New mini- zincin structures provide a minimal scaffold for members of this metallopeptidase superfamily

Abstract

Background: The Acel_2062 protein from Acidothermus cellulolyticus is a protein of unknown function. Initial sequence analysis predicted that it was a metallopeptidase from the presence of a motif conserved amongst the Asp-zincins, which are peptidases that contain a single, catalytic zinc ion ligated by the histidines and aspartic acid within the motif (HEXXHXXGXXD). The Acel_2062 protein was chosen by the Joint Center for Structural Genomics for crystal structure determination to explore novel protein sequence space and structure-based function annotation.

Results: The crystal structure confirmed that the Acel_2062 protein consisted of a single, zincin-like metallopeptidase-like domain. The Met-turn, a structural feature thought to be important for a Met-zincin because it stabilizes the active site, is absent, and its stabilizing role may have been conferred to the C-terminal Tyr113. In our crystallographic model there are two molecules in the asymmetric unit and from size-exclusion chromatography, the protein dimerizes in solution. A water molecule is present in the putative zinc-binding site in one monomer, which is replaced by one of two observed conformations of His95 in the other.

Conclusions: The Acel_2062 protein is structurally related to the zincins. It contains the minimum structural features of a member of this protein superfamily, and can be described as a "mini- zincin". There is a striking parallel with the structure of a mini-Glu-zincin, which represents the minimum structure of a Glu-zincin (a metallopeptidase in which the third zinc ligand is a glutamic acid). Rather than being an ancestral state, phylogenetic analysis suggests that the mini-zincins are derived from larger proteins.

Figure 1

Crystal structure. The crystal structure of the Acel_2062 protein from Acidothermus cellulolyticus, shown in magenta, reveals a single zincin-like domain. The putative zinc ligands (His95, His99 and Asp105), the putative catalytic Glu96, and Tyr113 are shown as sticks.

Figure 2

Sequence alignment of the Acel_2062 protein and a selection of its homologues. The UniProt accession and the range of the peptidase domain are shown on the left. The zincin motif is boxed in red. Conserved residues shown in white text highlighted in red. Key to sequences (ordered locus name, species): A0LWM4[UniProt:A0LWM4] (Acel_2062, Acidothermus cellulolyticus), F2NA85[UniProt:F2NA85] (Corgl_0144, Coriobacterium glomerans), C0ACH6[UniProt:C0ACH6] (ObacDRAFT_6101, Diplosphaera colitermitum), E6JBL2[UniProt:E6JBL2] (ES5_13138, Dietzia cinnamea), A5P6T1[UniProt:A5P6T1] (ED21_26213, Erythrobacter sp. SD-21), A1B0F0[UniProt:A1B0F0] (Pden_0883, Paracoccus denitrificans), E8L598[UniProt:E8L598] (Met49242DRAFT_2641, Methylocystis sp. ATCC 49242), Q1PUY8[UniProt:Q1PUY8] (kustc0300, Candidatus Kuenenia stuttgartiensis), F8CQR3[UniProt:F8CQR3] (LILAB_30480, Myxococcus fulvus), D0LZA2[UniProt:D0LZA2] (Hoch_3865, Haliangium ochraceu), H8MHL2[UniProt:H8MHL2] (COCOR_02006, Corallococcus coralloides), Q08VI2[UniProt: Q08VI2] (STAUR_2801, Stigmatella aurantiaca), E2T6Y2[UniProt:E2T6Y2] (TMBG_02375, Mycobacterium tuberculosis), Q5SLR6[UniProt:Q5SLR6] (TTHA0227, Thermus thermophilus), F2NR95[UniProt:F2NR95] (Marky_2224, Marinithermus hydrothermalis), F9MVY9[UniProt:F9MVY9] (HMPREF9130_1347, Peptoniphilus sp. oral taxon 375 str. F0436), D3PNK3[UniProt:D3PNK3] (Mrub_0627, Meiothermus ruber), B0TE39[UniProt:B0TE39] (Helmi_16090, Heliobacterium modesticaldum), E4U5S7[UniProt:E4U5S7] (Ocepr_0229, Oceanithermus profundus).

Figure 3

Electrostatic surface of the Acel_2062 protein. The electrostatic surface potential representation of the active pocket was made with a Delphi embedded JCSG script, (kindly provided by Qingping Xu) and displayed using Pymol. Negative electrostatic potential predominates over the entire surface of Acel_2062. The putative active site residues (Glu96 and Tyr113) and zinc ligands are (His95, His99 and Asp105) are shown in sticks. The color scale is in units of kT/e ranging from −10 to +10.

Figure 4

Superposition of the active sites of the Acel_2062 protein and Bap1 peptidase from Bothrops asper. The comparison between the active centres containing the HEEXH motif; Coot ssm superposition of the two PDB entries (3E11 and 2 W14), has been displayed using Chimera; 3E11-A monomer is shown in turquoise, B in pink, 2 W14 is shown in magenta.

Figure 5

Domain architectures for the Acel_2062 protein and its homologues. The different domain architectures for proteins containing an M94 metallopeptidase domain is shown. The UniProt identifier and source organism are given for an example of each domain architecture. Key to domains: DUF1025 is the metallopeptidase domain;TPR, tetratricopeptide repeats; TM, transmembrane region; Septum form, a domain found in proteins predicted to play a role in septum formation during cell division; S, signal peptide.

Figure 6

Superposition of structures for the Acel_2062 and TTHA0227 proteins. The structure for the Acel_2062 protein (chain A from PDB entry 3E11) is shown in magenta, and the structure for the TTHA0227 protein (chain B from 2EJQ) is shown in cyan. The putative zinc ligands (His95, His99, Asp105 and Asp108) and active site residues (Glu96 and Tyr113) are shown as sticks. Note that the terminal helices do not superimpose, and that Asp108 from the 2EJQ structure is not interacting with the other putative zinc ligands.

Figure 7

Secondary structure topologies for the Acel_2062 protein and other metallopeptidases. Helices are shown as red barrels, strands are shown as pink arrows and connecting regions of random coil and turns are shown as thin blue arrows. “N” and “C” indicate the N- and C-terminus. Numbers indicate the residue range of each structural element. The positions of the zinc ligands and catalytic Glu are indicated by the letters “H”, “D” and “E”. Some images have been rotated and/or flipped to that these active site residues are in equivalent positions. Key to structures: 3E11[PDB:3e11], mini- zincin, Acidothermus cellulolyticus; 4JIU[PDB:4jiu], mini-Glu-zincin, Pyrococcus abyssi; 3LMC[PDB:3lmc], archaelysin, Methanocorpusculum labreanum; 1KUH[PDB:1kuh], snapalysin, Streptomyces caespitosus; 2EJQ[PDB:2ejq], mini- zincin, Thermus thermophilus; 1AST[PDB:1ast], astacin, Astacus astacus.