Introduction

ADAMs (a disintegrin and metalloproteinase) or MDC (metalloproteinase/disintegrin/cysteine-rich) proteins comprise an emerging class of mammalian metalloproteinases with potential regulatory roles in cell–cell and cell–matrix adhesion and signalling (Becherer and Blobel, 2003; Seals and Courtneidge, 2003; White, 2003; Blobel, 2005). To date, over 30 ADAMs have been identified in a variety of species from fission yeast to human. Roughly, half of these are believed to function as active metalloproteinases and thus to constitute major membrane-bound sheddase that can proteolytically release cell-surface-protein ectodomains including growth factors and cytokines, their receptors and cell adhesion molecules. For example, ADAM17 (TACE, TNF- converting enzyme) releases many cell-surface proteins including TNF- precursor (Black et al, 1997; Moss et al, 1997) and ADAM10 (kuzbanian), which dictates lateral inhibition of Drosophila neurogenesis (Rooke et al, 1996), releases Notch ligand Delta (Qi et al, 1999) and Notch itself (Pan and Rubin, 1997). With regard to cellular interactions, fertilin and (ADAM1 and ADAM2, respectively) have been identified as sperm surface molecules essential for fertilization (Primakoff et al, 1987; Blobel et al, 1990, 1992) and meltrin (ADAM12) is implicated in myogenesis (Yagami-Hiromasa et al, 1995). ADAMs have been associated with numerous diseases including arthritis, Alzheimer's disease, and cancer (Duffy et al, 2003; Moss and Bartsch, 2004). ADAM33 has been genetically linked with asthma (Van Eerdewegh et al, 2002). ADAMs uniquely display both proteolytic and adhesive activities on the cell surface, however, most of their physiological targets and the adhesion mechanisms remain unclear.

Disintegrins are small proteins (40–90 aa) isolated from snake venom typically with an Arg-Gly-Asp (RGD) recognition sequence on an extended loop (disintegrin-loop) that inhibit platelet aggregation via integrin binding (Huang et al, 1987; Calvete et al, 2005). ADAMs are unique among cell surface proteins in possessing a disintegrin (D-) domain and thus it has been suggested that integrins might be common receptors for ADAMs (Blobel et al, 1992; Evans, 2001; White, 2003). However, the RGD sequence in the ADAMs' disintegrin-loop is usually replaced by XXCD and therefore, its adhesive potential has been controversial. Both the ADAMs' D- and cysteine-rich (C-) domains are involved in the protein–protein interactions (Myles et al, 1994; Almeida et al, 1995; Zolkiewska, 1999; Iba et al, 2000; Gaultier et al, 2002; Smith et al, 2002), however, the details of the interactions have remained elusive. This is because high-resolution structures have been available only for isolated domains (Maskos et al, 1998; Orth et al, 2004; Janes et al, 2005) and no structural information has been available for the C-domain of the canonical ADAMs. To clarify the molecular mechanisms of target recognition for shedding by and of cellular adhesion via ADAMs, elucidation of the atomic structure of the ADAMs' MDC domains is indispensable.

To obtain structural data on an ADAM family member, we exploited the fact that hemorrhagic P-III snake venom metalloproteinases (SVMPs) share the ADAMs' MDC architecture (Jia et al, 1996; Evans, 2001; Fox and Serrano, 2005). Most ADAMs possess additionally, EGF-like, transmembrane and cytoplasmic domains and therefore are primarily membrane-associated, whereas SVMPs are secreted. Vascular apoptosis-inducing protein-1 (VAP1) is a disulfide-bridged homodimer P-III SVMP isolated from Crotalus atrox venom (Masuda et al, 1998, 2000). VAP1's stability and intrinsic two-fold symmetry enabled us to solve the crystal structures at 2.5-Å resolution. The structure reveals the residues that are important for stabilizing the MDC architecture are strictly conserved throughout the primary structure among all the known ADAMs. Therefore, the present structure represents the general architecture of ADAMs' MDC domains and provides insights into the molecular mechanism of the ADAMs' target recognition.

Results

Structure determination

VAP1 yielded crystals readily, and initial phases were determined by molecular replacement method using the structure of P-I SVMP, acutolysin-C (1QUA) (Zhu et al, 1999) as a starting model. Although the initial model, with 99 identical residues out of 197, represented less than 50% of the total molecule, two distinct local noncrystallographic two-fold symmetry (NCS) operations (see below) allowed us to completely model the whole molecule. The native structures were determined from the crystals with two distinct space groups, P2₁2₁2₁ and P4₁2₁2, both at 2.5-Å resolution (Table I). Orthorhombic crystals were used for inhibitor soaking and the GM6001 ((3-(N-hydroxycarboxamido)-2-isobut
yl-propanoyl-Trp-methylamide))-boun
d structure was determined at 3.0-Å resolution (Table I). In either crystal forms, the asymmetric unit contained one dimer molecule. The four monomers in the two crystal forms have almost identical structures, except for slight variations in their domain orientations, terminal residues, surface loops and active-site GM6001-binding region.

MDC architecture

The MDC architecture of VAP1 is shown in Figure 1A and B. The metalloproteinase (M-) domains in the dimer are related by NCS such that their active sites point in opposite directions and an intermolecular disulfide bridge is formed between symmetry-related Cys365 residues (Figure 1A). The M-domain is followed by a disintegrin (D-) domain that is further divided into D_s- and D_a-domains (see below). The D_s-domain protrudes from the M-domain close to the Ca²⁺-binding site I (see below) opposing the catalytic site. The D-domain forms a C-shaped arm, together with the cysteine-rich (C-) domain, with its concave surface toward the M-domain. There are no direct interactions between the arm and the M-domain. Notably, the distal portion of the C-domain comes close to and faces toward the catalytic site in the M-domain. The C-terminus Tyr610 is located proximal to the boundary between the D_a- and C-domains (Figure 1A and B). Aside from Cys365, each monomer contains 34 cysteinyl residues, all of which are involved in disulfide bonding, and their spacings are strictly conserved among ADAMs (Figure 2 and Supplementary Figure 1) except within the substrate-binding (between the helices H4 and H5) and the HVR (see below) regions. Figure 2 provides a selected subset of the sequence alignments and the entire alignments of VAP1 and 39 ADAM sequences, including all 23 human ADAMs so far available, can be found as Supplementary Figure 1.

M-domain

Each VAP1 M-domain corresponds to a very similar structure to that of ADAM33 (Orth et al, 2004), with a flat ellipsoidal shape having a central core made up of five stranded -sheets and five -helices and a conserved methionine (Met-turn) below the active site histidine residues, which bears the typical structural feature of metzincin family of metalloproteinases (Bode et al, 1993). However, they differ in the dimer interface and the loop structure around the substrate-binding site (Figure 1C) that corresponds to the variable region in the primary structure (between the helices H4 and H5, see Figure 2). The N-terminal helix (H0) is also unique in VAP1. The dimer interface is best characterized by the recognition sequence QDHSK (residues 320–324, see Figure 1C and Supplementary Figure 2A–C) and by Cys365, however these are not conserved among ADAMs; therefore, none of the ADAMs' M-domains are suggested to form a stable dimer as VAP1. A peptide-like hydroxamate inhibitor GM6001 binds to VAP1 (Figure 1A and B, and Supplementary Figure 2D and E) in exactly the same manner as in the marimastat-ADAM33 M-domain complex (Orth et al, 2004), suggesting that the catalytic sites of VAP1 and ADAM33 share a common substrate recognition mechanism. The ADAM33 M-domain structure suggests that most ADAMs have a Ca²⁺-binding site (designated Ca²⁺-binding site I) opposing the active-site cleft; however, in VAP1, the distal ammonium group of Lys202 substitutes for the Ca²⁺ ion (Figure 1D). Replacement of the calcium-coordinating glutamate residue with lysine also occurs in ADAM16, ADAM25 and ADAMs38–40 (Supplementary Figure 1).

C-shaped arm

The D-domain follows the M-domain, with a short linker that allows slightly variable domain orientations at V405 as a pivotal point (Figure 3C). The D-domain is further divided into two structural subdomains (Figure 3), the 'shoulder' (D_s-domain, residues 396–440) and the 'arm' (D_a-domain, residues 441–487). The D_s- and D_a-domains constitute a continuous C-shaped arm, together with the following N-terminus region of the C-domain which we designate the 'wrist' (C_w-domain, residues 488–505). There are three disulfide bonds in the D_s-domain, three in the D_a-domain and one in the C_w-domain. The subdomains are connected by single disulfide bridges (Figures 2 and 3A) with slightly variable angles (Figure 3B).

Both the D_s- and D_a-domains contain structural calcium-binding sites. In the D_s-domain, the side-chain oxygen atoms in residues Asn408, Glu412, Glu415 and Asp418, and the carbonyl oxygen atoms of Val405 and Phe410 are involved in pentagonal bipyramidal coordination and constitute Ca²⁺-binding site II (Figures 2 and 3A). Notably, these residues are strictly conserved among all known ADAMs (Supplementary Figure 1). However, the side-chain oxygens of Asp469, Asp472 and Asp483, and carbonyl oxygens of Met470 and Arg484 form the corners of a pentagonal bipyramid to the calcium ligand and constitute the D_a-domain Ca²⁺-binding site III (Figures 2 and 3A) and these residues are highly conserved among ADAMs except ADAM10 and ADAM17 (Supplementary Figure 1). Because of the few secondary-structural elements, bound calcium ions and the disulfide bridges are essential for the structural rigidity of ADAM's C-shaped arm. The RGD-containing disintegrin trimestatin (Fujii et al, 2003) has a similar structure with the D_a-domain (r.m.s.d of 1.24 Å, Figure 3B); however, no disintegrins have been shown to bind Ca²⁺ ions.

Using isolated D-domains or portions thereof, numerous ADAMs and P-III SVMPs have been shown to interact specifically with particular integrins (Evans, 2001; White, 2003; Calvete et al, 2005). However, the disintegrin-loop is packed against the C_w-domain and a disulfide bridge (Cys468–Cys499) further stabilizes the continuous structure (Figure 3A). Therefore, the disintegrin-loop is inaccessible for protein binding.

Hand domain

The 'hand' domain (C_h-domain, residues 505–610) follows the C_w-domain. The C_h-domain, together with the C_w-domain, constitutes a novel fold (Figure 4A). In either crystal form, VAP1 dimers interact with molecules of neighboring units through the C_h-domains such that the molecules form a handshake (Figure 4B). Each C_h-domain interacts with its counterpart through a relatively large complementary surface of 860 Ų forming another NCS at the center, although VAP1 exists as dimers, not as oligomers, and is mono-dispersed in solution (data not shown).

HVR as a potential adhesive interface

C_h-domain residues 562–583 are predominantly involved in the handshake (Figure 4B). This is the region in which the ADAM sequences are most divergent and variable in length (16–55 aa) (Figure 2 and Supplementary Figure 1). We have designated this as the hyper-variable region (HVR). The HVR is subdivided into two structural elements. The N-terminal portion (residues 562–572) fits into an extended loop, filling the gap between the M-domain and the neighboring molecule's C_h-domain and thus fixing the position of the arm (Figure 4B). The variable structures and less-specific interactions suggest that this loop is stabilized by crystal packing. Some ADAMs possess a putative fusion peptide in this segment typical of viral fusion proteins (Blobel et al, 1992; Yagami-Hiromasa et al, 1995), although their role in the actual fusion process has not been demonstrated. However, the remainder of the HVR (residues 572–583) interacts extensively with its counterpart by forming an antiparallel strand at the center (Figure 4C and D). Although the ability to form strand is predictable from the sequence, this strand is stabilized mainly by interchain interactions (Figure 4D). There are no intrachain hydrogen bonds between residues 574–577 and the remainder of the C_h-domain; however a water-mediated hydrogen-bond network stabilizes this segment (Figure 4D). Therefore, it appears, that this strand might be formed by the induced-fit mechanism upon the association of the C_h-domains and that the conserved disulfide bond (Cys526–Cys572, see Figure 4D) may stabilize the structure when the HVRs are isolated in solution. In addition to the main-chain hydrogen bonds, side-chain atoms (particularly residues I574, Y575, Y576 and P578) in the HVR strand contribute numerous von der Waals interactions with their counterparts. Aside from the HVR, aromatic residues located at both sides of the strand in close proximity to the NCS axis create additional interaction surfaces: residues Phe515, Gly516, His535 and Tyr536 in the loop regions form hydrophobic ridges that fit complementarily into the NCS region (Figure 4C). The hydrophobic ridges are highly conserved among ADAMs (Figure 2 and Supplementary Figure 1), thus, in part, they may also constitute binding surfaces.

Discussion

The VAP1 structures reveal highly conserved structural calcium-binding sites and the numbers and the spacings of cysteinyl residues that are essential for maintaining structural rigidity and spatial arrangement of the ADAMs' MDC domains. The C-shaped MDC architecture implies meaningful interplay between the domains and their potential roles in physiological functions.

The HVR creates a novel interaction interface in collaboration with the conserved hydrophobic ridges. Different ADAMs have distinct HVR sequences, which result in distinct surface features, thus, they may function in specifying binding proteins. The HVR is at the distal end of the C-shaped arm and points toward the M-domain catalytic site, with a distance of 4 nm in between them. Collectively, these observations suggest that the HVR captures the target or associated protein that is processed by the catalytic site (Figure 5). The disintegrin portion is located opposit to and apart from the catalytic site and, thus, might play a primary role as a scaffold that allocates these two functional units spatially. The C-shaped structure also implies how the ADAMs' C-domains cooperate with their M-domains (Reddy et al, 2000; Smith et al, 2002). In membrane-bound ADAMs, the EGF-like domain (60 aa) follows the C_h-domain (Figure 2) and presumably works as a rigid spacer connecting the MDC-domains with and orientating against the membrane-spanning region (Figure 5A). Many ADAMs are proteolytically inactive (because of the defects in the catalytic HEXXHXXGXXHD sequence or the post-translational removal of the M-domain), and several of these are important developmentally. Therefore, the HVR may also work to modulate cell–cell and cell–matrix interactions. There is some experimental evidence for C-domain-mediated adhesion. Peptides encompassing the HVR and the hydrophobic ridge from P-III SVMPs interfere with platelet interaction and collagen binding (Kamiguti et al, 2003). A recombinant atrolysin-A C-domain specifically binds collagen I and von Willebrand factor (vWF) and blocks collagen–vWF interaction (Jia et al, 2000; Serrano et al, 2005). ADAM12 interacts with cell-surface syndecan through its C-domain and mediates integrin-dependent cell spreading (Iba et al, 2000). The D/C-domain portion of ADAM13 binds to the ECM proteins laminin and fibronectin (Gaultier et al, 2002). However, most of these studies do not assign specific regions of the C-domain to these interactions and the molecular recognition mechanisms are to be elucidated.

ADAM10 and ADAM17 lack the Ca²⁺-binding site III and show less sequence similarities in the C-domain with other canonical ADAMs (Supplementary Figure 1). Comparison of the recently solved ADAM10 D/C-domain partial structure (ADAM10_D+C) (Janes et al, 2005) and that of VAP1 reveals that the atypical ADAM10 shares the continuous D_a/C_w structure and the C_h-domain scaffold with VAP1; however, it has an disordered D_s-domain and an alternate HVR structure and a different orientation between C_w- and C_h-domains (Figure 6). The locations of four of the five disulfide bridges within the C_h-domain are conserved between VAP1 and ADAM10 (Figure 6B and C) and thus, they enabled us to align the two sequences (Figure 6E). Based on this alignment, we completed entire alignments (Supplementary Figure 1) including 38 sequences of mammalian ADAMs and Schizosaccharomyces pombe Mde10 (Nakamura et al, 2004), presumably the founding member of the ADAM family in evolutionary terms. The ADAM10_D+C structure lacks the eight residues (583–590 in ADAM10) that may form a flexible loop. However, VAP1 (Figure 6E) and the canonical ADAMs except for ADAM8 (Supplementary Figure 1) have extra 16 residues in this segment that, in part, forms a variable loop, flanked by the adjacent cysteinyl residues (Cys539 and Cys549 in VAP1) and protrudes from the main body of the C-domain (Figures 4A and 6B). The variable loop has highest temperature factor in the molecule and resembles to the disintegrin-loop, thus can be an additional protein-binding interface. The six VAP1 monomer molecules represent almost the same C_w/C_h domain orientation (data not shown), however that is distinct from that of ADAM10 (Figure 5A). Thus, the possibility whether different ADAMs have distinct C_w/C_h domain orientation remains to be established. Janes et al (2005) have shown that the three glutamate residues outside of HVR are essential for ADAM10-mediated ephrin proteolysis in trans, however, roles of the ADAM10 HVR has not been examined. An extensive molecular surface of the elongated arm structure (12 000 Ų for the VAP1 D/C-domains) might reveal additional protein–protein interaction interfaces other than the HVR. Multiple charged residues in the D-domain are essential for ADAM28 binding to 41 (Bridges et al, 2003) and the RX₆DLPEF motif has been proposed for integrin 91 binding (Eto et al, 2002). However, the D-domain portion of the C-shaped scaffold is away from the catalytic site; thus, those additional sites might not directly serve as target recognition interfaces for catalysis.

Uniquely among cell-surface proteins, ADAMs display both proteolytic and adhesive activities. The VAP1 structure reveals that these functions are spatially allocated to the ends of the unique C-shaped scaffold and face each other. This spatial allocation of the functional sites provide us insights into the molecular mechanism of ADAMs' target recognition, which ADAMs shed which key substrates in specific biological events. Since ADAMs are potential therapeutic targets, the distinct surface feature created by the HVR of the individual ADAMs might also provide insights into the future design of drugs with higher specificity for each member of ADAMs. We suggest that the HVR, not the disintegrin domain, should be the focus of searches for physiological targets of ADAMs.

Materials and methods

Protein preparation and crystallization

The details of the preparation, crystallization and preliminary X-ray analysis of VAP1 will be described elsewhere (T Igarashi et al, in preparation). VAP1 was isolated from the crude snake Crotalus atrox venom (Sigma-Aldrich, USA) and subjected to sitting- or hanging-drop vapor diffusion crystallization. Two distinct crystal forms (P2₁2₁2₁ and P4₁2₁2) were obtained with the reservoir solution containing 15% polyethyleneglycol 8000 and 100 mM sodium cacodylate at pH 6.5, with (orthorhombic form) or without (tetragonal form) 20 mM cobaltous chloride hexahydrate. GM6001-bound crystals were prepared by adding GM6001 (CALBIOCHEM) to the drop with the orthorhombic crystal at a final concentration qof 0.33 mM (twice the protein concentration) followed by a 12-h incubation. Crystals were flash-frozen under the nitrogen flow at 90 K.

Diffraction data collection

All the diffraction data were collected at SPring-8 beamlines using either ADSC quantum 310R CCD (for the inhibitor-bound crystal at the beamline BL41XU with =1 Å), Rigaku R-axis V imaging plate (for orthorhombic native crystal at the beamline BL45PX with =1 Å) or Jupitor CCD (for the tetragonal crystal at the beamline BL45PX with =0.98 Å) detectors at 90 K. The images were reduced using HKL2000. Both orthorhombic and tetragonal native data sets were collected to 2.5-Å resolution and inhibitor-bound crystal data sets were collected to 3.0 Å resolution (Table I).

Structural analysis

All structures were solved by the molecular replacement method by MOLREP in the CCP4 suite (CCP4, 1994) by using acutolysin-C (1QUA) (Zhu et al, 1999) as a starting model. Initially, the MR solution obtained from the orthorhombic crystal data set, assumed two M-domains in the asymmetric units. After manual rebuilding by TURBO-FRODO, the model was subjected to tortional molecular dynamic refinements with restrained NCS averaging of the M-domains using CNS (Brunger et al, 1998) and iterative refinements and manual rebuilding of the model improved the electron-density map and enabled us to extend the model. First, we found the electron densities associated with the pieces of helical segments of the molecules and modelled them as poly-alanine chains. After cycles of refinements, we assigned those segments as the parts of helices H7 and H8, where the secondary structures are predicted to be helices, judging from the electron densities associated with the side chains. At this stage, four tyrosine residues, Tyr575 and Try576 within the central strands of the HVRs were clearly defined, and we noticed that there was another NCS-axis between the C-domains. After iterative rounds of refinements with restrained NCS averaging of the C-domains and manual model building, we completed modelling of the C-domains. From this stage onward, no NCS averaging was included in the refinements. Next, we modelled the D-domains with the help of automated chain tracing using the program ARP/wARP (Perrakis et al, 1999) and with the structural model of trimestatin (1J2L) as a guide. After completely modelling the polypeptide chains, we noticed that isolated lobes of high electron densities surrounded by oxygen atoms occurred both in the D_s- and D_a-domains. For these sites, calcium ions fit optimally to the electron density with a refined occupancy of 100% and reasonably low B-values, thus, we included calcium ions in the model. We also assigned a cobalt ion, which was supplemented in the crystallization buffer for the orthorhombic crystal form, located between the M- and D_s-domains in the A molecule. The part of the carbohydrate chain linked to residue Asn218 (two N-acetyl-glocosamine (NAG) moieties) was modelled. Then, water molecules were assigned. The VAP1 cDNA encodes a protein with 610 amino-acid residues; however, the N-terminus is processed by post-translational modification (Masuda et al, 1998, 2000). Here, protein sequencing of the de-blocked VAP1 molecule clarified that the Glu184 side chain was modified into a pyro-form. The electron densities associated with almost the entire molecule except for the first pyroglutamic acid were defined in either monomer within the orthorhombic crystal. In the final model, 86.1% of the residues lay in the most favorable region, 13.3% in the additionally allowed region and 0.7% in the generously allowed region of the Ramachandran plot. The tetragonal crystal and inhibitor-bound crystal were solved by MR with the domains of the refined orthorhombic apo-form as a starting model. In the final model, 83.6% (80.6%) of the residues lay in the most favorable region, 15.7% (18.9%) in the additionally allowed region and 0.7% (0.5%) in the generously allowed region for tetragonal (inhibitor-bound) crystals in the Ramachandran plot. In either crystal form, the asymmetric unit contained one dimer molecule. All six monomers had almost identical structures. Refinement statistics are shown in Table I.

PDB accession codes

Atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 2ERO, 2ERP and 2ERQ for the orthorhombic native, GM6001-bound form and tetragonal-form, respectively.

Supplementary data

Supplementary data are available at The EMBO Journal Online.

Acknowledgements

We thank Yuko Oishi and staff in SPring-8 beamlines for assistance with data acquisition and Junichi Takagi for discussions and critical reading of the manuscript. This work was partly supported by Grant nano-001 for Research on Advanced Medical Technology from the Ministry of Health, Labor, and Welfare of Japan, and by grants from the Takeda Science Foundation, from the Kao Foundation for Arts and Science and from Senri Life Science Foundation. The authors declare no competing financial interests.

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