Fig 1.
Schematic representations of the primary structures of ChiW proteins.
ChiW denotes the full-length protein of Paenibacillus sp. str. FPU-7 ChiW; ChiW-SLHd (CBM-54, Ig-1, Cat-1, Ig-2 and Cat-2 domains), ChiW-CD (Cat-1, Ig-2 and Cat-2 domains) and CBM-54 of ChiW (CBM-54 domain) denote the truncated versions of ChiW. Boxes denote annotated structural elements. The numbers indicate the amino acid residue numbers. ChiW is specifically cleaved between Asn282 and Ser283. The position is pointed out as the cleavage site. Glu691 and Glu1177 are predicted to be catalytic residues and act as generic acids.
Table 1.
Data collection and refinement statistics for ChiW structures.
Fig 2.
Overall structures of ChiW-SLHd.
(A and B) The structures are represented as a ribbon model (A) and a molecular surface model (B). The structure is divided into three regions and six domains, a CBM-54 domain, a GS-rich loop and a catalytic region (Ig-1, Cat-1, Ig-2 and Cat-2 domains), with overall dimensions of approximately 130 × 120 × 70 Å.
Fig 3.
Close-up views of the structure of the catalytic region of ChiW.
(A) The overall catalytic region is shown as a ribbon model; front view (left figure) and side view (right figure). (B) Left figure, top view of the ChiW Cat-1 domain. The secondary structure elements (α-helices and β-strands) of the β8/α7-barrel are indicated by numbers. Right figure, side view of the ChiW Cat-1 domain. The secondary structure elements (α-helices and β-strands) of the extra subdomains (ID-1 and ID-2) are indicated by numbers. (C) Structural comparison of Cat-1 (orange) and Cat-2 (cyan). The three-dimensional structures are very similar (rmsd = 1.0 Å) with high amino acid sequence similarity (56% identity). (D) A comparison of the catalytic cleft of ChiW Cat-1 (orange ribbon model), and Cat-2 (cyan ribbon model). The amino acid residues (stick models), Trp568/Trp1055, Trp652/Trp1138, Asp687/Asp1173, Asp689/Asp1175, Glu691/Glu1177, Tyr766/Tyr1252, Trp772/Trp1258, Trp905/Trp1396, are near identical in the two active clefts. The amino acid residues (stick models), Lys573/Asn1060, Asp610/Glu1097, Ile613/Phe1098, Lys620/Gln1106, Trp698/Phe1184, Lys743/Pro1229 and Leu790/Phe1278, are located at the edge of the clefts. (E) Structural comparison of ChiW (orange) and BaChiA1CD (PDB ID: 1ITX; cyan). Their β8/α7-barrel core structures and ID-1 are similar, but significant differences exist in ID-2. The ChiW ID-2 forms the wall of the cleft, whereas the identical area of BaChiA1CD is positioned outside of the cleft.
Fig 4.
(A and B) The Ig-1 (A), and Ig-2 (B) domains are shown as ribbon models. (C) Structural comparison of ChiW-CD (left figure) and Micromonospora viridifaciens sialidase (PDB ID: 2BQ9) (right figure). The Ig-2 domain and the bacterial sialidase linker domain are very similar structures (rmsd, 2.1 Å).
Fig 5.
(A and B) The reaction product is located in the binding cleft of ChiW Cat-1. Electron density of the reaction product (GlcNAc)2 (stick model: carbon atoms, yellow; oxygen atoms, red; and nitrogen atoms, blue) in the omit (Fo–Fc) map (cyan) (A) was calculated without the substrate and contoured at the 3.0-σ level. The ChiW residues (B) that interact with the product are represented by pink stick models (oxygen atoms, red and nitrogen atoms, blue). The numbers (−2 and −1) indicate the subsite positions. Other numbers indicate the amino acid residues. (C) The comparison of the catalytic cleft residues at the 5 subsites (−3 to +2) of ChiW Cat-1 (pink), Cat-2 (green) and SerChiA (cyan). The numbers indicate the amino acid residues of ChiW Cat-1.
Fig 6.
Surface structure of ChiW catalytic domain.
(A-C) The surface models of SmChiA (A), Cat-1 of ChiW (B) and BaChiA1CD (C). The side chains of the aromatic residues (Trp, Phe and Tyr) are shown in magenta. Electrostatic potentials at pH 7 are also represented. The +8 to –8 kT/e potential isocontours are shown as blue to red surfaces, respectively. ChiW catalytic domains have characteristic subdomains (ID-1 and ID-2) that form deep and short clefts surrounded by negative charges.
Fig 7.
Properties of the CBM-54 of ChiW.
(A) The CBM-54 structure is shown as a ribbon model, front view (left figure) and top view (right figure). The right-handed β-helix structure consists of three distorted faces (SB1, SB2 and SB3). The cleavage site is located at one-third of the total distance from the bottom and between the blue and cyan ribbon models. (B) A close-up structure of the limited proteolysis site. The nearest residues of the cleavage site are shown as stick models (carbon atoms, yellow; oxygen atoms, red; and nitrogen atoms, blue), which form hydrogen bond networks. (C) The amino acid sequence of the cleavage site. Identical amino acid residues among the conserved proteins are indicated by asterisks, whereas colon and period characters indicate highly conserved residues. The sequence motif can be recognized as G-G-G-X1-X2-S-X3-X4 (the cleavage site is between X2 and S; X1: anything; X2: N, Q, D, or H; X3: V or I; X4: H, K, L, N, Y, or V). (D) SDS-PAGE analysis of the bound CBM-54 of ChiW (10 μg) with insoluble polysaccharides (2 mg). Protein bands were stained with CBB R-250. Lane M, molecular mass standards (25, 20, 15 and 10 kDa); lanes 1 and 2, bound or unbound CBM-54 with chitin; lanes 3 and 4, with chitosan; lanes 5 and 6, with β-1,3-glucan; lanes 7 and 8, with xylan; lanes 9 and 10, with cellulose; lanes 11 and 12 with no polysaccharide; lane 13, the purified CBM-54 domain (0.25 μg). The upper arrow corresponds to a CBM-54 domain fragment of ~18 kDa and the lower arrow to a ~9 kDa fragment. (E, F) The surface models of CBM-54 ChiW. The side chains of the aromatic residues (Trp, Phe and Tyr) are shown in magenta. The molecular surface has no distinctive cleft or patch surrounded by aromatic residues. Electrostatic potentials at pH 7 are also presented (F). The +12 to –12 kT/e potential isocontours are shown as blue to red surfaces, respectively. The negatively charged patch exists on the central part of the β-helix structure and is indicated by a black arrow.