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• W2065402743 abstract "Metalloproteases are involved in metabolic regulations across all kingdoms of life by either extensive protein degradation or very selective hydrolysis of specific peptide bonds.1 Metzincins form a subclan of Metalloproteases that are mostly zinc-dependent peptide-bond hydrolases. Families within the metzincin clan comprise, besides others, astacins, ADAMS, serralysins, and archaemetzincins.2-4 They all possess the zinc binding consensus sequence HEXXHXXGXXH/D with a conserved glutamate residue as the catalytic base and three histidine (or two histidine and one aspartate) residues coordinating the Zn2+ ion. The name giving, conserved methionine residue is located 6–53 amino acids downstream of this core motif.5 Archaemetzincins were first identified in archaeal genomes and based on sequence similarity, predicted to be zinc metalloproteases. In addition to the zinc binding consensus sequence of the metzincins, an extended core motif HEXXHXXGX3CX4CXMX17CXXC that contains four strictly conserved cysteine residues is observed for all archaemetzincins. The archaeal sequences show similarity to some mammalian sequences, that have been named archaemetzincin-1 and archaemetzincin-2 and also a bacterial sequence from Myxococcus xanthus, where the active site glutamate has been replaced by a glutamine.3 Human archaemetzincin-1 and archaemetzincin-2 were shown to have aminopeptidase activity.6 On the other hand, archaemetzincin from Mycococcus xanthus presents an interesting example of a protein with the metzincin fold that has lost its ability to hydrolyze peptide bonds and has evolved into a zinc-binding transcription factor with the metal fulfilling only a structural role.7 Seven of the known metzincin families have been structurally characterized to date. Those are the astacins, ADAMs/adamalysins/reprolysins, serralysins, matrix metalloproteases, snapalysins, leishmanolyisns, and pappalysins.4 With archaemetzincin from Methanopyrus kandleri, we describe for the first time the crystal structure of a member of the archaemetzincin family. Interestingly, we found in addition to the zinc ion in the active site, a second zinc ion coordinated by the four cysteine residues from the consensus sequence, thus resembling a zinc-finger motif. A synthetic DNA fragment optimized for E.coli codon usage (MrGene, Regensburg, Germany) encoding the full length AmzA from M. kandleri (accession number Q8TXW1) was subcloned into pET28-(a) Vector (Novagen) with a thrombin cleavable N-terminal His6-tag. The plasmid was transformed into E. coli BL21 (DE3) cells. Cells were grown in Luria-Bertani broth at 37°C to OD600∼1. After induction with 0.1 mM IPTG, protein was expressed over night at 20°C. Cells were disrupted by sonication in lysis buffer (300 mM NaCl, 25 mM Tris HCl pH 8.0, 10 mM imidazole). After centrifugation at 20,000g, the supernatant was purified by Ni-NTA affinity column (Ni-Sepharose, Qiagen, Germany). The eluted protein was treated with thrombin protease (1U per mg target protein) overnight at 20°C and passed again over Ni-NTA resin followed by size exclusion chromatography (Superdex S75). The protein was concentrated to 10 mg/mL and stored in 20 mM Tris-HCl pH 7.5, 300 mM NaCl, 4 mM DTT at −80°C. Crystals for the high resolution data were received from protein without cleaved His6-tag, as there was no difference in crystal quality. Crystals belonging to the space group P212121 were grown at 20°C by vapor diffusion in hanging drops mixing 1 μL of protein with 1 μL reservoir solution containing 0.04 M citric acid/0.06 M Bis-Tris Propane pH 7.6 and 20% PEG3350. Crystals were cryoprotected by raising the PEG3350 concentration of the crystallization solution to 37.5% and flash cooled in liquid nitrogen. The zinc K-edge peak diffraction data for phasing was collected at beamline X06SA, the higher resolution data set usend for refinement at beamline X06DA at the Swiss Light Source, Paul Scherrer Institute, Villigen. Processing was performed using the program XDS.8 The structure was solved by single wavelength anomalous diffraction (SAD). Zinc positions were found with SHELXD, the initial model was build by SHELXE9 and ARP/wARP.10 Structure refinement and model building was performed using iterative cycles of phenix.refine11 and COOT.12 The final model consists of residues 1–173 and 7 residues belonging to the N-terminal His-Tag. Residues 174–175 are disordered. The coordinates were deposited in the RCSB Protein Data Bank13 with accession number 2 × 7 M. The chemical identities of the two Zn2+ ions were established by an anomalous difference map. Data collection and refinement statistics are listed in Table I. Figures were prepared with PYMOL (http://www.pymol.org). The overall structure model shows the classical metzincin architecture [Fig. 1(A)]; namely the so-called “upper N-terminal” and “lower C-terminal” domain with respect to the active site cleft.3 The catalytic zinc ion is located at the bottom of the active site groove. The N-terminal domain features a five-stranded β-sheet with Richardson15 topology (−1X 2X 2 −1), where all strands, except βIV, are parallel to each other. β-strand IV limits the active site at one side and is positioned for fixing a substrate in an extended conformation via backbone interactions. Furthermore, the upper N-terminal domain possesses the α helices A and B. An additional α helix αA′ comprising residues 56–65 is inserted between strands βII and βIII. This helix is otherwise only observed in the ADAMS family of the metzincin clan. αB is the active site helix and composes the first half of the zinc-binding metzincin motif (HEXXHXXGXXH). Three residues after the second zinc-ligand histidine the polypeptide chain undergoes a sharp downward turn refereed by a glycine residue that is highly conserved amongst the metzincins and starting the lower domain. The third zinc-ligand (His135) follows this turn two residues later. The lower domain features two conserved secondary structure elements, the helix αC at the end of the polypeptide chain and the well conserved 1,4-β-turn containing a methionine residue. (A) Ribbon cartoon of the AmzA structure from Methanopyrus kandleri. (B) Close view on the active site with the three zinc coordinating histidine residues (His125, His129, and His135), the nucleophilic water (red), and the two phenylalanines blocking the active site cleft (Phe88 and Phe90). All archaemetzincins possess four conserved cysteine residues additionally to the core metzincin motif, such that the sequence fingerprint reads HEXXHXXGX3 CX4CXMX17CXXC, comprising the metzincins' methionine and the four cysteines. The latter are located in the lower C-terminal domain of the protein and coordinate a Zn2+ ion in a tetrahedral geometry. The distances between Zn2+ and the thiol groups vary between 2.29 and 2.39 Å and are in good agreement with literature values where the reported ideal distance is 2.34 Å (CSD16 version 5.31). Two aromatic residues, Phe88 and Phe90, are positioned to block the putative S′ sites of the active site cleft [Fig. 1(B)] if an antiparallel orientation of the substrate to strand ßIV is assumed, as observed in other metzincins.17 These two hydrophobic residues are conserved among most archaemetzincins, an exception being CarG from Mycococcus xanthus (see Fig. 2). As the active site cleft is sterically restrained, binding of longer peptides seems unlikely. This finding could be in agreement with the aminopeptidase activity that has been reported by Diaz et al.6 but would then require a parallel binding mode to strand ßIV for substrates longer than two amino acids. Multiple sequence alignment of AmzA homologs. Highly conserved residues, namely the HEXXHXXGX3CX4CXMX17CXXC core motif, are printed in white on a red background. Residues with conservative substitutions are indicated with red letters. Alignment tools used are multialign18 and ESPript.19, 20 Surprisingly, under all tested conditions we were not able to detect any endo- or exopeptidase activity. Assays were performed with different substrates, among them resorufin labeled casein, p-nitroanilide (pNA), amido-methylcoumarin (AMC) (one to three amino acids in length), and Hippuryl-aminoacid substrates, at temperatures ranging from 20°C to 90°C in low and high salt buffers21 under different pH conditions. Noteworthy, AmzA is also inactive against the aminopeptidase substrates Arg/Lys-pNA, and Arg/Lys-AMC, which were reported to be turned over by the human homolog archaemetzincin-2.6 This complete absence of activity is quite unexpected, as all the features of a functional metzincin protease are present. It is possible however, that this archaemetzincin is highly specific and does not recognize the substrates employed in our study. With respect to the lack of activity, a conspicuous feature of the active site cleft is the restriction imposed by the aromatic side-chains of Phe88 and Phe90 mentioned above. To remove the steric hindrance at the S′ sites, these two residues were mutated to alanines. The double mutant FF88,90AA behaved with respect to expression and purification similar as the wildtype, but remained inactive in proteolytic assays. The function of the zinc-Cys(4) cluster is not clear. It might just have a structural role, as AmzA shows high structural similarity to Acutolysin A from the venom of the chinese moccasin snake (PDB ID: 1BSW), where at the equivalent position of the zinc-Cys(4) cluster two disulfide bridges are formed.22 An other possibility could be, that the zinc-Cys(4) cluster acts as a regulatory element similar to a redox switch.14, 23 Hypothetically, the oxidation of the cysteine residues to cystines might lead to release of the zinc ion, causing a conformational rearrangement and thereby opening up the active site cleft. The authors thank Dr. Anselm E. Oberholzer, Christian Trachsel and Dr. Jürg Hauser for fruitful discussions and suggestions." @default.
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• W2065402743 title "Crystal structure of archaemetzincin amza from Methanopyrus kandleri at 1.5 Å resolution" @default.
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