Characterization of Diverse Internal Binding Specificities of PDZ Domains by Yeast Two-Hybrid Screening of a Special Peptide Library
Gao Y (2014) Characterization of Diverse Internal Binding Specificities of PDZ Domains by Yeast Two-Hybrid Screening of a
Special Peptide Library. PLoS ONE 9(2): e88286. doi:10.1371/journal.pone.0088286
Characterization of Diverse Internal Binding Specificities of PDZ Domains by Yeast Two-Hybrid Screening of a Special Peptide Library
Yi Mu 0
Pengfei Cai 0
Siqi Hu 0
Sucan Ma 0
Youhe Gao 0
Olivier Kocher, Harvard Medical School, United States of America
0 1 National Key Laboratory of Medical Molecular Biology, Department of Physiology and Pathophysiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College , Beijing , P.R. China , 2 MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College , Beijing , P.R. China
Protein-protein interactions (PPIs) are essential events to play important roles in a series of biological processes. There are probably more ways of PPIs than we currently realized. Structural and functional investigations of weak PPIs have lagged behind those of strong PPIs due to technical difficulties. Weak PPIs are often short-lived, which may result in more dynamic signals with important biological roles within and/or between cells. For example, the characteristics of PSD-95/Dlg/ZO-1 (PDZ) domain binding to internal sequences, which are primarily weak interactions, have not yet been systematically explored. In the present study, we constructed a nearly random octapeptide yeast two-hybrid library. A total of 24 PDZ domains were used as baits for screening the library. Fourteen of these domains were able to bind internal PDZ-domain binding motifs (PBMs), and PBMs screened for nine PDZ domains exhibited strong preferences. Among 11 PDZ domains that have not been reported their internal PBM binding ability, six were confirmed to bind internal PBMs. The first PDZ domain of LNX2, which has not been reported to bind C-terminal PBMs, was found to bind internal PBMs. These results suggest that the internal PBMs binding ability of PDZ domains may have been underestimated. The data provided diverse internal binding properties for several PDZ domains that may help identify their novel binding partners.
Funding: This work was supported by the National Basic Research Program of China (2012CB517606, 2013CB530805), 111 Project (B08007), the National Natural
Science Foundation of China (31200614) and the Beijing Natural Science Foundation (5132028). 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.
The human genome encodes 20,000,25,000 protein-coding
genes, which is much lower than previous estimates (,100,000 in
2001) . The number was only is approximately 30% more than
that of the roundworm Caenorhabditis elegans (,18,000), which once
led to the speculation that alternative splicing may occur more
frequently in humans than invertebrates. Further investigation
revealed no major differences in the frequency of alternatively
spliced genes among humans and any of the other animals tested,
including C. elegans . Thus, the phenomenon may denote the
presence of more diverse modes of protein-protein interactions
(PPIs) than currently known to exert complex physiological
functions in higher organisms. Although a lot of information on
PPIs has been released publicly on the benefit of advances in
proteomics technologies , the identification of promiscuous
weak or transient protein interactions, which may play critical
roles in cellular physiology, remains a challenge .
Protein domains, such as SH2 (Src Homology 2), SH3 (Src
Homology 3), PDZ (PSD-95/Dlg/ZO-1), and WW domains, are
abundant PPI modules that mediate a variety of biological and
cellular functions . These domains have different recognition
motifs for their target ligands; for example, SH2 can recognize
tyrosine-phosphorylated (pTyr) motifs, SH3 and WW can
recognize proline-rich sequences containing PxxP or PPxY motifs,
in which x denotes any amino acid , and PDZ domains can
usually bind to 4,5 C-terminal amino acids of target proteins
. Besides, non-canonical interaction modes have also been
reported , which could contribute to the diversity and
complexity of PPIs. And our understanding of non-canonical
modes of interaction can be facilitated by systematically exploring
the binding properties of those domains.
Most PDZ domains are involved in the recognition of the
extreme C-terminal PDZ-domain binding motifs (PBMs) of target
ligands; however, accumulating evidences have revealed that some
of them can also bind internal PBMs of partner proteins (Table 1).
Efforts have been made to develop safe and effective drugs to treat
diseases by targeting the internal binding sequences of PDZ
domains [17,18]. However, as limited information about the
internal ligand binding specificities of PDZ domains is available,
more efforts are needed to systematically explore this
noncanonical mode of interaction. Usually, conventional cDNA
libraries full of high-abundance and diverse C-terminal ligands
were screened against the PDZ domains, which might be
unfavorable for the discovery of the internal binding pattern.
DTX1, STAU1 S/T-X-V/L-D
PDZ No. Partner
*: Based on the N-terminal phage display peptide library screening.
Here, we speculate that more internal recognition patterns might
be found once the diverse C-terminal motifs in the system are
For some PDZ domains, no C-terminal ligands were screened
out in the C-terminal phage-display peptide system , raising
the possibility that these PDZ domains may have a preference for
binding internal motifs. The yeast two-hybrid (Y2H) system is
another powerful and high-throughput method for studying PPI,
which can determine one-to-one interactions, that means when
one weak interaction occurs in a single yeast cell, it will not be
inhibited by other strong ones. Typically, the main consensus
motif for PDZ domains is limited to the three , five residues, so a
library with internal octapeptide may cover these core binding
motifs. To ensure that the interactions occur between the internal
motifs and PDZ domains, all clones in the library should bear an
identical C-terminal sequence that is not any type of C-terminal
motif for PDZ domains. Based on these criteria, an internal
octapeptide library was designed as shown in Figure 1. To avoid
pre-termination, the inserted internal DNA fragment was designed
as (NNY)8, (N = A/G/T/C, Y = C/T), which will not encode any
of the three stop codons: TAA, TAG, and TGA. Further, we
symmetrically explore the potential internal sequence binding
properties of a panel of PDZ domains by screening this special
library in the Y2H assays.
Materials and Methods
Preparation of bait plasmids
The information of 24 PDZ domains from 18 proteins was
shown in Table S1. The boundaries of the PDZ domains were
mainly determined in the UniProt database. The human ZO-1
PDZ1-pMBa, PDZ2-pMBa, and PDZ3-pMBa plasmids were
kindly provided by Dr. Ben Giepmans (The Netherlands Cancer
Institute). For cloning other domains, the gene fragment encoding
each domain was respectively amplified by Phusion DNA
polymerase (New England Biolabs, NEB) from commercial
plasmids purchased from ProteinTech Group and Source
BioScience. All sequences of PCR primers were listed in Table
S2. The PCR was performed with an initial denaturation for
1 min at 98uC. Ten PCR cycles were performed: 98uC for 5 s,
47uC for 30 s, and 72uC for 5 s, followed by twenty PCR cycles:
98uC for 5 s, 54uC for 30 s, and 72uC for 5 s, and the final
extension was 3 min at 72uC. The PCR product was digested with
restriction enzymes, and cloned into GAL4 BD vector, pBridge
(Clontech Laboratories, Palo Alto, CA). The recombinant
plasmids were transformed into DH5a (DE3) Escherichia coli and
positive clones were selected for DNA sequencing.
Construction of random octapeptide internal sequence
An oligonucleotide 59-TAG GGG GAATTC GCT (NNY)8 TAC
GGGATCCAT CGA-39 (N is an equal mixture of A, G, T, and C;
Y is an equal mixture of C and T), encoding 8 random codons in
the middle, which flanked by EcoR I and BamH I restriction sites,
was synthesized to construct a random peptide library. PCR was
performed with 240 pmol of the oligonucleotide and 240 pmol of
the primer 59-TCGATGGATCCCGTA-39 in a total volume of
120 ml. The complementary chain of the template was synthesized
by program included denaturation at 94uC for 1 min; followed by
annealing at 42uC for 10 min, and extension at 50uC for 10 min.
After double digestion with EcoR I and BamH I, the products were
recovered by short DNA fragment rapid recovery kit and ligated
into pGADT7 AD vector, which predigested with the same
enzymes. A negative control, in which no insert DNA fragments
were added to the ligation reaction, was performed to monitor
vector self-ligation. The overnight ligation products were desalted
and transformed into electrocompetent cells at 1.8 kv with E. coli
Pulser Apparatus (BioRad). The transformation product was
recovered in 1 mL of LB broth in 37uC for 1 h at 200 rpm.
Transformation product (1 ml) was diluted 1:1000 times. The
diluted product was spread on a pre-warmed LB-Amp plate and
incubated at 37uC overnight until colonies appeared. The
numbers of the colonies were counted and the transformed clones
calculated with: Transformed clones = (Colony number/Volume
plated (ml)) 6dilute times61000 ml/ml . To evaluate the
library, fifty clones were randomly selected for colony PCR with
primer pairs (forward primer,
59-CTATTCGATGATGAAGATACCCCA-39, and reverse primer,
59CTTGCGGGGTTTTTCAGTATCTAC-39). The plasmids
extracted from these clones were further sequenced.
against 10 PDZ domains (Table 2). Sequence information for all
internal PBMs was shown in Table S3.
Y2H screening of the internal peptide library
Each GAL4 BD-fusion bait plasmid was transformed into the
yeast strain CG1945 using the lithium acetate protocol. The
transformants were grown on SD/-Trp plates and lacZ assays
were performed to examine self-activation. The transformants that
had no background growth or had background growth but could
be inhibited by 3-amino-1,2,4-triazole and were also negative for
the LacZ assay were selected for the subsequent screening. The
random octapeptide library was screened following the
MATCHMAKER Two-Hybrid System protocol (Clontech).Approximate
107 Trp+Leu+ transformants were selected on plates with SD
-TrpLeu-His medium in the primary screening and then tested by the
improved LacZ assay in the second screening. 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 .
Analysis of the consensus-binding sequences
Internal PBMs for each positive PDZ domain were aligned with
ClustalX1.83, respectively, and further refined by GeneDoc
software. Consensus sequences were deduced from sequence
Results and Discussion
Construction and evaluation of the nearly random
Y2H screening is usually used as a tool for studying
proteinpeptide and domain-peptide interactions. Different kinds of
libraries were constructed according to the characteristics of the
objective peptide ligands. To explore the internal binding
properties of PDZ domains in the Y2H system, a random library
with highly abundant and diverse internal sequences should be
constructed. To ensure the diversity of the library, the
complementary chain of the template was directly synthesized, but not via
PCR procedure. The double strand cDNA template appeared as a
clear 54-bp band on 15% PAGE gel (Figure S1). The ligation and
subsequent electrotransfection generated ,1.06107 transformed
clones. We evaluated the recombination frequency and diversity of
our library. Fifty clones were randomly selected from the library
and identified by colony PCR for further sequencing. The library
may have a negligible number of empty vectors; however, in some
clones, the insertion sequence was elongated due to multiple DNA
fragments self-ligation (Figure 2). No repetitive insertion sequences
were detected among the fifty clones, and their deduced peptide
sequences were unique. However, as (NNY)8 was designed as the
inserted internal sequence, five amino acids M, K, E, Q, and W
were excluded from their corresponding peptide sequences.
Y2H screening of the internal binding sequences of 24
A total of 24 PDZ domains derived from different proteins that
participate in various biological processes and mediate different
functions were chosen to screen the octapeptide library (Table S1).
Fourteen of the PDZ domains exhibited an ability to bind internal
sequences, of which eight were previously reported and six were
reported here for the first time. No positive clone was probed
Binding specificities of internal sequences for each
positive PDZ domain
(a) ZO1-PDZ1: ZO-1 is a scaffold protein of the tight junction
complex that is known to bind a-actinin-4  and the integral
tight junction proteins occludin and claudins [45,46]. The ZO1-1
domain has been suggested to bind the NS5 proteins of dengue
virus (DENV) via an internal sequence recognition mechanism
. After alignment of the internal PBMs of ZO1-1, a consensus
can be deduced as: [I/V]-[T/S/V]-T-[Y/C]-F, where bold
indicates high frequency (Figure 3A). The most important feature
of the consensus is that the aromatic amino acid F and polar
amino acid T were relatively conserved. The internal sequence
binding specificity of ZO1-1 was similar to its core C-terminal
PBM obtained from phage peptide screening, W-[T/S/R/K]-[S/
T]-[Y/W]-[V/I/L]COOH (where W is a hydrophobic residue)
[41,48], but some differences were found between them at
particular sites. For example, the last position of the internal
motif preferred the aromatic amino acid F, but not the aliphatic
amino acids V, I, and L. Also, within the internal motif, the polar
amino acid T, rather than S, was preferred. The nature of the
hydrophobic residue at b2-1 was previously suggested to be a key
determinant of the specificity of the last position (P0) of the
Cterminal motifs of PDZ domains . As ZO1-1 contains an Ile at
b2-1, it can likely accommodate the large Phe at P0 with a less
buried surface. For C-terminal PBMs of ZO1-1, both W and Y
were preferred at P21, which was shown to interact with the Asp in
position b3-5 . As our system ruled out the aromatic residue
W, the amino acid with similar properties, Y, appeared at this
position of the internal consensus. Alternatively, ZO1-1 readily
accepted another internal peptide, AAAAYIIS (not shown in
Figure 3A), which differed from the core consensus and presented
as a non-consensus internal ligand for this domain. The
Nterminal side of four internal sequences (#2, #3, #4, and #6)
contained charged amino acid residues (H, D, R), indicating that
they might interact with other charged residues within the loops
between b2 and b3, or b4 and a2 of the PDZ domain, similar to
the Par-6 PDZ-Pals internal ligand interaction .
(b) GOPC-PDZ: GOPC has been reported as a canonical class I
PDZ protein that can bind to SSTR5 (-QTS[K/R][I/L]*),
cadherin 23 (-ITEL*), hFrizzled 8 (-LSQV*), and neuroligin
(STTRV*) (* represents the stop codon of the peptide) via its PDZ
domain . In the present study, GOPC-PDZ exhibited the
ability to interact with internal sequences, which can be described
as an apparent consensus:
[VILS]-T-C-[FL]-C-[SH]-R-C(Figure 3B). Similarly, within the consensus, an analogous class I
PBM, T-C-[FL], was observed. The amino acid composition was
relatively conservative at several positions, such as T other than S,
and L or F, but not other hydrophobic amino acids, were
respectively preferred at the second and fourth positions of the
internal consensus. In addition, residue C appeared frequently,
interspersed in the internal sequences. At P21, the relatively large
amino acids K, R, E, and Q were selected in canonical C-terminal
ligands of GOPC-PDZ, whereas the small amino acid C was
strictly preferred at the corresponding site of the putative internal
binding motif T-C-[FL]. Similar to ZO1-1, we found that
GOPCPDZ also contained an Ile at b2-1, which may be beneficial for
accommodating a large amino acid, such as Phe or Leu, at
position 4 of the internal motifs.
(c) RIMS2-PDZ: RIMS2 (Rab-3-interacting molecule 2) is a
scaffold protein of the pre-synaptic active zone, which is involved
in exocytosis and synaptic plasticity. It has previously been
Figure 2. Library quality control by colony PCR. M: DNA ladder 2000 plus; lanes 124: PCR product of 24 randomly selected transformants from
the internal octapeptide library, respectively.
suggested that the hScrib-4, ZO1-1, and RIMS2-1 PDZ domains
can recognize internal sequences due to their larger flexible
carboxylate-binding loop . The RIMS2-PDZ domain was
found to associate with the NS5 protein of tick-borne encephalitis
virus (TENV) via an internal PBM . Here, we further
demonstrated the internal sequence binding ability of RIMS2-1.
The consensus was deduced to be [VIT]-F-X-[VT]-X-W, where X
is any type of amino acid (Figure 3C). The internal sequences
appeared to be interspersed with hydrophobic amino acids F, V,
and W. Unlike ZO1-1, no C-terminal sequence was screened out
from our system against this PDZ domain (Table 3).
(d) Dvl2-PDZ: Previous studies have revealed that the
Dvl2PDZ binding site is inherently flexible and can accommodate
different types of ligands . The C-terminal PBMs of this
Reported to bind internal PBMs
Confirmed to bind internal PBMs
domain can be summarized as VWGWF (V is an aromatic amino
acid) , and its internal PBMs (i.e. WKDYGWIDGK,
SGNEVWIDGP, etc.) were also characterized by phage display
screening . The preference of W in both types of ligands
indicated that this aromatic amino acid may play a crucial role in
recognition. Here, we identified another two internal binding
patterns of Dvl2-1 (Figure 3D). The consensus of the first pattern
was W-[VTI]-L-Y-[SG]-S-W (Figure 3D, upper panel), in which
the two amino acids L and Y were conserved and two hydrophilic
residues ([SG]-S) flanked by hydrophobic residues. The consensus
of the second pattern was L-X-[HD]-X-X-D-H-I (Figure 3D,
lower panel). Both patterns differed from the internal PBMs
screened from the phage display library , further
demonstrating the flexibility of the ligand binding site of the Dvl2-PDZ
(e) LNX2-PDZ1: Ligand of numb protein-X2 (LNX2) protein
contains four PDZ domains that might target specific substrates to
mediate ubiquitin ligase activity . The ligand binding
# of internal PBMs
# of C-terminal PBMs
specificity of the PDZ domains of LNX2 has not been studied
previously. In this study, for the first time, a total of 17 unique
internal PBMs were identified for the first PDZ domain of LNX2,
from which a consensus was deduced as W-Tx-W-Tx-W (Figure 3E),
which can regarded as two canonical class II C-terminal PBMs in
(f) HtrA2-PDZ: The serine protease HtrA2/Omi helps maintain
mitochondrial function by handling misfolded proteins in the
intermembrane space. The PDZ domain within the protein
recognizes both C-terminal and internal PBMs of extended,
hydrophobic tri-peptides in the phage-display system . In the
present study, the internal PBMs of HtrA2-PDZ were also
composed mainly of hydrophobic polypeptides (Figure 3F).
Though hydrophobic sequences were favored by HtrA2-PDZ in
both the phage-display and yeast systems, the amino acid W might
play a key role in the internal PBMs when screened in the
phagedisplay system, whereas the residues of I, L, V, and Y were more
preferred in the internal PBMs probed here. This difference may
be caused by the loss of the amino acid W in our library.
Nevertheless, the internal PBMs were relatively diverse, and no
consensus was deduced, indicating that HtrA2-PDZ may have
relatively strong plasticity, exhibiting low specificity for internal
(g) Syntenin1-PDZ2: Syntenin is a multifunctional intracellular
adaptor protein with two adjacent PDZ domains. The PDZ
domains were not restricted to binding a unique sequence, but
were capable of binding multiple peptide motifs . The binding
specificity of the syntenin1-2 PDZ domain was determined by the
combination of three binding pockets that accommodate different
motif structures via an induced-fit mechanism . Here, two
types of internal PBMs were detected against the PDZ domain,
with preference for hydrophobic amino acids, such as A, I, V, and
L. The consensus of the first group was deduced as
W-A-I-X[ALV]-[IV] (Figure 3G, upper panel), which includes two tandem
canonical class II PBMs, i.e. W-A-I and I-X-[ALV]. The consensus
of the second group was defined as W-R-W-W-W, which can be
regarded as two tandem canonical class II PBMs, i.e. W-R-W and
W-W-W (Figure 3G, lower panel).
(h) Par6A: Par6 is a cell polarity protein containing a single PDZ
domain that has been shown to bind both C-terminal (-XEXLV/
I-COOH) and internal (-XEXAVDX-) motifs , which denotes
that the negatively charged amino acid E may be important for
both recognition events. However, this amino acid was not
encoded by internal fragment in our system. Nevertheless, the
internal PBMs were detected for Par6A-PDZ, which contains
extensively hydrophobic tri-peptides that end with the conserved
aromatic amino acid F (Figure 3H). One of these internal binding
sequences, #1 (-SDRGPFFP-), exhibited stronger activity in the
LacZ assay than any other sequences, indicating that a strong
interaction may occur between this PBM and the Par6A-PDZ
(i) Whirlin PDZ3: Whirlin is a multi-PDZ scaffold protein, and
defects in this protein have been shown to be involved in deafness
and short cochlear hair cell stereocilia in mice and recessive
deafness (DFNB31) in humans . The third PDZ domain of
whirlin has been suggested to interact with MPP1 via an atypical
C-terminal PBM (-PQWVPVSWVY*) as well as an additional
internal interaction mechanism . We confirmed that this
domain could bind the internal sequences with strong preference.
The core of the consensus was W-T-[FLV]-I (Figure 3I), which can
be regarded as two classes of tandem PBMs: W-T-[FLV] belongs
to class II motif, whereas T-[FLV]-I can be viewed as a class I
motif. Based on the observations that the atypical C-terminal PBM
derived from MPP1, -PQWVPVSWVY*, which can be viewed as
a class I motif if the last two amino acids were treated as one large
hydrophobic residue, and the C-terminal PBM of this PDZ
domain from Myo15A (-ITLL*), which is also a class I motif ,
we postulated that the motif -T-[FLV]-I- is the main interface for
the internal recognition.
(j) Harmonin PDZ1: The harmonin-1 PDZ domain binds the
C-terminal PBM (-LTFF*) of harmonin and an internal PBM of
cadherin 23 . Although this domain prefers a class I
Cterminal PBM similar to that of GOPC-PDZ, it is capable of
binding more diverse internal PBMs than the GOPC-PDZ
domain. Two types of binding specificities have been observed
for the harmonin-1 PDZ domain in this study. In the first group, a
conserved aromatic amino acid (i.e., F) was located in the middle,
three hydrophilic or negatively charged amino acids (e.g., S or D)
on the N-terminal side of F, and four hydrophobic residues (e.g.,
V, I, L, and F) predominantly on the C-terminal side of F
(Figure 3J, upper panel). A mimic Class I PBM (-S/T-X-F-) was
present in sequences #1, #2, and #5, and another mimic Class I
PBM (-SIV-) located at the extreme N-terminus of sequence #3.
In the second group, two similar internal PBMs differed from
group I but still contained a mimic Class I PBM (-S-A/G-C-) in
the middle of these motifs (Figure 3J, lower panel), suggesting that
harmonin-PDZ1 may be flexible enough to accommodate other
kinds of internal PBMs.
(k) Only one internal sequence was screened out from the
library when the PDZ domains NHERF1-1, NHERF1-2, DlG5-3,
and Par3L-1 were used as bait sequences, respectively (Figure 3K).
For the NHERF1-1 PDZ domain, four C-terminal motifs tailed
with -S/T-R-L* were probed (Table 3 and Table S3).
Characteristics of the internal binding recognition of PDZ
According to the intrinsic properties of the internal PBMs, they
can be divided into three major categories: cryptic class I, such as
those from ZO1-1, GOPC-1, harmonin-1, and whirlin-3, in which
obvious motifs similar to C-terminal Class I PBMs could be found;
cryptic class II, such as those from RIMS2-PDZ, HtrA2-PDZ, and
syntenin1-2 in which the composition of the sequences were
almost hydrophobic amino acids, or similar to those from LNX2-1
in which the hydrophobic amino acids were separated by
hydrophilic residues (all of the internal PBMs may inherently
mimic the canonical Class II C-terminal PBMs); cryptic class III,
such as those from DVL2-1 and Par6A-1, in which diverse internal
PBMs were detected.
C-terminal PBMs were probed against several PDZ domains
(Table 3) due to the unexpected errors that occur during
oligonucleotide synthesis or multiple DNA fragments being cloned
into one vector; both situations may lead to pre-termination or
elongation of the peptides. However, these unexpected results
provided an opportunity for us to compare the preference or
strength of recognition between the C-terminal and internal PBMs
of PDZ domains. For example, the ZO1-1 domain can bind both
C-terminal and internal PBMs with similar peptide numbers
(Table 3 and Table S3). As internal PBMs were predominant in
this special library, this result suggests that ZO1-1 may prefer to
bind C-terminal PBMs. More importantly, this result also hints
that uncovering the internal binding pattern for PDZ domain is
difficult if a conventional cDNA or genome library with
highabundance, diverse types of unique C-terminal ligands is screened.
The proportion of hydrophobic residues in the internal PBMs
was higher than that of hydrophilic ones, indicating that, for
internal recognition, hydrophobic amino acids may be preferred
by the binding sites of PDZ domains. In contrast, some charged
amino acids (e.g., D, R, and H) are usually located at the
Nterminus or C-terminus of the internal PBMs, such as those from
the ZO1-1, syntenin-1, and harmonin-1 PDZ domains. The
presence of these amino acids may be consistent with the
Par-6PDZ domain recognizing the internal sequence HREMAVDCP
from the Pals1-PDZ domain, in which the negatively charged
amino acid Asp forms salt bridge with the positively charged Lys
residue from the carboxylate binding loop of Par-6-PDZ, and the
salt bridges induces a conformational change of the carboxylate
binding loop, resulting in the interaction . Thus, the charged
amino acids within the internal PBMs may play a similar role as
Asp does in the sequence of HREMAVDCP.
No internal motifs were probed for some PDZ domains, which
could be caused by the deficiency of five amino acids in our system
as mentioned above. The Scrib-PDZ4 domain has shown an
ability to bind to TBEV-NS5 via a predominant internal motif,
219EMYYS223 , but no positive clones were probed in our
system. It might be lacking of some specific types of residues, such
as E and M, which led to the failure. Based on the C-terminal
binding properties of ZO1-3 and CASK-1, we tried to explain why
no internal PBM was detected against these two domains from our
library. The C-terminal consensus of ZO1-PDZ3 screened out
from the phage system (WV[S/T]DWY-COOH) belongs to a Class
3a motif  in which the amino acid W was overwhelmingly
preferred at the P21 and P25, indicating that the interactions with
some the binding sites within the pocket of PDZ domains were
highly specific. The C-terminal binding properties of the
CaskPDZ domain (WXVFDV-COOH) were determined in the phage
display system, in which the P25 site was ready to accommodate
the amino acid W. Thus, the absence of particular amino acids,
such as W, may be an obstacle for probing the internal PBMs for
these domains. Another reason may be that these PDZ domains
may prefer longer internal PBMs, so a library with internal
peptides of 10 or 12 residues may suit for screening these PDZ
Although the C-terminal binding properties of two PDZ
domains tend to be similar, such as ZO1-1 and HtrA2-1, or
GOPC-PDZ and harmonin-1, their internal binding properties
may be different in regards to several aspects, which indicates that
the diversity in PDZ domain binding properties can be more easily
revealed by studying their preferred internal sequences.
Using the SP score to evaluate the internal binding ability
of PDZ domains
The specificity potential (SP) score was used to measure the
position specificities of C-terminal ligands in a previous study .
The SP score ranged from least specific (0, any amino acid is
recognized) to most specific (1, only a single amino acid is
recognized). The SP score could be an indication of the flexibility
of the binding groove of the PDZ domain. The lower SP score
suggests higher plasticity of the PDZ pocket, which accommodates
more kinds of ligand residues. We introduced the SP score for
Cterminal PBMs determined in the phage-display system  to
assess the internal sequence binding ability of PDZ domains. From
the SP score for human PDZ domains (Table S4), the SP score for
P0 and P21 of the C-terminal ligands of CASK-1 was 0.93 and
0.89, respectively; the SP score at the same ligand position of
ZO13 was 0.80 and 0.98, respectively. No internal PBM was probed for
these two domains in our system. In contrast, some PDZ domains
that can bind internal PBMs, such as DVL2-1, ZO1-1, SNTA1-1,
HtrA1-1, and HtrA2-1, had low SP scores (,0.70) at the P0 and
P21 positions of their C-terminal PBMs (Table S4). However, the
SP score of P22 might not correlate with the plasticity of the PDZ
domain, such as the C-terminal MPBs of DVL2-1 and CASK-1,
which both had SP scores of 0.98 at this position. Therefore, an SP
score for the P0 and P21 ligand position of ,0.70 might be a
threshold for PDZ domains binding to internal PBMs. Based on
this hypothesis, some PDZ domains, such as DLG4-3, Shank3-1,
MPDZ-3, PTPN13-4, and MLLT4-1 may bind internal PBMs.
Here, we focused on the interaction patterns between PDZ
domains and internal peptide motifs of their ligands. A systematic
strategy based on Y2H screening of a special octapeptide library
was developed for characterization of the internal sequences
binding properties of a panel of PDZ domains. Among them, 14
PDZ domains were confirmed to bind to the internal sequences,
most of them exhibited obvious consensus against one particular
PDZ domain, and showed that the internal binding properties
were diverse among different PDZ domains. These data suggest
that the internal PBMs binding ability of PDZ domains may have
been underestimated. The specificity potential (SP) score has
potential to predict the internal PBMs binding ability of PDZ
Figure S1 Double strand cDNA template separated on
15% PAGE gel. M: GeneRuler Ultra Low Range DNA Ladder;
lane 1: 54-bp double strand cDNA template for library
General information for 24 PDZ domains.
Primers used for molecular cloning in this
Table S3 Detailed sequence information of the
Cterminal and internal PBMs screened from the special
peptide library against 14 PDZ domains.
Conceived and designed the experiments: YM SH YG. Performed the
experiments: YM PC SM. Analyzed the data: YM PC YG. Wrote the
paper: YM PC YG.
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