Figure 1.
Schematic structures and multi-alignment of PDZ domains of different Scribble orthologs.
(A). Diagram of the structures of SjScrib, SmScrib, hScrib, and DmScrib, which consist of an N-terminal leucine-rich repeat (LLR), and four PDZ domains. Sequence identities of full-length protein, as well as LRR and PDZ domains between SjScrib and the orthologs from S. mansoni, H. sapiens, and D. melanogaster are shown between schematic structures. (B). Structure-based sequence alignments of four PDZ domains of Scrib orthologs. Secondary structure elements are indicated above the alignment and refer to the structure of hScrib-PDZ1 domain (PDB code 1X5Q). Each fundamental residue that mediates ligand recognition is denoted and numbered according to its position in an element of secondary structure; and within loops, the residues are numbered according to the sequence of SjScrib-PDZ4. The black arrowheads under the sequence indicate residues that are potentially involved in ligand recognition. Sequence alignment showed that a stretch of 27 amino acids in the second PDZ domain of SmScrib is missing in the sequence of CCD76510 (GenBank accession number). The missing fragment (-AIYVSKITEGGAAHKQGQLRVGDQIIS-) was retrieved from another orthologous sequence (GenBank accession number: XP_002581546).
Figure 2.
Superimposition of the structures of SjScrib and hScrib PDZ domains.
Molecular models of the first (A), second (B), and fourth (C), PDZ domain of SjScrib (blue ribbon) were respectively superimposed on the template structure of the corresponding hScrib-PDZ domain (red ribbon). Superimposition was produced by the program Swiss PDB Viewer [51] and refined by PyMol Viewer program. RMSD (root mean square deviation) value for the first, second, and fourth PDZ domain is 0.41 Å, 1.36 Å, and 0.64 Å, respectively.
Figure 3.
Transcriptional analysis of the SjScrib gene at different developmental stages by qRT-PCR.
Transcriptional levels were calibrated according to the comparative 2−ΔΔCt method using the housekeeping gene SjPSMD4 as an endogenous control. The expression of the SjScrib gene was normalized relative to the cercarial stage. Error bars represent the standard deviations of the mean from the three replicates.
Figure 4.
The unique ligand carboxyl termini for each PDZ domain of SjScrib.
The unique ligand carboxyl termini for SjScrib-PDZ1 (A), SjScrib-PDZ2 (B), SjScrib-PDZ3 (C), and SjScrib-PDZ4 (D), are shown respectively. The extreme carboxyl termini (8 amino acids) of unique ligands interacted with each SjScrib-PDZ domain were aligned by ClustalX 2.0, respectively, and the alignments were refined with GeneDoc software. Amino acids that are identical or similar are shaded in black and grey, respectively.
Table 1.
Validation the interaction between the mutants and the SjScrib-PDZ domains by Y2H assays.
Figure 5.
Ligand binding specificities of SjScrib-PDZ1, SjScrib-PDZ3, and SjScrib-PDZ4.
Each aligned C-terminal and internal PBMs set was used to create a position weight matrix (PWM) for SjScrib-PDZ1 (A), SjScrib-PDZ3 (B), SjScrib-PDZ4 (C), respectively. Two irregular PBMs (-TFPCGASW* and -KSYLPSGF*) were excluded from the analysis of the ligand binding specificity of SjScrib-PDZ4. The percentages of each type of the amino acids at a particular position (from P−5 to P0) are presented in the tables on the right panel.
Figure 6.
Modeling of ligand binding specificities for the SjScrib-PDZ domains.
The predicted structures of the first (A), second (B), third (C), and fourth (D) PDZ domain of SjScrib are shown and colored to highlight the functional residues (shown as a partial molecular surface model) involved in ligand recognition. The position in an element of secondary, name, and the potential ligand subsite involved in recognition for each key residue are denoted. Color schemes: large hydrophobic residues (magenta); small hydrophobic residues (green); small polar residues (orange); aromatic residues (cyan); positively charged residues (red); negatively charged residues (blue). The ligand specificities for each PDZ domain are shown under each panel. Structural figures were produced with PyMOL.
Table 2.
Validation of the predicted ligand candidates of SjScrib-PDZ1 and SjScrib-PDZ4 in the Y2H system.