Crystal structures of archaemetzincin reveal a moldable substrate-binding site

Abstract

Background: Archaemetzincins are metalloproteases occurring in archaea and some mammalia. They are distinct from all the other metzincins by their extended active site consensus sequence HEXXHXXGXXHCX(4)CXMX(17)CXXC featuring four conserved cysteine residues. Very little is known about their biological importance and structure-function relationships.

Principal findings: Here we present three crystal structures of the archaemetzincin AfAmzA (Uniprot O29917) from Archaeoglobus fulgidus, revealing a metzincin architecture featuring a zinc finger-like structural element involving the conserved cysteines of the consensus motif. The active sites in all three structures are occluded to different extents rendering the enzymes proteolytically inactive against a large variety of tested substrates. Owing to the different ligand binding there are significant differences in active site architecture, revealing a large flexibility of the loops covering the active site cleft.

Conclusions: The crystal structures of AfAmzA provide the structural basis for the lack of activity in standard proteolytic assays and imply a triggered activity onset upon opening of the active site cleft.

Figure 1. Structure of AfAmzA.

Overall structure of AfAmzA in cartoon representation. The N-terminal domain (NTD) is colored in slate, the active site helix α2 in orange and the C-terminal domain (CTD) in green. The N- and C-termini, the edge strand β4 (cyan), the backing helix α1, the S-loop (yellow), the bulge edge segment (red), the S1′-wall forming segment (magenta) and the specificity loop (purple) are labeled. The residues involved in zinc ion binding, the catalytic base and the structurally important methionine are shown as sticks and the zinc ions as spheres.

Figure 2. Structural alignment of archaemetzincins and metzincins.

(A) Superposition of the three known AmzA structures AfAmzA (yellow), MkAmzA (green) and MlAmzA (slate). (B) Close-up view of the Cys₄ zinc finger domains of all three AmzA structures from (A). (D) Superposition of AfAmzA (yellow) with selected metzincins Bap1 (orange), H2-proteinase (grey), acutolysin A (red) and ADAM33 (blue). In place of a Cys₄ zinc finger found in AfAmzA (yellow), the superposed metzincins exhibit two disulfide bonds as shown in (C) using the example of Bap1 (orange). Individual cysteine residues are labeled in accordance with the amino acid sequence of AfAmzA (yellow) in (B) and Bap1 (orange) in (C), respectively. Important secondary structure elements are labeled.

Figure 3. Multiple sequence alignment of the amino acid sequences of AfAmzA with other archaemetzincins and metzincins.

Archaemetzincins from M. kandleri (AMZA_METKA), M. labreanum (AMZA_METLZ), H. sapiens (AMZ2_HUMAN), non-catalytic archaemetzincin from M. xanthus (Q50888_MYXXA) and metzincins H. sapiens ADAM33 (ADA33_HUMAN), P. flavoviridis H2-proteinase (VMHR2_PROFL), B. asper Bap1 (VMBP1_BOTAS) and A. acutus acutolysin A (VMACA_DEIAC). Sequences were aligned using Chimera , and visualized with ESPript . 3₁₀-Helices are indicated by η, β-turns by TT. Conserved residues in all sequences are highlighted in red. Similar sequences are in red letters, orange color indicates residues similar in each group (1, 2 or 3) but significantly different from the other groups.

Figure 4. Structural differences between NHis-AfAmzA and nat-AfAmzA.

Simulated annealing F_o–F_c omit map of (A) the N-terminus of a symmetry-related molecule (Gly-3*-Ser-2*) in NHis-AfAmzA, (B) a malonate molecule (MLI) in nat-AfAmzA::malonate and (C) a citrate molecule (FLC) in nat-AfAmzA::citrate, contoured at 2σ level. The maps were generated using phenix.omit_map and converted to the ccp4 format with FFT (V6.1) . Important residues are shown as sticks. The zinc ions and water molecules are shown as grey and red spheres, respectively. (D) Surface representation of NHis-AfAmzA active site cleft with the N-terminus of a symmetry related molecule bound to the catalytic zinc ion (primed site in closed conformation). (E) Surface representation of nat-AfAmzA active site cleft with the zinc-bound malonate molecule (MLI; primed site in open conformation). Important residues and the catalytic water molecule (H₂O_cat) are labeled. The surface is transparent to allow a view on the residues involved in zinc ion and ligand binding.

Figure 5. The two conformations of AfAmzAs substrate-binding site.

Superposition of NHis-AfAmzA (yellow) and nat-AfAmzA (green) substrate binding site. Changes in the position of side chains in the bulge edge segment (Met78, Phe80,82), side chains and the main chain of the S1′-wall forming segment (Phe136, Asn138) and in the position of the catalytic water molecule (H₂O_cat) are indicated by the arrows.