian proteins ADAM family ADAM17(TACE) ADAM33 ADAM10 ADAM22 ADAMTS family ADAMTS5 ADAMTS1 ADAMTS4 ADAMTS5 ADAMTS13

Source

Domains

PDB code

Reference

C. adamanteus C. atrox A. acutus B. asper T. flavoviridis T. mucrosquamatus A. acutus A. acutus B. moojeni

M M M M M M M M M

1IAG, 2AIG, 3AIG 1ATL, 1HTD 1BSW, 1BUD 1ND1 1WNI 1KUF, 1KUG, 1KUI, 1KUK 1QUA 1YP1 3GBO

[13] [125] [126] [127] [128] [129] [108] [130] [131]

C. atrox B. jararaca A. acutus N. atra N. atra C. atrox D. russelli

MDC MDC MDC MDC MDC 2 (MDC) MDC + 2 snaclec

2DW0, 2DW1, 2DW2 3DSL 3HDB 3K7L 3K7N ERO, 2ERP, 2ERQ 2E3X

[53] [88] [132] [54] [54] [50] [56]

T. flavoviridis E. carinatus E. carinatus

D 2 D (homodimer) 2 D (heterodimer)

1J2L 1RMR 1TEJ

[67] [133] [134]

Human Human Bovine Human

M M DC MDCE

1BKC 1R54, 1R55 2AO7 3G5C

[135] [136] [24] [23]

Human Human Human Human Human

M MD* MD* MD* D*TCS

3B8Z 2JIH, 2V4B 2RJP, 3B2Z 2RJQ, 3GHM, 3GHN

[137] [59] [60] [60] [61]

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Fig. 2. Structure of the M domain. (A) Ribbon structure of adamalysin II (PDB ID: 1IAG), a structural prototype of P-I SVMPs. Zinc and calcium ions and a catalytic water molecule are represented as magenta, dark gray and gray spheres, respectively. All structural figures were prepared using PYMOL [102]. (B) Superimposition of the M domains of the P-I SVMP adamalysin II (PDB ID: 1IAG, in light salmon), the P-III SVMP catrocollastatin/VAP2B (PDB ID: 2DW0, in cyan) and human ADAM33 (PDB ID: 1R54, in gray). (C) Close up view of the catalytic site of BaP-1 bound with the inhibitor WR2 (PDB ID: 2W12). WR2 (in magenta) forms hydrogen bonds (represented by yellow dotted lines) with the adjacent β4 strand and the part of the loop in the irregular region between the α4 and α5 helices in BaP-1. The peptide-like portion of the inhibitor binds to the catalytic site cleft, closely mimicking the C-terminal part (P1′ to P3′) of an enzyme-bound substrate from left to right.

primary specificity toward substrates with more amino acids than pentapeptides. As mentioned above, peptide bond specificities for all known SVMPs show a clear preference for Leu residues or for residues with a bulky side chain, such as Met in the P1′ site [52]; however, preferences in the other P4-P4′ sites appeared to be less significant than in the P1′ site [48]. There are large differences in hemorrhagic activities even among the P-I SVMPs, indicating that structural features in the M domain itself may play a role. Structural comparisons among SVMPs have revealed differences in the features of the substratebinding cleft in the M-domain, particularly in the S′ sites, although these data have proved insufficient to fully explain the structural basis of hemorrhage. 5.3. Structures of P-III SVMPs and mammalian ADAMs Fig. 3A depicts the crystal structure of catrocollastatin/VAP2B from C. atrox venom, a structural prototype of the P-III class of SVMPs [53]. All the P-III SVMP structures determined thus far show that the MDC domains fold into a C-shaped configuration in which the distal portion of the C domain comes close to the catalytic zinc ion. Comparison of the available P-III SVMP structures reveals that the structural variations of the D and C domains themselves are relatively small, with the exception of peripheral loop regions, as in the case of the M domain. However, there is a substantial diversity in the relative orientation between the M and D domains, thus resulting in variability in the spatial alignment of the M and C domains (see below). The kaouthiagin-like (K-like) proteinase from Naja atra adopts a more elongated configuration than typical P-III SVMPs because of the absence of a 17-amino acid segment and a different disulfide bond pattern in the D domain [54] (Fig. 3B). The structure of VAP1, a representative of the dimeric class (P-IIIc) of SVMPs [50], is shown in Fig. 3C. The M domains in the dimer are related by 2-fold symmetry, such that their active sites point in opposite directions. In the middle of the molecule, an inter-chain disulfide bond is formed between Cys374 residues conserved among the P-IIIc SVMPs. HV1 isolated from T. flavoviridis venom is another example of a P-IIIc SVMP and has also been shown to possess apoptotic activity [55]. Alignment of the amino acid sequence of 40 P-III SVMPs revealed that five members of SVMPs other than VAP1 and HV1 have this unique cysteine residue and the additional consensus sequence (Q-D-H-S(N)-K, residues 320–324 in VAP1) in

the M domain [50] and were therefore shown to be candidate P-IIIc SVMPs [53]. However, exactly how this unique dimeric structure correlates to its function remains to be elucidated. Fig. 3D depicts the structure of RVV-X [56], which is a representative P-IIId SVMP. RVV-X has a unique cysteine residue in the middle of the HVR (see below) in the C domain. This cysteine residue forms a disulfide bond with the C-terminal cysteine residue of one of the two light chains (LA). Carinactivase-1 and multactivase, potent prothrombin activators isolated from E. carinatus and Echis multisquamatus, respectively, also contain snaclec-type light chains within the molecule [32,57], and thus represent other examples of the P-IIId class of SVMPs. These two SVMPs, however, do not have a disulfide bond between the heavy chain and the light chain, and their 3D structures remain to be elucidated. Recently, the crystal structure of the entire ectodomain of human ADAM22 was determined, which revealed that the C-shaped configuration of the MDC domains observed in the P-III SVMPs is also conserved in mammalian ADAMs [23] (Fig. 3E). The additional EGF domain in ADAM22 is tightly associated with both the D and C domains, forming a rigid spacer that can properly allocate the M and D domains against the membrane. The MDC domain of P-III SVMPs and ADAMs contains three potential calcium-binding sites. One is in the M domain in opposition to the catalytic site (Fig. 3A). This calcium-binding site (site I) is highly conserved among all classes of SVMPs, ADAMs and also in ADAMTSs [50,58,59], with the exception of some SVMPs in which the distal ammonium group of the lysine sidechain substitutes for the Ca2+ ion [50,58]. Two other calcium-binding sites (sites II and III) are located in the D domain. 5.4. Structure of ADAMTS proteinases Recent crystallographic studies revealed that the D domain of the ADAMTS proteinases showed no structural homology to disintegrins, but were very similar in structure to part of the C domain of P-III SVMPs [59–61]. Thus, while the “disintegrin” nomenclature has been used to describe the ADAMTS proteinases, ADAMTSs actually have no disintegrin-like structures within the molecule. In addition to the D domain, the N-terminal portion of the C domain of ADAMTSs, which is designated the CA subdomain, was found to adopt the fold of Ch-subdomain modules as found in ADAMs (see Fig. 1 and Section 5.6) [61]. Therefore, ADAMTSs have two such modules, spatially separated

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Fig. 3. Structures of P-III SVMPs and mammalian ADAM/ADAMTS proteins. Each domain or subdomain is colored as in Fig. 1. The HVR regions are shown in blue. Bound zinc and calcium ions are shown by yellow and black spheres, respectively. (A) Two orthogonal views of the structure of catrocollastain/VAP2B (PDB ID: 2DW0), a structural prototype of P-III SVMPs [53]. The M, D and C domains are arranged in the C-shaped configuration. (B) The structure of an atypical P-IIIa SVMP, K-like protein from Naja atra (PDB ID: 3K7F) presents an elongated C-shaped structure because of the lack of a 17-amino acid segment and a different disulfide bond pattern in the D domain. (C) Structure of VAP1 (PDB ID: 2ERO), representative of the P-IIIc, homodimeric class of SVMPs. In this class of SVMPs, an inter-chain disulfide bond is formed at the center of the molecule. (D) Structure of RVV-X (PDB ID: 2E3X), a representative of the P-IIId, heterotrimeric class of SVMPs. The two light chains (LA and LB, colored in orange and light pink, respectively) of RVV-X show an intertwined dimer formation that is a typical structural feature of snake C-type lectin-like proteins (snaclecs). The C-terminal Cys133 in LA forms a disulfide bond with Cys389 in the HVR of the heavy chain of RVV-X. (E) Structure of the ecto-domain of human ADAM22 (PDB ID: 3G5C). The disordered 18-amino acid residues linker connecting the EGF domain and the transmembrane region is shown schematically. (F) Structural model of the MDTCS domain of ADAMTS13 [61].

by the TSP domain within the molecule. Fig. 3F depicts the structural model of the MDTCS domain, which is conserved among all members of ADAMTS family proteins, of ADAMTS13 constructed based on the crystal structures of the MD domain of ADAMTS4 and the DTCS domain of ADAMTS13 [61]. Due to the lack of a real disintegrin-like structure and an insertion of the elongated TSP domain, the MDTCS domain of ADAMTSs shows quite a different structure from that of the C-shaped P-III SVMPs. ADAMTSs possess an additional spacer (S) domain that is not present in the P-III SVMPs and ADAMs. The S domain has a β-sandwich structure with surface-exposed variable loops that are thought to bind with the target proteins [61].

5.5. Disintegrin and disintegrin-like domains in P-III SVMPs Disintegrins are small cysteine-rich proteins (40–90 amino acids) that are generated by the proteolytic processing of larger precursor P-II SVMPs [49,62,63], albeit with some exceptions [64]. Disintegrins typically possesses an Arg-Gly-Asp (RGD) recognition sequence on an extended loop (disintegrin (D)-loop) that can inhibit integrinmediated platelet aggregation and cell–matrix interactions [65,66]. As predicted by the amino acid sequence, the overall structure of the D domain of P-III SVMPs is very similar to that of the RGDcontaining disintegrin trimestatin [67], with the exception of the Dloop and two bound calcium ions (Fig. 4). These calcium-binding sites

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and apart from the M domain catalytic site. Thus, it may function primarily as a scaffold that spatially allocates two other functional domains. 5.6. Cysteine-rich domain The C domain of P-III SVMPs is structurally subdivided into “wrist” (Cw) and “hand” (Ch) subdomains [50]. The Cw subdomain is tightly associated with the D-loop and forms a continuous structure with the D domain. The Ch subdomain has a core of α/β-fold structure consisting of two antiparallel β strands packed against two of the three α helices, and five disulfide bonds. The Ch subdomain in the P-III SVMPs has a novel and unique fold with no structural homology to currently known proteins, with the exception of the corresponding segments of ADAM and ADAMTS family proteins [23,24,59–61]. The whole C domain of the P-III SVMPs and ADAMs has been deposited at the conserved domain database (CDD, http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd. shtml) [69], as a member of the ADAM_CR superfamily (cl02698). Fig. 5 represents a structural comparison of the Ch subdomains of SVMPs and their mammalian counterparts. Despite low amino acid sequence identities (e.g., ~15% between VAP and ADAM10, ~16% between VAP1 and ADAMTS13 (D*), and ~17% between the D* and CA domains of ADAMTS13), they share a similar core structure and topology. The peripheral loops, however, differ markedly in structure between these proteins, providing extended surface areas that are most likely involved in protein-binding. 5.7. Hyper-variable region

Fig. 4. Structures of disintegrin trimestatin (A) and catrocollastatin/VAP2B (B), and their superimposition (C). The Arg-Gly-Asp side chains in trimestatin and the disulfide bonds and the residues involved in the calcium-binding in catrocollastatin/VAP2B are shown in ball-and-stick representation. The calcium ions at sites II and III are coordinated by the oxygen atoms located at the corner of the pentagonal bipyramid.

were shown to have higher affinities for calcium ions than EDTA, indicating that the calcium ions in the D domain of P-III SVMPs and ADAMs likely stay unchanged once the subdomains are folded [22]. However, no such structural calcium ions have thus far been identified in isolated disintegrins in the crystal structures. In addition, the residues involved in the calcium-binding sites of the P-III SVMPs are not conserved in P-II SVMPs or disintegrins [53]. The residues in the D-loop of disintegrins are highly mobile and accessible to solvents (Fig. 4A). On the other hand, the D-loop in the P-III SVMPs usually contains an XXCD sequence instead of the typical RGD motif, and is packed against the next C domain. It is also less flexible because the bound calcium ion at site III forms a structural core (Fig. 4B). The disulfide bond between the D and C domains further stabilizes the continuous structure, suggesting that there is little inter-domain flexibility. These observations suggest that the disintegrin-loop of P-III SVMPs is unavailable for integrin-binding due to steric hindrance [50,68]. By contrast, the subsequent C domain has the surface features that are potentially suitable for protein–protein interactions (see below). The D domain can be further divided into two subdomains, the Ds and Da subdomains [50,58]. Each subdomain contains three disulfide bonds and one bound structural calcium ion (Fig. 4C) that stabilize the structure and ensure functional integrity in the oxidative extracellular environment. The number and spacing of cysteinyl residues and the residues that form calcium-binding sites II and III are strictly conserved in the primary structures of almost all known P-III SVMPs [50,58]. The D domain of the P-III SVMPs is located opposite to

The loop that encompasses residues 561–582 and extends across the central region of the Ch subdomain in catrocollastatin/VAP2B (residues 562–582 in VAP1) is the region in which the amino acid sequences are most divergent and variable in length among SVMPs (16–22 amino acids), mammalian ADAMs (27–55 amino acids) and ADAMTs (13–17 amino acids). Therefore, this region (shown in blue in Fig. 5) has been designated as the hyper-variable-region (HVR) [50]. The structures of the HVRs that have been determined to date are all involved-at least partially in crystal packing with the exception of RVV-X. They show a relatively small number of direct interactions with the remaining core regions of the Ch subdomain, which suggests that they are potentially flexible in solution. Different P-III SVMPs have distinct HVR sequences, which result in distinct surface features. They might, therefore, function in specific protein–protein interactions, explaining the diversity of biological activities characteristic of the P-III class of SVMPs. Because of its location, opposite to the catalytic site within the C-shaped MDC domain structure, the HVR has been assigned putative protein-binding and substrate recognition functions [50,68]. As described above, ADAMTSs lack the D/Cw domain, and the D domain is connected to the M domain by a loop (21–23 residues) that wraps around the back of the M domain, resulting in a drastically different position of the HVR-containing domain relative to the M domain as compared to P-III SVMPs (Fig. 3). The D domain of ADAMTSs is located in close proximity to the active site cleft and forms a continuous structure together with the M domain (Fig. 3F), thus forming, in part, the substrate-binding S3′ pocket. Of note, the HVR segment runs across the middle of the D domain. In this configuration, the region downstream of the P3′ residues of the substrate can bind directly to the HVR [68]. This is supported by the finding that the deletion of the 15 amino acid segment downstream of the P3′ residue of VWF abolished cleavage by recombinant MD-domain containing fragment of ADAMTS13 [70]. The structures of ADAMTS proteins support the idea that the HVR create a protein–protein interaction site. Comparison between structures further identified the variable (V)-loop (gray regions in Fig. 5) located beside the HVR, which shows

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Fig. 5. Ch subdomain structures of SVMPs and their mammalian counterparts. (A) Ribbon representation of the Ch subdomains of, from left to right and top to bottom, VAP1 [50], RVV-X [56], K-like [54], human ADAM22 [23], bovine ADAM10 [24], and the D* and CA domains of human ADAMTS13 [61]. The PDB ID for each protein is indicated in parentheses. The conserved N-terminal α-helix, C-terminal β-strands, and disulfide bonds are shown in red, yellow, and orange, respectively. The V-loop and the HVR are shown in gray and blue, respectively. Disordered regions within the crystals are shown by dotted lines. The N and C termini of the subdomains are indicated. The part of the light chain-A in RVV-X is shown in light pink.

a higher degree of variability among ADAMTSs and the subgroup of ADAMs (ADAM10 and ADAM17) than HVRs, as another putative protein-binding site [50,61,68]. The V-loop in the P-III SVMPs consists of an α helix and two solvent exposed loops. Although the tertiary structures are not so variable among the P-III SVMPs, these loop regions contain a variable segment and conserved aromatic residues, which are both exposed to the solvent and are thus suggested to be additional protein-binding sites [50].

5.8. Flexibility between the domains Comparison of the available structures of the P-III SVMPs and ADAM22 reveals that there is substantial diversity in the relative orientation of the M and D domains. For example, the MDC domains of ADAM22 form a closed C-shaped structure, unlike the open Cshaped structures of VAP1 and catrocollastain/VAP2B (Fig. 6A). Such variability is also observed even among the structures of the same SVMP in different crystals. Fig. 6B depicts one such example showing the superimposition of the M domains of the six molecules of catrocollastain/VAP2B determined in the three distinct crystal packing modes [53]. The flexibility of the molecule is reflected in the appearance of the various crystal forms of the same proteins, and vice versa[71]. The Leu408 side chain in the D domain of catrocollastatin/ VAP2B is located at the pivot point of the bending motion. The mainchain carbonyl oxygen atom of Leu408 directly coordinates the calcium ion of site II, whereas the side chain of Leu408 protrudes from the D domain and interacts with a small hydrophobic cavity on the surface of the M domain. A bulky hydrophobic residue at position 408 in the catrocollastatin/VAP2B sequence is highly conserved among the P-III SVMPs and ADAMs (either leucine, isoleucine or phenylalanine), and its side chain probably functions as a universal joint that allows the D domain to adopt various orientations with respect to the M domain in solution [53]. There is also diversity in the relative orientation between the Cw and Ch subdomains among the available structures. The intrinsic flexibility of the molecule may be

important for the fine tuning of substrate recognition in the P-III class of SVMPs, probably by adjusting the spatial alignment of the catalytic site and the exosite during catalysis (Fig. 6C).

6. C-domain-mediated protein–protein interactions The C domain may function to target the P-III class SVMPs to their specific substrates, and they have therefore been suggested as the key structural determinants of potent hemorrhagic activity or diverse biological activities of this class of SVMPs. Interestingly, a substantial amount of isolated DC-domain-containing fragments have been identified in the venoms that are probably the proteolytic products derived from the P-IIIb class of SVMPs. Jararhagin-C, catrocollastatin-C and leberagin-C, which are DC domain-containing fragments isolated from B. jararaca, C. atrox and Macrovipera levetina, respectively, inhibit collagen-induced platelet aggregation [72–75]. Alternagin-C from Bthrops alternatus has been shown to modulate α2β1 integrin-mediated cell adhesion, migration and proliferation [76]. The recombinant C domain of atrolysin A, another P-IIIa SVMP from C. atrox venom, specifically binds to collagen type-1 and the A domain of VWF, blocking collagen–VWF interactions [77,78] through binding to the VWF Adomain (VWA) [79]. The C domain of atrolysin A also binds to VWA-like domain-containing ECM proteins, such as collagen XII, collagen XIV, and matrilins 1, 3 and 4 [80]. The C domain of mammalian ADAMs has also been suggested to be involved in protein–protein interactions. ADAM12 interacts with cell-surface syndecan through its C domain and integrin-mediated cell spreading [81]. The fragment containing DC domains of ADAM13 has been implicated in cell migration and binds to the ECM proteins laminin and fibronectin [82]. Several studies have indicated that the C domain of ADAMs can influence proteolytic activity. The C domain of ADAM13 was also found to be a major determinant of specific developmental events that are mediated by the proteolytic activity of ADAM13 [83]. Shedding of the interleukin-1 receptor-II by ADAM17 requires the DC domains, whereas TNF-α and p75 TNF receptor shedding by ADAM17 requires only the M domain [84]. The

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Fig. 6. Flexibility between the domains in the P-III SVMPs. (A) Superimposition of the M domains of the RVV-X heavy chain (in light green), catrocollastain/VAP2B (in cyan), VAP1 (in gray) and ADAM22 (in light salmon). The HVR and the side chain of the residue located at the pivot point of the RVV-X heavy chain (Ile220), catrocollastatin/VAP2B (Leu408), VAP1 (Phe410) and ADAM22 (Phe451) are colored in green, blue, red and magenta, respectively. The zinc ion bound to catrocollastain/VAP2B is shown as a red sphere. (B) Superimposition of the M domains of the six crystallographically independent catrocollastatin/VAP2B molecules. The HVR and side-chain of Leu408 in each molecule are shown in blue and red, respectively. (C) Schematic model of substrate binding and cleavage by P-III SVMPs.

acidic surface pocket in the ADAM10 C domain serves as a binding site for the ephrin-A5/EphA3 complex in ADAM10-mediated ephrin-A5 proteolysis [24]. It should be noted, however, that most of these studies do not identify specific regions of the C domain involved in these interactions, and that the molecular mechanisms of recognition remain to be elucidated. Several reports suggest that the HVR region directly contributes to the function of P-III SVMPs. Peptides encompassing the HVR of jararhagin interfere with the interaction between platelet and collagen [85]. The peptides derived from the HVR of HF3, a hemorrhagic P-III SVMP from B. jararaca, promoted leukocyte rolling that was inhibited by the antibodies directed against integrin αMβ2 [86], and also inhibited collagen-induced platelet aggregation [87]. The peptide derived from the HVR region of atragin inhibited the migration activity of cultured cells [54]. Although these studies shed light on the functions of the HVR, it should be noted that short peptides do not always mimic their counterparts in the folded proteins. It has been suggested that P-III SVMPs can be classified into two or more sub-groups according to their HVR sequences [88], but the relationship between the different HVR sequence classes and their biological activities remains to be elucidated. Recently, the D domain HVR and the variable (V)-loop (gray regions in Fig. 5) adjacent to the HVRs of both the D and CA domain of ADAMTS13 were shown to function as VWF-binding exosites by structure-based site-directed mutagenesis experiments [61,89]. Additional structural and biochemical studies, including site-directed mutagenesis, will facilitate the identification of the key substrates of individual SVMPs

and enable a better understanding of the molecular mechanism of action of P-III SVMPs. 6.1. RVV-X The venom of Russell's viper Daboia russelli contains a potent blood coagulation factor X (FX) activator, RVV-X. Because of its high specificity, RVV-X is widely used both in coagulation research and in diagnostic applications [90–92]. RVV-X specifically activates FX by cleaving the same Arg194-Ile195 bond in FX that is cleaved by factors IXa or VIIa during physiological coagulation. The cleavage removes the heavily glycosylated N-terminal 52 residues of the FX heavy chain, resulting in the formation of the active-site catalytic triad in the serine proteinase domain (Fig. 7A). Activated FX (FXa), in turn, converts prothrombin to thrombin, which ultimately leads to the formation of a hemostatic plug. As described before, RVV-X belongs to the P-IIId class of SVMPs and is composed of an MDC domain-containing heavy chain and two light chains with a snaclec-fold [56,93–95] (Fig. 3D). Three peptide chains are assembled into a hook-spanner-wrench configuration, in which the MD domains constitute a hook, and the rest of the molecule forms a handle. The RVV-X heavy chain has a unique cysteine residue (Cys389 (H)) in the middle of the HVR. Cys389 forms a disulfide bond with the C-terminal cysteine residue of the light chain-A (Cys133 (LA)). In addition to this inter-chain disulfide bond, the surrounding residues of the HVR form multiple hydrophobic interactions and

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between the light chains may serve as a primary capture site for FX zymogens in the blood. This interaction also plays an essential role in the Ca2+-dependent activation of FX by RVV-X. The Ch/LA/LB portion, constituting the handle, may function as a scaffold to accommodate the elongated FX molecule, while separating the Gla domain and the scissile bond. This relatively large separation between the catalytic site and the Gla-domain-binding exosite may account for the high specificity of RVV-X for FX. Additionally, the mobile “hook” portion of RVV-X, the MD domain, not only exerts a catalytic effect but may also help regulate the binding affinity between molecules, thus driving the catalytic cycle; the M domain may associate with the AP region of the zymogen and release it upon activation. This scenario is a natural extension of the model depicted in Fig. 6C. The basis of the catalytic mechanism of FX activation by RVV-X is essentially consistent with the original proposal by Morita [90]. The RVV-X structure revealed that the light chain portion of RVV-X forms an exosite for binding to the substrate (FX) on the one hand, and directly interacts with the HVR in the heavy chain on the other. The RVV-X structure is a good illustration of evolutionary gain of specificity of the P-III SVMPs, which occurs through HVR-mediated binding to other proteins to create an exosite for binding ligands. 7. Concluding remarks

Fig. 7. Model of factor X (FX) activation by RVV-X. (A) A schematic diagram of FX activation by RVV-X. Cleavage of Arg194-Ile195 bond of FX by RVV-X removes the heavily glycosylated FX activation peptide, resulting in the exposure of the serine proteinase active site (left). The factor Xa (FXa) structure (PDB IDs: 1XKA, 1IOD) is shown in ribbon representation (right). Ile195 of FXa heavy chain is shown in magenta. (B) Two orthogonal views of the docking model. RVV-X is shown in surface representation.

hydrogen bonds, which further stabilize continuous the Ch/LA structure [56]. The RVV-X structure represents the first example of an HVRmediated protein–protein interaction.

The P-III SVMPs generally show higher hemorrhagic activity or more diverse biological functions than the P-I SVMPs. However, exactly how the modular architecture of the P-III class SVMPs relates to their functions remains to be elucidated. The resolution of the three-dimensional structures of some of the P-III SVMPs and their phylogenetically related mammalian proteins have shed new light on the structure–function properties of this class of proteinases, revealing potentially novel protein–protein interaction sites, and providing intriguing data for the development of working hypotheses. The HVR may constitute an exosite that captures the target molecules directly or through associated proteins. The RVV-X structure is consistent with this model and provides insights into the molecular basis of HVR-mediated protein–protein interactions and target recognition by the P-III class of SVMPs, as well as by mammalian ADAM/ADAMTS family proteins. Additional structural and biochemical studies of enzyme–substrate interactions are necessary to elucidate the molecular mechanisms of target recognition, identify the key substrates of each proteinase during specific biological events, and enable the design of selective inhibitors of this class of enzymes.

6.2. Factor X activation by RVV-X

Acknowledgments

The light chains of RVV-X form an intertwined pseudo-symmetrical dimer, in which the central portion of each chain projects into the adjoining subunit forming a loop-exchange dimer, which represents a hallmark of the structure of snaclecs [14,96]. In addition to the overall fold, the surface properties of the light chains of RVV-X are quite similar to those of the FX-binding protein (X-bp) from Deinagkistrodon acutus venom [97], whose crystal structure was solved in the complex with the gamma carboxylgulutamic acid (Gla) domain of FX [98]. These structural similarities suggest the intriguing possibility that RVV-X initially recognizes the FX Gla domain through an exosite, consisting of the concave cleft formed at the interface between the light chains. A 6.5-nm separation between the catalytic site and the putative Gla-domain-binding exosite in RVV-X suggest a docking model for FX [56] (Fig. 7B). In support of this hypothesis, when the properly folded Gla domain is absent from FX, the rate of FX activation by RVV-X is markedly diminished [99–101]. Moreover, RVV-X-catalyzed FX activation is inhibited by X-bp [93]. Based on the crystal structure and the docking model, a model explaining the activation mechanism of FX by RVV-X has been proposed [56,95]. The concave cleft formed

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