Fig 1.
Overall structure of BbtPGRP3.
(a) Domain organization of BbtPGRP3. (b) Structure of BbtPGRP3. The secondary structure elements are numbered in the order of appearance in the primary sequence. The N and C-termini are indicated. (c) Topology diagram of BbtPGRP3. The N- and C-terminal domains are shown in cyan and green, respectively. The linker is shown in red.
Fig 2.
The N-terminal CBD of BbtPGRP3.
(a) Cartoon representation of the BbtPGRP3 N-terminal CBD. The side chains of the conserved cysteine and putative functional residues are shown, with the conserved disulfide bridges colored in yellow. (b) Superposition of BbtPGRP3 CBD against UDA (PDB 1ENM). The CBD and UDA are colored in cyan and magenta, respectively. (c) The chitin-binding activity. Purified BbtPGRP3, CBD, PGRP domain and BbtPGRP3-W53A/W55A were incubated with insoluble chitin, respectively. Protein remaining in the supernatant (Unbound) and associated with the pellet (Bound) were analyzed by SDS-PAGE and Coomassie staining. (d) 3D structure-based sequence alignment of several chitin-binding proteins in plants against the Cys41–Thr64 region of BbtPGRP3. The proteins include Amaranthus caudatus antimicrobial protein 2 (Ac-AMP2, PDB code 1MMC), hevein from rubber tree (Hevein, PDB code 1T0W), Phytolacca americana Lectin-B (PL-B_I, UniProtKB accession number Q9AVB0.1), Phytolacca americana Lectin-C (PL-C_I, PDB code 1ULK), Phytolacca americana Lectin-D (PL-D_I, PDB code 1ULM), Urtica dioica agglutinin (UDA_I, PDB code 1IQB), and wheat germ agglutinin (WGA_I, PDB code 1WGC). The chitin-binding residues are aligned. The polar and hydrophobic residues are highlighted in blue and red, respectively.
Fig 3.
The C-terminal amidase domain of BbtPGRP3.
(a) Surface representation of the BbtPGRP3 C-terminal PGRP domain. The zinc ion and its coordinating water molecule are displayed as lightblue and red spheres, respectively. The active-site residues and putative residues related to PGN-binding specificity are shown as sticks and colored in green. (b) Reactions containing RBB-labeled E. coli PGNs were incubated with 50 nM wild-type BbtPGRP3, EDTA-treated BbtPGRP3 and BbtPGRP3-C238S, respectively. Undigested PGNs were pelleted by centrifugation and the absorbance of the supernatants at 595 nm were determined. Results of activity are expressed as the percentage to the wild-type BbtPGRP3. The values are expressed as mean values ± SD of three independent experiments. All data were analyzed by t-test, and the statistical significance of the differences between experimental and a control group is shown with asterisks (**P<0.005). (c) Superposition of PGRP domain from full-length BbtPGRP3 against the refolded PGRP domain. The full-length BbtPGRP3 and refolded PGRP domain are colored in green and red, respectively. The zinc ion and its coordinating water molecule are displayed as spheres and the active-site residues are shown as sticks. (d) Multiple-sequence alignment of specificity-determining residues of PGRPs. Residues corresponding to Gly150/Tyr151 and Arg170 of BbtPGRP3 are highlighted in yellow and red, respectively. The sequences are (NCBI accession numbers codes in parentheses) Branchiostoma belcheri tsingtauense BbtPGRP3 (KR136228), Asterias rubens ArPGRP-S2a (ABB04460.1), Crassostrea gigas CgPGRP-S1S (BAG31896.1), Drosophila melanogaster DmPGRP-LB (NP_650079.1), Danio rerio DrPGLYRP2 (NP_001038631.1), Euprymna scolopes EsPGRP-1 (AAY27973.1) and Homo sapiens HsPGLYRP1 (NP_005082.1).
Fig 4.
The G/Y/R motif and amidase activity.
Docking models of (a) TCT or (b) MPP binding to the PGRP domain. TCT and MPP are colored in white, whereas the interacting residues are colored in green. Reactions containing RBB-labeled (c) E. coli PGNs or (d) S. aureus PGNs were incubated with wild-type BbtPGRP3, the R170T and G151D/Y152F mutants, respectively. Undigested PGNs were pelleted by centrifugation and the absorbance of the supernatants at 595 nm were determined. Results of activity are expressed as the percentage to the wild-type. The values are expressed as mean values ± SD of three independent experiments. All data were analyzed by t-test, and the statistical significance of the differences between experimental and a control group is shown with asterisks (*P<0.01, **P<0.005).
Fig 5.
Cooperation of the N- and C-terminal domains of BbtPGRP3.
Reactions containing RBB-labeled (a) S. aureus PGNs or (b) E. coli PGNs were incubated with 50 nM PGRP domain, BbtPGRP3 in the absence or presence of chitin and BbtPGRP3-W53A/W55A, respectively. Undigested PGNs were pelleted by centrifugation and the absorbance of the supernatants at 595 nm were determined. Results of activity are expressed as the folds to that of individual PGRP domain. The values are expressed as mean values ± SD of three independent experiments.
Table 1.
Data collection and refinement statistics.