Crystal structure of an intracellular protease from Pyrococcus horikoshii at 2-A resolution

Abstract

The intracellular protease from Pyrococcus horikoshii (PH1704) and PfpI from Pyrococcus furiosus are members of a class of intracellular proteases that have no sequence homology to any other known protease family. We report the crystal structure of PH1704 at 2.0-A resolution. The protease is tentatively identified as a cysteine protease based on the presence of cysteine (residue 100) in a nucleophile elbow motif. In the crystal, PH1704 forms a hexameric ring structure, and the active sites are formed at the interfaces between three pairs of monomers.

Figure 1

Sequence alignment of PH1704 homologs. Abbreviations are: Pho, P. horikoshii; Pfu, P. furiosus (NCBI access ID, Q51732; percentage identity, 90%); Ape, Aeopyrum pernix (BAA79274.1, 61%); Afu, Archaeoglobus fulgidus (2649300, 53%); Eco, E. coli (P45470, 47%); Bsu, Bacillus subtilis (BAA23403, 43%); Sau, Staphylococcus aureus (Q53719, 39%); Aae, Aquifex aeolicus (2983230, 42%). Secondary structure analysis is based on the crystal structure of PH1704. “H” and “S” refer to helix and strand, respectively.

Figure 2

Electrophoretic migration of PH1704 and gelatin protease assay. (A) 10% SDS-PAGE gel of purified PH1704. Lane 1, molecular markers; lane 2, PH 1704 boiled for 20 min in sample buffer; lane 3, PH1704 in sample buffer without boiling. (B) Gelatin overlay of 8% SDS-PAGE gel of purified PH1704. Proteolytic activity is manifested by cleared zones in the dark background. Molecular weights indicated are based on molecular markers in gelatin-SDS-PAGE gel.

Figure 3

Structural features of PH1704. (A) Ribbon diagram of PH1704. “H” and “S” refer to helix and strand, respectively. Green, gold, and blue code for β-strand, α-helix, and 3₁₀ helix, respectively. (B) The superposition of the Cα trace of amidotransferase domain of GMP synthetase (green) and that of PH1704 (red). The three members of the catalytic triad, Cys-86, His-181, and Glu-183 of the amidotransferase and Cys-100 and His-101 of PH1704 are labeled. (C) The “nucleophile elbows” in PH1704 (Left) and amidotransferase domain of GMP synthetase (Right).

Figure 4

Hexameric structures and putative catalytic sites of PH1704. (A) Ribbon diagram of the hexamer with the threefold NCS axis (perpendicular to the plane of paper) and the three twofold axes (in the plane of paper) are shown. Axis 1 coincides with a crystallographic symmetry axis. Axis 2 and 3 are NCS symmetry axes. (B) Surface representation of the hexamer color-coded by electrostatic potential. The diameter of the center opening is ≈24 Å. (C) Ball and stick model of the putative active site. All of the residues surrounding Cys-100 are shown. (D) A stereogram of the proposed catalytical triad and Glu-15 with the corresponding solvent-flattened experimental map. The distances shown are in Å.