Architecture and function of metallopeptidase catalytic domains

Abstract

The cleavage of peptide bonds by metallopeptidases (MPs) is essential for life. These ubiquitous enzymes participate in all major physiological processes, and so their deregulation leads to diseases ranging from cancer and metastasis, inflammation, and microbial infection to neurological insults and cardiovascular disorders. MPs cleave their substrates without a covalent intermediate in a single-step reaction involving a solvent molecule, a general base/acid, and a mono- or dinuclear catalytic metal site. Most monometallic MPs comprise a short metal-binding motif (HEXXH), which includes two metal-binding histidines and a general base/acid glutamate, and they are grouped into the zincin tribe of MPs. The latter divides mainly into the gluzincin and metzincin clans. Metzincins consist of globular ∼ 130-270-residue catalytic domains, which are usually preceded by N-terminal pro-segments, typically required for folding and latency maintenance. The catalytic domains are often followed by C-terminal domains for substrate recognition and other protein-protein interactions, anchoring to membranes, oligomerization, and compartmentalization. Metzincin catalytic domains consist of a structurally conserved N-terminal subdomain spanning a five-stranded β-sheet, a backing helix, and an active-site helix. The latter contains most of the metal-binding motif, which is here characteristically extended to HEXXHXXGXX(H,D). Downstream C-terminal subdomains are generally shorter, differ more among metzincins, and mainly share a conserved loop--the Met-turn--and a C-terminal helix. The accumulated structural data from more than 300 deposited structures of the 12 currently characterized metzincin families reviewed here provide detailed knowledge of the molecular features of their catalytic domains, help in our understanding of their working mechanisms, and form the basis for the design of novel drugs.

Figure 1

Classification of mononuclear MPs. Within the MP class, mononuclear MPs are a subclass that is divided into tribes currently characterized at the structural level: inverzincins, zincins, relatives of hydrogenase maturating factor (HybD), LAS MPs, and αβα-exopeptidases. These tribes subdivide into clans, which in turn give rise to families. The metzincins are depicted and framed in blue. Metal-binding residues are shown in green, general base/acid residues in magenta, and residues/molecules occupying the position of the Met-turn or Ser/Gly-turn beneath the metal site are in orange. Other residues engaged in substrate binding, stabilization of the reaction intermediate, and/or catalysis are further shown in black, except for X, which stands for any residue and is here only used as a spacer within motifs. Within gluzincins, Z = A/F/S/G/T. Variability within motifs was considered if present only in more than one case. *Leishmanolysins have an extra domain inserted between the glycine and the third histidine metal ligand of the metzincin extended zinc-binding signature. **Igalysins have an extra glycine residue inserted just before the glycine of the metzincin signature. ***Dipeptidyl peptidase III has an extra residue inserted between the general base/acid glutamate and the second metal-binding histidine. This classification supersedes previous schemes.,

Figure 2

Generally accepted catalytic mechanism of monometallic MPs. The catalytic solvent molecule is bound first to the catalytic metal ion (white sphere) and the general base/acid in the active site in the absence of a peptidic substrate (I). Once the substrate is accommodated in the cleft and the Michaelis complex is formed (II), the polarized solvent molecule attacks the scissile carbonyl group, which leads to the tetrahedral reaction intermediate (III). The latter resolves in scissile bond breakage and double proton transfer to the newly formed α-amino group to render a double-product complex (IV).

Figure 3

Structure of metzincin catalytic domains. Ribbon-type Richardson plots of a representative member of each of the 12 structurally characterized metzincin families (see the sections on “Common structural features of metzincins” and “Metzincin family-specific features,” and Fig. 1) in standard orientation. Depicted are astacin (AST; PDB 1AST; 200 residues), aeruginolysin (SER; PDB 1KAP; 220 residues), leishmanolysin (LEI; PDB 1LML; 213 residues), snapalysin (SNA; PDB 1C7K; 132 residues), human neutrophil collagenase (MMP; PDB 1JAN; 164 residues), adamalysin II (ADA; PDB 1IAG; 203 residues), ulilysin (PAP; PDB 2CKI; 262 residues), Methanopyrus kandleri archaemetzincin AmzA (AMZ; PDB 2X7M; 173 residues), igalysin BACOVA_0063 (IGA; PDB 3P1V; 272 residues), toxilysin EcxA (TOX; PDB 4L63; 258 residues), fragilysin-3 (FRA; PDB 3P24; 188 residues), and cholerilysin StcE (CHO; PDB 3UJZ; 258 residues). The common β-strands and α-helices are shown in purple and cyan, respectively (see Fig. 4 for their nomenclature). Unique regular secondary structure elements of each family are in white. The N-and C-termini are labeled and the side chains of the zinc-binding histidines/aspartates (brown), general base/acid glutamates (pink), Met-turn methionines (blue), disulfide-linked cysteines (yellow), family-specific residues (red), and zinc-binding or substrate-stabilizing tyrosines (where present; in tan) are displayed as sticks. Preceding, inserted, and following (sub-)domains have been omitted for clarity. Catalytic metal ions—mostly zinc—are shown as magenta spheres, calcium cations as red spheres, and the potassium cation of IGA is in blue. Orange arrows pinpoint the anchor points for a domain inserted in LEI between the glycine of the extended consensus sequence and the third metal-binding histidine, PAP is unique in having an asparagine instead of the glycine. Black arrows pinpoint two disordered loops in TOX.

Figure 4

Topology of metzincin catalytic domains. Scheme showing the regular secondary structure elements (helices as rods, strands as arrows) of each metzincin prototype depicted for its structure in Figure 3. The common elements are in pink (strands) and turquoise (helices) and labeled (βI-βV and αA-αC). Hydrogen bonds and metal–ligand bonds are shown as dashed lines, disulfide bonds as orange solid lines. Disordered segments or points of insertion of extra domains are characterized by blue dashes. Family-specific residues are in red, B stands for bulky, hydrophobic residues, X for any residue.

Figure 5

Superposition of common structural elements. (A) Stereo image depicting the superposition of the 12 reference structures only around the common structural elements, β-sheet strands βI (turquoise), βII (brown), βIII (orange), βIV (yellow), and βV (blue), as well as backing helices αA (pink), active-site helices αB (white), and C-terminal helices αC (red). (B) Superposition of the active-site helix and the downstream chain segment until the family-specific residue plus the Met-turn of AST (white), ADA (gold), SER (pink), LEI (purple), MMP (turquoise), and SNA (green) in two orthogonal views. The catalytic metal ions have been omitted for clarity. (C) Same as (B) but showing AMZ (blue), FRA (red), IGA (orange), CHO (tan), PAP (salmon), and TOX (sienna).