Crystal structures of VAP1 reveal ADAMs' MDC domain architecture and its unique C-shaped scaffold - PubMed (original) (raw)
Crystal structures of VAP1 reveal ADAMs' MDC domain architecture and its unique C-shaped scaffold
Soichi Takeda et al. EMBO J. 2006.
Abstract
ADAMs (a disintegrin and metalloproteinase) are sheddases possessing extracellular metalloproteinase/disintegrin/cysteine-rich (MDC) domains. ADAMs uniquely display both proteolytic and adhesive activities on the cell surface, however, most of their physiological targets and adhesion mechanisms remain unclear. Here for the first time, we reveal the ADAMs' MDC architecture and a potential target-binding site by solving crystal structures of VAP1, a snake venom homolog of mammalian ADAMs. The D-domain protrudes from the M-domain opposing the catalytic site and constituting a C-shaped arm with cores of Ca2+ ions. The disintegrin-loop, supposed to interact with integrins, is packed by the C-domain and inaccessible for protein binding. Instead, the hyper-variable region (HVR) in the C-domain, which has a novel fold stabilized by the strictly conserved disulfide bridges, constitutes a potential protein-protein adhesive interface. The HVR is located at the distal end of the arm and faces toward the catalytic site. The C-shaped structure implies interplay between the ADAMs' proteolytic and adhesive domains and suggests a molecular mechanism for ADAMs' target recognition for shedding.
Figures
Figure 1
MDC architecture. (A) VAP1 dimer viewed from the NCS axis. The H0-helix, M-domain, linker, Ds-, Da-, Cw-, and Ch-domains and HVRs belonging to the one monomer are shown in red, yellow, gray, cyan, pink, gray, green and blue, respectively. The disulfide-linked counterpart is shown in gray. Zinc and calcium ions are represented as red and black spheres, respectively. The NAG (_N_-acetyl-glucosamine, in orange) moieties linked to Asn218, the calcium-mimetic Lys202 and the bound inhibitor GM6001 (GM, in green) are in ball-stick representations. (B) Stereo view of VAP1 monomer from the direction nearly perpendicular to (A). The helix numbers are labelled. (C) Superposition of the M-domains of ADAM33 (blue) and VAP1 (yellow). The calcium ion bound to site I and the zinc ion in ADAM33 are represented by black and red spheres, respectively. The disulfide bridges are indicated in black and blue letters for VAP1 and ADAM33, respectively. The QDHSK sequence for the dimer interface in VAP1 (residues 320–324) is in red. (D) Comparison of the calcium-binding site I structures of ADAM33 (blue) and VAP1 (yellow) in stereo. The residues in ADAM33 and in VAP1 are labelled in blue and black, respectively. A calcium ion and a water molecule bound to ADAM33 are represented as green and red spheres, respectively. The ammonium group of Lys202 in VAP1 occupies the position of the calcium ion in ADAM33. In ADAM33 (Orth et al, 2004), side-chain oxygen atoms of Glu213, Asp296 and Asn407, the carbonyl oxygen of Cys404 and a water molecule form the corners of a pentagonal bipyramid and ligand to the calcium ion.
Figure 2
Sequence alignments of VAP1 and human ADAMs. The cysteinyl residues and the conserved residues are shaded in pink and yellow, respectively. Disulfide bridges, secondary structures and domains are drawn schematically. The HVR, calcium-binding site I, catalytic site and disintegrin-loop (D-loop) are boxed in blue, red, green and cyan, respectively. The hydrophobic ridges (H-ridges) are indicated. Calcium-binding sites II and III and the coordinating residues (shaded in red) are indicated. The NCBI accession numbers for the sequences are indicated.
Figure 3
Arm structure. (A) Arm structure in stereo. The Ds-, Da-, and Cw-domains are in cyan, pink and light green, respectively. The calcium-coordinating residues and the disulfide bridges are shown in red and green, respectively. The residues with carbonyl oxygen atoms involved in calcium coordination are underlined. Calcium ions are represented as black spheres. The disintegrin-loop (DECD) is in blue. (B) Superimposition of the four Da-domains of VAP1 and trimestatin (1J2L). Trimestatin and its RGD loop are shown in red and blue, respectively. (C) Superimposition of the four Ds-domains. The linker between the M- and Ds-domains is shown in gray. Val405 at the pivotal point is indicated.
Figure 4
C-domain architecture and HVR. (A) The C-domain architecture in stereo. The Cw- and Ch-domains are in gray and light green, respectively. The disulfide bridges and the residues forming the hydrophobic ridges are indicated. The HVR and its NCS counterpart are shown in red and blue, respectively. The variable loop (residues 539–549), flanked by two adjacent cysteine residues, is in green. (B) Crystal packing in the orthorhombic crystal. The crystallographically equivalent molecules (HVRs) are in cyan (blue) and pink (red), respectively. The arrows indicate the directions of the HVR chains. Zinc and calcium ions are represented as red and black spheres, respectively. (C) Interactions between the HVRs (cyan and pink) in stereo. The molecular surface of the cyan molecule is shown with the electrochemical surface potential (red to blue). The residues constituting the hydrophobic ridges are in yellow. The residues are labelled in blue and red for cyan and pink, respectively. (D) Water-mediated hydrogen-bond network in the HVR. The HVR residues are in pink and cyan; non-HVR residues in the pink molecule are in gray.
Figure 5
Models for ADAM's shedding. The molecular surface of the VAP1 monomer, without VAP1's unique H0-helix, are colored as in Figure 1A. Hydrophobic ridges are in yellow. EGF-like, transmembrane and cytoplasmic domains are represented schematically. (A) Membrane-anchored substrate molecule ‘X' is directly recognized and captured by the HVR on the membrane-bound ADAM molecule. The distance between the center of the HVR (Tyr575) and the catalytic zinc ion is about 3.5 nm. (B) Substrate ‘X' is recognized by the ADAM HVR via binding with an associated protein ‘Y'. (C) ADAM cleaves substrate ‘X' in trans via binding with an associated protein ‘Y'.
Figure 6
Comparison of the VAP1 and ADAM10 D/C domains. (A) Superimposition of the Da-domains of ADAM10 and VAP1. The Ds/Da/Cw-domains and the H7 helix of VAP1 and those of ADAM10 are shown in blue and red, respectively. The Ch-domains of VAP1 and ADAM10 are shown in cyan and pink, respectively. The arrow indicates the pivotal point between the Cw- and Ch-domains. Bound Ca2+ ions in VAP1 are shown as black spheres. (B) Ribbon representation of the Ch-domain of VAP1. The HVR is shown in blue. The common scaffold between the VAP1 and ADAM10 Ch-domains are shown in cyan and the segment lacking in ADAM10 is shown in light green. Disulfide bridges are indicated. (C) Ribbon representation of the Ch-domain of ADAM10. The HVR is shown in red. Disulfide brides are indicated. (D) Superimposition of the Ch-domains of VAP1 and ADAM10 in stereo with the colors as in (B, C). The N- and C-termini of the Ch-domains are indicated. (E) Structure-based alignments of VAP1, bovine ADAM10 (cADAM10), human ADAM17 (hADAM17) and S. pombe Mde10 (Mde10) Cw/Ch-domains. Secondary structures and the disulfide bridges are represented schematically. The HVR sequences and the missing segment in the ADAM10 structure are boxed in blue and green, respectively.
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