Discovery and Confirmation of Ligand Binding Specificities of the Schistosoma japonicum Polarity Protein Scribble
et al. (2014) Discovery and Confirmation of Ligand Binding Specificities of the Schistosoma japonicum Polarity Protein
Scribble. PLoS Negl Trop Dis 8(5): e2837. doi:10.1371/journal.pntd.0002837
Discovery and Confirmation of Ligand Binding Specificities of the Schistosoma japonicum Polarity Protein Scribble
Pengfei Cai 0
Yi Mu 0
Xianyu Piao 0
Nan Hou 0
Shuai Liu 0
Youhe Gao 0
Heng Wang 0
Qijun Chen 0
Kenji Hirayama, Institute of Tropical Medicine (NEKKEN), Japan
0 1 MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College , Beijing , The Peoples Republic of China, 2 National Key Laboratory of Medical Molecular Biology, Department of Physiology and Pathophysiology, School of Basic Medicine, Peking Union Medical College, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences , Beijing , The Peoples Republic of China, 3 Department of Microbiology and Parasitology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & School of Basic Medicine, Peking Union Medical College , Beijing , The Peoples Republic of China, 4 Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, Jilin University , Changchun , The Peoples Republic of China
Background: Schistosomiasis is a chronic debilitating parasitic disease that afflicts more than 200 million individuals worldwide. Long-term administration of chemotherapy with the single available drug, praziquantel, has led to growing concerns about drug resistance. The PSD-95/Dlg/ZO-1 (PDZ) domain is an important module found in many scaffolding proteins, which has been recognized as promising targets for the development of novel drugs. However, the parasitederived PDZ domains and their associated functions are still largely unknown. Methodology/Principal Findings: The gene encoding the Schistosoma japonicum Scribble protein (SjScrib) was identified by homologous search with the S. mansoni Scrib sequence. By screening an arbitrary peptide library in yeast two-hybrid (Y2H) assays, we identified and confirmed the ligand binding specificity for each of the four PDZ domains of SjScrib. Both SjScrib-PDZ1 and SjScrib-PDZ3 recognize type I C-terminal PDZ-domain binding motifs (PBMs), which can be deduced as consensus sequences of -[W][x][E][TS][x][ILF] and -[x][RKx][ETS][T][WW][ILV], respectively. SjScrib-PDZ2 prefers stringent type II C-terminal PBMs, which significantly differs from that of its human ortholog. SjScrib-PDZ4 binds to typical II C-terminal PBMs with a consensus sequence -[x][FW][x][LI][x][LIV], in which the aromatic residue Phe is predominantly selected at position -4. The irregular and unconventional internal ligand binding specificities for the PDZ domains of SjScrib were confirmed by point mutations of the key amino acids within the ligand binding motifs. We also compared the differences in ligand specificities between SjScrib-PDZs and hScrib-PDZs, and explored the structural basis for the ligand binding properties of SjScrib-PDZs. Conclusions/Significance: In this study, we characterized and confirmed the ligand binding specificities of all four PDZ domains of SjScrib for the first time. We denoted the differential ligand binding specificities between SjScrib-PDZs and hScrib-PDZs as well as the structural basis for these properties. This work may provide a fundamental basis for the rational design of novel anti-schistosomal drugs.
Funding: This study was supported by the National Natural Science Foundation of China (NSFC 81270026 and 30901254), the National S & T Major Program
(Grant No. 2012ZX10004-220), and the intramural grant from Institute of Pathogen Biology, CAMS (2012IPB207). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to the work.
Schistosomiasis, caused by members of the genus Schistosoma,
afflicts more than 200 million people living in the endemic areas
of 77 countries worldwide, representing a major health and
economic burden in tropical and developing nations . The
treatment of schistosomiasis is largely based on the long-term
application of the single available drug, praziquantel (PZQ).
However, concerns were raised about the problem of drug
resistance [2,3]. The advances of schistosome genomics have
provided a valuable basis for dissecting the parasite biology and
identifying novel drug targets against the parasite .
Protein-protein interactions (PPIs) mediate multiple biological
processes and physiological functions in organisms, and offer a
variety of opportunities for exploring potential drug targets .
The PSD-95/Dlg/ZO-1 (PDZ) domain is one of the most crucial
modules for PPI, and is emergently recognized as a novel target of
drug discovery . PDZ domains consist of approximately 80
90 amino acids, that typically form a packed structure composed
of six b-strands (b1-b6) and two a-helices (a1, a2) . A
Schistosomiasis japonica remains a major public health
problem in China and Southeast Asia. The long-term of
treatments with the only available drug, praziquantel, has
raised the concerns about drug resistance. Protein-protein
interactions (PPIs), for highly discriminating specificities,
are thought to be the innovative targets for a generation
of new drugs. The PDZ domain is one of the most
important modules for PPIs. A number of compounds
screened based on binding specificities of PDZ domains
have shown their potential therapeutic power in several
disease models with less side effects. Although domain
loss events are widespread in S. japonicum, a panel of PDZ
domains is conserved in this species. So far, however, little
is known about ligand binding specificities and the
molecular functions of parasite-derived PDZ
domaincontaining proteins. In this study, by yeast two-hybrid
screening of a random library, we confirmed the ligand
binding properties of a multiple PDZ domain-containing
protein Scribble of S. japonicum for the first time.
Divergent ligand specificities between the homologous
PDZ domains of S. japonicum and human Scribble
orthologs were revealed. Internal motif recognition and
irregular ligand interaction models for the SjScrib-PDZ
domains were identified. These results provide an
important basis for the rational discovery of anti-schistosomal
hydrophobic pocket formed with the carboxylate-binding loop
(b1-b2 loop), b2 strand and the a2 helix of the PDZ domain can
accommodate canonical four to five amino acids at the C-termini
of partner proteins . The PDZ-domain binding motifs (PBMs)
have been conventionally classified into four categories based on
the extreme C-terminus: Class I, -[S/T]-X-W*; Class II, -W-X-W*;
Class III, -[D/E/K/R]-X-W*; and Class IV, -X-y-[D/E]*; where
X is any amino acid, W is a hydrophobic residue, y is an aromatic
residue, and * represents the stop codon of the peptide .
Although most of PDZ domains prefer binding of the extreme
Ctermini of their target ligands, several of them can also recognize
internal motifs of their partner proteins [15,16].
Scribble is a member of the evolutionarily conserved LAP (LRR
(leucine-rich repeats) and PDZ) protein family. The N-terminal
LRR domain is necessary for targeting to the basolateral
membrane of epithelial cells [17,18], and can interact with the
PH domain leucine-rich-repeat protein phosphatase 1 (PHLPP1)
to negatively regulate Akt signalling , while the PDZ domains
of Scribble mediate interactions with a number of partners, such as
b-PIX (PAK-interacting exchange factor b) , Vangl2, a core
planar cell polarity (PCP) protein , NOS1AP , zyxin
, and ZO-2 , as well as proteins from pathogenic viruses
. Scribble has been implicated to be involved in
establishing and maintaining membrane polarity in epithelia,
neurons, and T cells , mediating PCP signalling
[21,34,35], regulating cell migration [36,37] and act as a tumor
suppressor . Also, it has been shown that Scribble plays an
important role in clustering synaptic vesicles at developing
synapses via interaction with b-catenin . The binding
specificity profiles for a number of PDZ domains of LAP proteins,
including human Scribble (hScrib), have been investigated by
screening phage display library, most of which were found to
prefer typical I C-terminal PDZ binding motifs (PBMs) .
In this study, by yeast two-hybrid (Y2H) screening of a random
peptide library and introducing point mutations, we revealed and
confirmed the ligand binding specificities, including the canonical
C-terminal and non-canonical internal binding specificities for all
four PDZ domains of the S. japonicum cell polarity protein Scribble
(SjScrib). The binding specificity profiles were compared with
those of human Scrib ortholog. The potential ligands of SjScrib
were further predicted based on the ligand binding specificities of
the PDZ domains, which were validated in the Y2H system. This
work will facilitate the identification of novel drug targets against
All procedures carried out on animals within this study were
conducted following animal husbandry guidelines of the Chinese
Academy of Medical Sciences and with permission from the
Experimental Animal Committee of Chinese Academy of Medical
Sciences with the Ethical Clearance Number IPB-2011-6.
Parasites and animals
Schistosoma japonicum-infected Oncomelania hupensis were purchased
from Jiangxi Institute of Parasitic Diseases, Nanchang, China.
Cercariae were freshly shed from the infected snails. New Zealand
White rabbits were percutaneously infected with ,1,000 S.
japonicum cercariae. Hepatic schistosomula and adult worms were
recovered from the rabbits by hepatic-portal perfusion at 2 and 6
weeks post-infection (p.i.), respectively. Male and female adult
worms were manually separated with the aid of a light microscope.
Eggs were isolated from the liver tissues of the infected rabbits at 6
weeks p.i. by the sieving and enzymatic digestion method .
Cloning of the S. japonicum Scribble gene
Two EST sequences (GenBank accession number AY809835
and AY815909), respectively encoding the N-terminal and
Cterminal fragment of SjScrib, were identified and retrieved directly
from NCBI using the S. mansoni Scribble cDNA sequence (GenBank
accession number: XM_002581163) as a bait. Forward
(59-GGCTCATAATGTTCAAGTGTTTGCCAATTATAGG-39) and reverse
were designed based on these EST sequences and used to
amplify the full-length ORF of SjScrib from adult worm pair
cDNA templates with high fidelity Phusion DNA polymerase
(New England Biolabs, NEB, UK). The resulting PCR fragment
was cloned into T-vector and sequenced. The amino acid
sequence of SjScrib (GenBank accession number: AHC92618)
was deduced from the obtained cDNA sequence (GenBank
accession number: KF730248) for further bioinformatic analysis.
Bioinformatical analysis of SjScrib
The amino acid sequences of the Scrib orthologs of S. mansoni
(GenBank accession number: CCD76510), Drosophila melanogaster
(GenBank accession number: NP_733154) and Homo sapiens
(GenBank accession number: AAL38976) were retrieved from
GenBank. The domain boundaries of SjScrib and hScrib were
analyzed using the following public domain tools: the NCBI CDD
(http://www.ncbi.nlm.nih.gov/cdd) , PFAM , and
SMART . Peptide sequences of all PDZ domains from
SjScrib, SmScrib, and hScrib were aligned by ClustalX 2.0. The
structure-based alignment of these PDZ domains was produced by
ESPript . The three-dimensional (3D) structures of
SjScribPDZ1, SjScrib-PDZ2 and SjScrib-PDZ3 were predicted by the
online service Phyre 2 (http://www.sbg.bio.ic.ac.uk/phyre2/
html/page.cgi?id = index) . The 3D structure of
SjScribPDZ4 was predicted based on the NMR structures of
hScribPDZ1 (PDB code 1X5Q), hScrib-PDZ2 (PDB code 1WHA), and
hScrib-PDZ4 (PDB code 1UJU) using the program EasyModeller
4.0 . The structural alignments of PDZ domains between
SjScrib and hScrib were performed by using the Swiss PDB
Viewer (SPDBV) . The solution NMR structures of
hScribPDZ1 (PDB code 1X5Q), hScrib-PDZ2 (PDB code 1WHA), and
hScrib-PDZ4 (PDB code 1UJU) were used as templates for
superposition of the predicted structures of SjScrib-PDZ1,
SjScribPDZ2, and SjScrib-PDZ4, respectively. The superimposed
structures were further refined with PyMOL Viewer program (DeLano
Scientific, San Carlos, CA, http://www.pymol.org/). Free, open
source Java software for visualizing ligand binding specificity
profiles is available from http://baderlab.org/Software/LOLA.
Transcriptional characterization of the SjScrib gene by
Total RNAs of S. japonicum at different developmental stages
(cercariae, hepatic schistosomula, separated male and female adult
worms, and eggs) were extracted using Trizol reagent (Invitrogen,
CA, USA). The possible contaminating genomic DNA was
completely removed from RNA samples with TURBO DNA-free
kit (Ambion, CA, USA). RNA quantification and quality control
was conducted by 1% agarose gel electrophoresis and the
Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies,
Wilmington, DE). For each sample, 1 mg total RNA was reverse
transcribed into first-strand cDNA using SuperScript III Reverse
Transcriptase Kit (Invitrogen). The synthesis procedure was
performed as follows: 25uC for 5 min, 50uC for 1 h, 70uC for
15 min. Each PCR reaction contained 12.5 ml of 26Brilliant II
SYBR Green QPCR Master Mix (Agilent, USA), 1 ml diluted
cDNA (206), 1 ml of the forward and reverse primer pair (Table
S1), and 10.5 ml of sterile water. The PCR program included 40
cycles with denaturation at 95uC for 30 s, followed by annealing
and extension at 60uC for 1 min. Quantification of the
transcriptional level of the SjScrib gene was performed by normalizing
against the PSMD4 transcript (26S proteasome non-ATPase
regulatory subunit 4, GenBank Accession Number: FN320595)
[52,53] and applying the comparative 22DDCt method, according
to the SDS 1.4 software.
Construction of bait plasmids and Y2H screening of a
random peptide library
Primer pairs were designed based on the boundaries of PDZ
domains (shown in Table S1). The DNA fragments encoding PDZ
domains of SjScrib were respectively amplified from the adult
worm cDNA templates using Phusion DNA polymerase. The PCR
was performed with an initial denaturation for 1 min at 98uC,
followed by thirty cycles: 98uC for 5 s; 50uC for 30 s and 72uC for
15 s. The final extension was 5 min at 72uC. The PCR products
were digested with EcoR I and BamH I, and cloned into the EcoR
I/BamH I sites of the GAL4 BD vector, pBridge (Clontech).
Each GAL4 BD-fusion bait plasmid was transformed into the
yeast strain CG1945 using the lithium acetate procedure. The
transformants were grown on SD/-Trp plates and lacZ assays were
performed to examine self-activation. The transformants were
further spread on SD/-His/-Trp plates with different
concentration of 3-amino-1,2,4-triazole for leakage test. A random peptide
library (constructed with Tsp509I-digested human genomic DNA
fragments)  was screened following the MATCHMAKER
Two-Hybrid System protocol (Clontech). The transformants were
screened on SD/-Trp/-His/-Leu plates with different
concentration of 3-amino-1,2,4-triazole (2.5 mM for the SjScrib-PDZ1,
SjScrib-PDZ2, and SjScrib-PDZ4 bait transformants, and 7.5 mM
for the SjScrib-PDZ3 bait transformant) and further verified by
the improved LacZ assays. After rescue, the potential positive
plasmids were isolated and retransformed into the yeast strain
CG1945 containing corresponding bait plasmid. Only the clones
that were positive for all the reporter assays and confirmed by at
least two independent tests were selected for specific interactions
and sequenced .
Confirmation of the potential binding site within
For each irregular ligand, a putative binding site was deduced
from the pattern of regular C-terminal PDZ binding motifs.
Point mutation was induced by PCR against key residues within
the putative binding site. DNA fragments produced by self
primer template PCR reactions or amplified from ligand plasmids
were digested with EcoR I and BamH I, and cloned into the GAL4
BD vector, pBridge. Each clone was subjected to sequencing
to make sure that the mutation was correctly introduced. Each
of the mutated plasmids was co-transformed with the
corresponding PDZ domain bait plasmids into yeast strain CG1945,
respectively. The interactions between the mutants and the
corresponding PDZ domain were validated by Y2H assays as
Prediction of native ligand candidates
The consensus C-terminal binding sequences for each
SjScribPDZ domain were deduced from sequence alignments of positive
clones from Y2H screening. The S. japonicum predicted peptide
sequences were downloaded from SDSPB (http://lifecenter.sgst.
cn/schistosoma/cn/schdownload.do). The Tailfit software 
was used to search against the S. japonicum peptide dataset to
retrieve the potential ligands whose carboxyl termini matched the
consensus-binding sequences. These peptide sequences were then
manually BLAST in NCBI to filter the truncated fragments
lacking of the C-terminus. Further, the most promising candidates
were selected based on biological information such as subcellular
localization and potential molecular function, as well as the
Cterminal homology between S. japonicum and S. mansoni orthologs
Confirmation of candidate SjScrib-ligand interactions
The GAL4 AD plasmids expressing the carboxyl-terminus of
the candidate ligands of SjScrib-PDZ1 and SjScrib-PDZ4 were
first constructed. The self primer template PCR reactions were
performed to produce DNA templates encoding the 15 amino
acids of extreme carboxyl-terminus of each ligand candidate
(The primer pairs were listed in Table S1). The PCR procedure
included thirty cycles as follows: 98uC for 5 s; 50uC for 25 s,
and 72uC for 1 s, with a final extension at 72uC for 2 min. The
resulting DNA fragments were recovered, and further digested
with EcoR I and BamH I endonucleases, and cloned into the
EcoR I/BamH I sites of GAL4 AD vector, pGADT7 followed by
sequencing. Each constructed plasmid was co-transformed
with the corresponding SjScrib-PDZ1 and SjScrib-PDZ4
bait plasmids into yeast strain CG1945, respectively. The
positive interactions were selected by Y2H assays as mentioned
PDB: 1X5Q, PDB: 1WHA, PDB: 1UJU, GenBank: AY809835,
GenBank: AY815909, GenBank: XM_002581163, GenBank:
AHC92618, GenBank: KF730248, GenBank: CCD76510,
GenBank: NP_733154, GenBank: AAL38976, GenBank: XP_
002581546, GenBank: FN320595.
Results and Discussion
Molecular characteristics of S. japonicum Scribble
The S. japonicum Scribble gene encodes a protein of 1,486 amino
acids, with a theoretical molecular weight of 168 kDa. Homology
blast showed that SjScrib shares a relatively high degree of
sequence homology with the orthologous S. mansoni scribble,
SmScrib (GenBank accession number: CCD76510) (Figure 1A). A
leucine-rich repeat (LRR) domain within the N-terminus of
SjScrib is relatively conserved with orthologs of Drosophila and H.
sapiens, indicating that the function mediated by this domain may
be evolutionarily conserved. Four PDZ domains are located in the
relatively extreme C-terminal region of schistosome Scribs
(Figure 1A). It is obvious that the carboxyl-terminal (CT) domain,
which has previously been suggested not being essential for any
aspect of Scrib localization or function , is missing in SjScrib
and SmScrib when compared with DmScrib and hScrib
(Figure 1A). Among the four PDZ domains of SjScrib, the third
PDZ domain shares the highest degree of homology (60%) with
hScrib-PDZ3 (Figure 1A). The peptide sequences of the
SjScribPDZ domains were aligned with homologous sequences of
SmScrib and hScrib (Figure 1B). The secondary structures of the
PDZ domains were determined using the NMRs structure of
hScrib-PDZ1 (PDB codes 1X5Q) as a template. It is notable that
the loop between b2 and b3 in the fourth PDZ domain of
schistosome Scribs is longer than that of hScrib-PDZ4, which may
increase the flexibility of this domain (Figure 1B). Totally, SjScrib
and SmScrib possess the general primary characteristics as that of
the homologous proteins of other organisms, indicating that they
may play analogous roles in schistosome parasites. The Scribble
polarity module is composed of Scribble, lethal giant larvae (Lgl)
and discs large (Dlg), which are well conserved across species from
worms and flies to mammals [41,58]. Recently, it has been shown
that the S. japonicum Lgl protein is localized in the worm tegument
and knockdown of this gene can significantly deform the surface
structure of adult worm and impair egg hatching . To some
extent, these data may reveal the potential function of the Scribble
polarity module in S. japonicum.
Homology modeling reveals a different degree of
structural divergence between SjScrib-PDZs and
To assess the structural divergence between SjScrib and hScrib
PDZ domains, we further superimposed the structures of
SjScribPDZs with three available NMR structures of hScrib PDZ
domains. Although the primary sequence identity of the second
PDZ domain between SjScrib and hScrib is higher than that of the
first and fourth PDZ domains (Figure 1A), the structural similarity
of the second PDZ domain between SjScrib and hScrib is the
lowest among the three PDZ domains (Figure 2). The root mean
square deviation (RMSD) value for the first, second, and fourth
PDZ domain is 0.41 A, 1.36 A, and 0.64 A, respectively,
indicating that the ligand binding specificity for SjScrib-PDZ2
may be more likely to diverge from that of hScrib-PDZ2 than
those of SjScrib-PDZ1 and SjScrib-PDZ4 from the corresponding
hScrib PDZ domains, and the ligand binding specificity may be
relatively conserved between SjScrib-PDZ1 and hScrib-PDZ1.
Transcriptional analysis of the SjScrib gene at different
developmental stages of S. japonicum
QRT-PCR was performed to determine the transcriptional
profiles of SjScrib at different developmental stages and between
sexes of the parasite. As a result, we observed that the SjScrib gene
was ubiquitously expressed during different developmental stages,
but in a stage-biased pattern. The transcription levels of the SjScrib
gene were similar in cercariae and schistosomula at a medium
level. In adult worms, the transcriptional level was significantly
higher in male than that in female worms. In the egg stage, the
expression of SjScrib was relatively higher than that in any other
stages detected (Figure 3). The data suggest that SjScrib is a
necessity for all developmental stages of the parasite, particularly
during the development of egg embryo. The reason that SjScrib
was less transcribed in the female parasites is puzzling, but it could
be postulated that in female adult worms, a relatively low number
of cells need to maintain membrane polarity when compared to
other developmental stages of the parasite.
Y2H screening against each of SjScrib-PDZ domain
To determine the binding properties of SjScrib-PDZs, an
arbitrary peptide library was screened by Y2H assays with a
similar approach for the determination of the binding specificity of
SjGIPC3-PDZ . For SjScrib-PDZ1, a total of 35 positive
clones were obtained, which encodes 25 unique carboxy-termini.
Among them, 16 belong to Class I PBM, 6 belong to irregular
Class I PBM (which can be viewed as a projection of an amino
acid at C-terminus of regular PBMs), and 3 cannot be classified
(Figure 4A). For SjScrib-PDZ2, only 2 positive clones were
probed. One is a Class II PBM, and the other is an irregular Class
II PBM (Figure 4B). For SjScrib-PDZ3, 37 positive clones which
encode 30 unique carboxy-termini were obtained. Among them,
16 belong to Class I PBM, 2 belong to irregular Class I PBM, and
12 are irregular (Figure 4C). For SjScrib-PDZ4, 58 positive clones
encoding 56 unique carboxy-termini were obtained. Among them,
53 belong to Class II PBM, 1 belongs to Class I PBM, and 2 are
irregular (Figure 4D).
Determination of the specific binding sites within
For each PDZ domain of SjScrib, ligands with irregular
Ctermini were subjected to further analysis. Consequently, putative
binding sites were inferred within the internal sequences of these
ligands according to the pattern of regular C-terminal PBMs
(Table 1, bold and underline characters). To confirm these
putative binding motifs, point mutations were introduced into the
key amino acid residues and those mutants were further validated
in the Y2H system (Table 1).
For SjScrib-PDZ1, a core binding sequence was deduced from
its canonical C-terminal PBMs, -[E][TS][x][LIF]* (Figure 4A). A
putative binding motif, [E][TS][x][IF], was inferred for each
irregular ligand. Substitution of the hydrophobic residue Ala for
the polar residue Thr/Ser totally interrupted the interactions
between SjScrib-PDZ1 and the irregular ligands. For
SjScribPDZ2, only two unique sequences were obtained, probably due to
the stringent recognition at particular binding sites. We
hypothesized that the typical II C-terminal PBM, -WELSI* and the
atypical II C-terminal PBM, -FELMLE* with one amino acid Glu
projected at the C-terminus of the binding site, are likely to be the
core binding motifs for SjScrib-PDZ2. This hypothesis was
confirmed by a series of point mutations introduced into each
amino acid of the putative PBMs (Table 1). For SjScrib-PDZ3, a
putative core binding sequence, -[E][T][W][W] was deduced based
on its canonical typical I C-terminal PBMs (Figure 4C). Potential
PBM was postulated for each irregular ligand of SjScrib-PDZ3.
Substitutions the conserved polar residue Thr with the
hydrophobic residue Ala led to the failure of the interactions between the
PDZ domain and these mutants (Table 1). For SjScrib-PDZ4, a
consensus sequence -[FWY][x][W][x][W]* was inferred from most
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).
of the typical C-terminal PBMs (Figure 4D). The binding sites
within the five irregular ligands were located based on the
consensus sequence. Two of them, P4-L32 and P4-L39 were found
to be particularly unusual, as they potentially bear 7 and 6 amino
acids, respectively, for interacting with SjScrib-PDZ4. However, as
residues of small size are dominant within both PBMs (PCGAS
in P4-L32 and PSG in P4-L39), it is reasonable to speculate that
they can be well accommodated within the binding groove of
SjScrib-PDZ4. To confirm our prediction, three point mutations
were introduced in each of the five putative binding sites (Table 1),
and as a result, these mutants were found to be unable to bind to
SjScrib-PDZ4. Together, these data support our speculation that
SjScrib-PDZ1, SjScrib-PDZ3, and SjScrib-PDZ4 have the ability
to bind both C-terminal and internal PBMs, which will be further
Determination of ligand binding specificities of the
SjScrib-PDZ domains based on the statistical analysis of
amino acid composition within C-terminal and internal
SjScrib-PDZ1 showed particular preference for hydrophobic
residues at the carboxyl terminus of its ligand binding site (P0, not
the extreme carboxyl terminus of the ligand), especially Ile
(44.0%), Leu (36.0%), or Phe (20.0%). At P21, no clear preference
was observed. At P22, only the polar amino acids Thr (80.0%) and
Ser (20.0%) were selected. At P23, Glu (100%) was absolutely
preferred. At P25, the hydrophobic residues, such as Leu (28.0%),
Phe (24.0%) and Tyr (16.0%), along with a rare occurrence of
other hydrophobic residues were selected (Figure 5A). Based on
the statistical analysis of its unique PBMs, a consensus-binding
sequence was determined as -[W][x][E][TS][x][ILF] for
Hydrophobic residues, especially Leu (36.7%), Ile (26.7%), or
Val (26.7%), were also predominantly preferred at P0 site of
SjScrib-PDZ3 ligands. At P21, the preference for Trp (36.7%) was
observed, followed by other hydrophobic residues Val (13.3%),
Phe (10.0%) and Tyr (10.0%). The P22 site demonstrated an
absolute preference for the polar residue Thr (100%). At P23, Glu
(60.0%) was predominantly selected, along with a rare selection of
residues Thr (16.7%) and Ser (13.3%). While the positive charged
residues, Arg (23.3%) and Lys (20.0%) were slightly prone to be at
P24 site. At P25 site, no obvious preference was observed
(Figure 5B). Based on these results, a consensus-binding sequence,
-[x][RKx][ETS][T][WW][ILV], was deduced for SjScrib-PDZ3.
For the ligands of SjScrib-PDZ4, hydrophobic residues,
especially, Ile (25.9%), Leu (25.9%), or Val (16.47%), were
preferred at P0, with a weak preference for the residues of Ala
(11.1%) and Cys (9.3%). The P22 site was also occupied by the
hydrophobic residues of Ile (44.4%) and Leu (27.8%). At P24, Phe
(81.5%) was predominantly selected, with a less occurrence of Trp
(14.8%). No clear preference was observed for the sites of P21, P23
and P26 (Figure 5C). Therefore, a consensus-binding sequence,
[x][FW][x][IL][x][ILV], was inferred for SjScrib-PDZ4 based the
amino acid occurrence at particular positions.
Ligand specificity analysis by comparison of SjScrib-PDZs
and other LAP family PDZ domains according to the key
residues for ligand recognition
The comparative structural analysis of human LAP family
members ZO-1-PDZ1 and Erbin-PDZ has provided a valuable
insight for ligand recognition across the entire binding site .
To better understand the molecular basis for ligand recognition of
SjScrib-PDZ domains, comparative analyses were conducted for
ligand specificities of the SjScrib-PDZ domains according to the
key residues for ligand recognition. It has been suggested that the
hydrophobic residue at b2-1 is a key determinant of the P0
specificity of ZO-1-PDZ1 and Erbin-PDZ. The PDZ domains that
show a stringent preference for a Val0 side chain contain a Phe at
position b2-1, whereas those that can accommodate a larger Leu/
Ile at site 0 contain a Leu/Ile at the same position . Here,
however, it seems that SjScrib-PDZ1, SjScrib-PDZ3 and
SjScribPDZ4, but not SjScrib-PDZ2, complies with this rule, suggesting
that other residues within the hydrophobic pocket (i.e. b2-3, a2-5,
and a2-8) may also affect the P0 specificity of LAP family PDZ
domains (Figure 6) .
The PDZ domains of LAP family that contain His and Val at
a2-1 and a2-5, respectively, usually have a strong preference for
Thr/Ser at P22 . Strikingly, SjScrib-PDZs obey this rule quite
well, i.e., SjScrib-PDZ1 and SjScrib-PDZ3 that bear His and Val
at a2-1 and a2-5, respectively prefer Thr at P22 (Figure 6A and
C), whereas SjScrib-PDZ2 that has an Ile at a2-5 and
SjScribPDZ4 that has a Leu at a2-1 prefer Ile and Leu at P22,
respectively (Figure 6B and D). Also, it has been suggested that the
positively charged residue Arg or Lys at position b3-5 of the LAP
family PDZ domains favors the electrostatic interactions with a
negatively charged ligand side chain at P23 . A similar
situation was observed in regarding of the selectivity of P23 of
SjScrib-PDZs. SjScrib-PDZ1, SjScrib-PDZ2, and SjScrib-PDZ3
that contain an Arg or a Lys at position b3-5, exhibit a strong
preference for a Glu at P23 (Figure 6A, B, and C). In contrast,
SjScrib-PDZ4 that has a Trp at position b3-5 displays promiscuity
for residues at P23 (Figure 6D).
In addition, SjScrib-PDZ2 and SjScrib-PDZ4 display a
preference for ligands that contain an aromatic side chain at
P24. Both recognitions are predominantly mediated by residues
within the b2:b3 loop. In the case of SjScrib-PDZ2, the side chain
of Phe/Trp24 may interact with residues in b2 (Ala (b2-4)) and the
b2:b3 loop (Pro (b2:b3-9) (Figure 6B). While in the case of
SjScribPDZ4, the aromatic side chain at site -4 of its PBMs may
potentially interact with Thr (b2-4) and Phe (b2:b3-17), based on
the structural analysis (Figure 6D). However, the interactions
between the PDZ domains and the artificial ligands may not
completely reflect the nature of the molecular recognitions in vivo,
thus approaches such as co-immunoprecipitation with native
SjScrib will be needed to further address the binding capacity of
Divergence of ligand specificities of SjScrib-PDZs and
Zhang et al. have previously investigated the binding specificity
profiles of a panel of PDZ domains of LAP proteins by phage
display screening . They found that all four C-terminal
residues of the ligands constituted a core recognition motif for
interactions, while the more upstream residues also supported the
core binding sites. Here, we performed a comparative analysis to
figure out the convergent and divergent ligand specificities
between SjScrib-PDZs and hScrib-PDZs. The ligand binding
profile for SjScrib-PDZ1 (-[W][x][E][TS][x][ILF]) was found to be
similar with that of hScrib-PDZ1 (-[W][W][E][TS][x][VIL]*), but
not exactly the same. For example, hScrib-PDZ1 prefers a
hydrophobic residue at P-4, whereas this is not the case for
SjScrib-PDZ1. Except that both PDZ domains prefer the
hydrophobic residues Leu and Ile at P0, SjScrib-PDZ1 exhibits a
preference of the aromatic residue Phe; whereas hScrib-PDZ1
prefers an aliphatic residue Val at this site. The binding specificity
pattern for SjScrib-PDZ3 (-[x][RKx][ETS][T][WW][ILV])
resembles that of hScrib-PDZ3 (-[W][W][EDQH][TS][x][LIV]*) ,
but with certain distinctions for SjScrib-PDZ3. For instance,
SjScrib-PDZ3 shows no clear preference of hydrophobic residues
at P25. At P24, unlike hScrib-PDZ3 that prefers hydrophobic
residues, SjScrib-PDZ3 exhibits a preference for the positively
charged residues, Arg and Lys, which may form electrostatic
interactions with the negatively charged residue Asp at a2-2 or
b2:b3-3 (Figure 6C). Further, there is a strong preference for
hydrophobic residues, particularly the aromatic residue Try, at
P21 for SjScrib-PDZ3, which was not the case for hScrib-PDZ3.
The most significant divergence was observed when comparing
the binding preferences of SjScrib-PDZ2 with that of
hScribPDZ2. In contrast to hScrib-PDZ2, which prefers typical I
Cterminal PBMs, -[W][W][ED][TS][W][V]* , SjScrib-PDZ2
displays a preference for a typical II PBM, -WELSI*, and an
irregular typical II PBM, -FELMLE*. Also, SjScrib-PDZ2 exhibits
no preference of hydrophobic residues and Tyr at P25 and P21,
respectively, in contrast to that observed in those of hScrib-PDZ2.
These results are consistent with that of homology modeling, i.e.
the structural similarity between SjScrib-PDZ2 and hScrib-PDZ2
is poor (Figure 2B). As no binding preference of hScrib-PDZ4 is
available so far, we cannot directly compare the binding specificity
profile of SjScrib-PDZ4 with that of hScrib-PDZ4. However,
given the relatively low homology between SjScrib-PDZ4 and
hScrib-PDZ4 (Figure 1A), as well as the difference in several key
residues involved in ligand recognition, such as b3-5, a2-1,
b1:b27, b2:b3-7, b2-4, and a2-8 between the two domains (Figure 1B)
, we postulate that the binding specificity of SjScrib-PDZ4
may be highly divergent from that of hScrib-PDZ4. Totally, these
different ligand binding characteristics between the homologous
PDZ domains of SjScrib and hScrib can be potentially utilized for
the drug design against SjScrib.
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.
The internal ligand binding ability of SjScrib-PDZs
For the first time, the internal motifs binding ability of the three
SjScrib-PDZ domains, SjScrib-PDZ1, SjScrib-PDZ3, and
SjScribPDZ4 was confirmed in this study. Based on the ratio of internal
PBMs to C-terminal PBMs obtained from each SjScrib-PDZ, we
can infer that SjScrib-PDZ3 (12:18) displays a relatively strong
preference for internal motif binding, followed by SjScrib-PDZ1
(3:22); while SjScrib-PDZ4 (1:55) shows a weak ability of internal
motif recognition. Interestingly, preferences at P23 between the
Cterminal and internal PBMs of SjScrib-PDZ3 are different. The
Cterminal PBMs of SjScrib-PDZ3 display a strong preference for
Glu23; whereas its internal PBMs show a preference for Thr/
Ser23, which may interact with the residue Cys at b2-4 (Figure 6C)
as previously suggested . Also, the negatively charged residues
(Glu and Asp) frequently appear at the position +1 (P+1) (the
residue beyond the core binding motif) in these irregular and
internal PBMs of SjScrib-PDZ1 (Table 1). In contrast, the
hydrophobic residues (Leu, Ile, and Val) or the aromatic amino
acids (Phe and Tyr) are preferred at this position in the irregular
and internal PBMs of SjScrib-PDZ3. The structural analysis
indicated that the P+1 residue may interact with the residues of the
carboxylate-binding loop or a2-9 (Figure 6). In the case of
SjScribPDZ1, the Glu/Asp+1 may form electrostatic interactions with the
positively charged residue Arg at position a2-9 (Figure 6A), while
the hydrophobic residues at P+1 of the irregular PBMs of
SjScribPDZ3 potentially interact with Pro (b1:b2-6) and Ile (a2-9)
(Figure 6C). Similar to the case of SjScrib-PDZ1, the Glu+1 of the
atypical PBM of SjScrib-PDZ2 (P2-L2) and the Asp+1 in the
internal ligand of SjScrib-PDZ4 (P4-L4) can potentially interact
with Arg (b1:b2-4) (Figure 6B) and Arg (a2-9) (Figure 6D),
respectively. These results indicate that SjScrib-PDZs may favor
the unconventional interactions with a mode of Par6-Pals internal
In Drosophila and zebra fish, it has been shown that some PDZ
domains of Scribble can interact with ligands via internal
recognition [24,62]. These data together with ours suggest that
the internal interaction pattern of the Scrib-PDZ domains may be
more common than currently appreciated. The internal binding
model of PDZ domains can dramatically increase the diversity of
its native ligands. Thus, it is possible that SjScrib may mediate
multiple biological pathways through a manner of internal
recognition. So far, several compounds screened based on the
internal binding specificities of PDZ domains have shown their
therapeutic potential against several diseases with less side effects
. The finding of the internal binding preferences of
SjScribPDZs has raised an opportunity for designing small molecular
drugs against this target.
Potential native ligand prediction and validation for the
To identify the native ligand candidates of SjScrib-PDZs, the
Tailfit program was employed to search potential ligands in the
Ligand sequence and point mutation1
Ligand sequence and point mutation1
1: The potential PBMs within the irregular ligands of each PDZ domain of SjScrib are shown in bold and underline. The substituted residues are indicated in brackets.
2: !: positive in the Y2H system; 6: negative in the Y2H system.
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 P25 to P0) are presented in the tables on the right panel.
S. japonicum predicted proteomic database (sjr2_protein.fasta) with
less stringent core binding motifs as baits (Table 2). A panel of
protein sequences was retrieved. After filtering, 2 and 9 proteins
were selected as potential ligand candidates for SjScrib-PDZ1 and
SjScrib-PDZ4, respectively (Table 2). However, only one protein,
Prepro-cathepsin C, was confirmed to be potential ligand of
SjScrib-PDZ4 in the Y2H system. Although the C-terminal tails of
most of the candidates fit the ligand consensus sequence quite well,
they are not native partners of SjScrib. One possibility is that the
interaction between PDZ domain and ligand partner was not
simply determined by the consensus sequence. For example, the
consensus sequence ([x][FW][x][IL][x][ILV]) of SjScrib-PDZ4 is
#: !: positive in the Y2H system; 6: negative in the Y2H system.
just a primary, but not tertiary determinant of specificity. The
characteristics, such as the charge and size of the residues at
position -1 and -3, may also affect the ligand recognition. These
residues must coordinate with the ones at position 0, -2 and -4 and
function as a whole determinant for recognition. Vice verse, this
fact may explain why some unusual PBMs (e.g. P4-L32,
FPCGASW* and P4-L39, -YLPSGF*) could interact with
SjScrib-PDZ4, although they do not fit the typical consensus
sequence well. As it has been suggested that each PDZ domain is
capable of interacting with 17 partners on average , it is
obvious that more potential native ligands of SjScrib are still under
discovery, which probably due to the poor quality of the proteomic
database, or in the other case, these PDZ domains may interact
with the native ligands via internal recognition. Also, it is worth
noting that this positive ligand confirmed in Y2H assay is merely a
potential partner of SjScrib at current stage, since we expressed
the two proteins (the PDZ domain and the tail of Cathepsin C),
which may be from different tissues of the parasite under native
condition, into one yeast cell. Co-localization assay of the two
proteins in different developmental stages of the parasite may
further address whether this interaction indeed takes place in vivo.
In summary, for the first time, we presented the molecular
characterization of the PDZ domain-containing protein Scribble
Lysosomal protective protein precursor
putative TatD DNase domain containing 1
putative phosphate transporter
putative zinc finger protein
from S. japonicum. We defined the ligand binding specificities of the
SjScrib-PDZ domains by screening a random peptide library in
the Y2H system. The convergent and divergent ligand specificities
between the SjScrib-PDZ domains and those of its human
ortholog were denoted. The confirmation of internal ligand
specificities and the identification of irregular ligands for the
SjScrib-PDZ domains will assist in the rational design of novel
drugs against the parasite. The strategy used here can also be
generically applied to the determination of the ligand binding
specificities of other parasite-derived PDZ domains.
Primer sequences used in this study.
Conceived and designed the experiments: PC YM QC. Performed the
experiments: PC YM XP NH SL. Analyzed the data: PC YM QC.
Contributed reagents/materials/analysis tools: YG HW. Wrote the paper:
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