Table 1.
X-ray diffraction data collection and refinement statistics.
Fig 1.
Expression and refolding of Pro21717-PCD.
(A) Schematic diagrams of the Pro21717 domain structures. Pro21717 includes a precursor sequence (expressed from a 2,127-bp coding sequence) composed of four domains: a signal peptide, an N-terminal pro-sequence, a catalytic domain (CD), and two pre-peptidase C-terminal domains (PD1 and PD2). Pro21717 activation requires self-cleavage via its own catalytic domain to unmask the N-terminal pro-sequence. (B) SDS-PAGE analysis of the recombinant Pro21717 protease. Lane M, protein size marker; lane 1, Pro21717-PCD in inclusion bodies after washing; lane 2, self-cleaved Pro21717-CD after 48 h of refolding and purification through gel-filtration chromatography. (C) Time courses of cell densities (open circle) and protease activities (black square). Cells were grown in a 5-L jar fermenter using 2 L of modified R medium, with IPTG induction at a DCW of 4.
Fig 2.
Multiple-sequence alignment and crystal structure of Pro21717-CD.
(A) Structure-based alignment of the amino acid sequences of serine proteases from Pseudoalteromonas arctica PAMC 21717 (Pro21717), Dichelobacter nodosus (AprV2), Dichelobacter nodosus (BprB), Bacillus licheniformis (subtilisin Carlsberg), and Bacillus sp. strain AK1 (AkP), using ClustalX [29]. Residues constituting the catalytic triad (Asp185, His244, and Ser425) are indicated by filled circles. Cysteine residues (Cys207–Cys254, Cys296–Cys333, and Cys439–Cys442), joined by red-dotted lines, represent intramolecular disulfide bond formation. (B) Ribbon diagram of Pro21717-CD showing active site residues and secondary structure elements. The 2Fo–Fc electron density map (contoured at 1σ) around the co-purified peptide is shown in blue. The bound calcium ions are presented with yellow balls, and disulfide bonds are shown with yellow sticks.
Fig 3.
Structural comparison of Pro21717-CD with other serine proteases.
(A) Superimposition of Pro21717-CD (orange color) onto AprB2 protease (an extracellular protease from Dichelobacter nodosus; PDB code: 3LPC; gray color) revealed that Pro21717-CD adopts a novel conformation in the capping-loop region of the active site. (B) Structural comparison of Pro21717-CD (orange) and subtilisin Carlsberg (green) complexed with the protease inhibitor OMTKY3 (cyan). Notably, the conformation of the co-purified peptide in the active site of Pro21717-CD is identical to that of the residues (Ala15[P4], Cys16[P3], and Thr17[P2]) in the subtilisin Carlsberg inhibitor (OMTKY3) (right panel).
Fig 4.
Substrate-binding mode of Pro21717-CD.
(A) Stereoview of the substrate-binding site in Pro21717-CD. The bound, co-purified peptide (magenta) facilitated the characterization of the substrate-binding pocket and the substrate-binding mode of Pro21717. The side chains of the catalytic triad (Asp185, His244, and Ser425) and the residues in the nearby S2 and S4 pockets are presented as a stick model. (B) Ribbon diagram showing superposition of Pro21717-CD (orange) and Carlsberg subtilisin (cyan) at the active site. The A–A–P–F peptide in Pro21717-CD and the A–C–T–L peptide in subtilisin Carlsberg are shown as sticks. The catalytic triad residues Asp185, His244, and Ser425 (Pro21717-CD) and Asp32, His64 and Ser221 (subtilisin Carlsberg) are also shown as sticks. (C) Schematic view of the A–A–P–F peptide-binding mode in Pro21717-CD. The key hydrogen and hydrophobic interactions involved in peptide binding are presented using Ligplot.
Table 2.
Proteolytic activities of Pro21717-CD on various substrates.
Fig 5.
Activity and stability assays for Pro21717-CD.
(A–D) Thermal stability and the effect of pH on Pro21717-CD activity (black circles), compared with those for subtilisin Carlsberg (open circles). The optimal temperature (A) and pH (C) for activity were measured, and the thermal stability of Pro21717-CD and subtilisin Carlsberg (B), and the activity in various pH values (D) were examined as described in the Materials and Methods section. Briefly, the thermal melting profile of Pro21717-CD and subtilisin Carlsberg were recorded using CD spectroscopy. The signal at 220 nm was recorded while the temperature was increased from 5 to 95°C at intervals of 2.5°C. (E) The effects of metal ions and detergents on Pro21717-CD activity and stability. (F) Effect of recombinant protease Pro21717-CD on milk/blood/ink stain removal. The washing test was performed at 15°C in LAS detergent in water (control) or LAS detergent containing 13 mg of Pro21717-CD in water (reaction).