From the Departments of Physiology and
¶ Biology, and the
Macromolecular Interactions Facility,
University of North Carolina at Chapel Hill, Chapel Hill, North
Carolina 27599 and ** Department of Pharmacology, University of
Wisconsin-Madison, Madison, Wisconsin 537060
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ABSTRACT |
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Syntrophins, a family of intracellular peripheral
membrane proteins of the dystrophin-associated protein complex (DAPC),
each contain a single PDZ domain. Syntrophin PDZ domains bind
C-terminal peptide sequences with the consensus
R/K-E-S/T-X-V-COOH, an interaction that mediates
association of skeletal muscle sodium channels with the DAPC. Here, we
have isolated cyclic peptide ligands for syntrophin PDZ domains from a
library of combinatorial peptides displayed at the N terminus of
protein III of bacteriophage M13. Affinity selection from a library of
X10C peptides yielded ligands with the
consensus
X-(R/K)-E-T-C-L/M-A-G-X--C,
where
represents any hydrophobic amino acid. These peptides contain
residues (underlined) similar to the C-terminal consensus sequence for
binding to syntrophin PDZ domains and bind to the same site on
syntrophin PDZ domains as C-terminal peptides, but do not bind to other
closely related PDZ domains. PDZ binding is dependent on the formation
of an intramolecular disulfide bond in the peptides, since treatment
with dithiothreitol, or substitution of either of the two cysteines
with alanines, eliminated this activity. Furthermore, amino acid
replacements revealed that most residues in the phage-selected peptides
are required for binding. Our results define a new mode of binding to
PDZ domains and suggest that proteins containing similar
conformationally constrained sequences may be ligands for PDZ
domains.
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INTRODUCTION |
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PDZ1 domains are 80-90-amino acid modules present in numerous eukaryotic proteins. They were first described as a series of three internal, repeated elements within the post-synaptic density (PSD)-95 protein (1). In fact, the name PDZ is derived from three proteins first recognized to contain repeats of this domain: PSD-95; the Drosophila discs-large tumor suppressor protein, Dlg; and the mammalian tight-junction protein zona occludens-1, ZO-1 (2-5). PDZ domains have since been identified in a large number of multifunctional proteins, many of which are associated with specialized regions of cell to cell contact such as tight junctions, septate junctions, and synaptic junctions (6). The PDZ domain may be an evolutionarily old domain, as it has been detected in mammalian, nematode, yeast, plant, and bacterial genomic sequences by computer analysis (7).
PDZ domains mediate protein-protein interactions by at least two
distinct mechanisms. Certain PDZ domains bind directly to specific
recognition sequences at the C terminus of transmembrane proteins. For
example, the second PDZ domain of PSD-95 interacts with an
S/T-X-V-COOH motif in N-methyl-D-aspartate
receptor 2B subunits (8, 9) and in Shaker-type potassium channels (10). PDZ domains can also form heterotypic dimers with other PDZ domains. For instance, the N-terminal region of nNOS, which itself contains a
PDZ domain, binds directly to PDZ domains in both PSD-95 and 1-syntrophin, a component of the dystrophin-associated
protein complex (DAPC) (11). A third possible mode of interaction, in which the consensus binding sequence is located internally, is suggested by the observation that the PDZ domain in the
Drosophila InaD photoreceptor protein interacts with a
S/T-X-V sequence near, but not at, the C terminus of the
transient receptor potential Ca+2 channel (12). More
recently, the PDZ domain in the actinin-associated LIM protein has been
shown to bind to unidentified sequences within the internal
spectrin-like repeats of
-actinin (13). Thus, PDZ domains may take
part in several diverse types of interactions.
The structures of several PDZ domains, alone and complexed with
peptides, have been deduced (14-16). The PDZ domain has an overall
structure very much like the phosphotyrosine binding domain, even
though they are unrelated in function (17, 18). Essentially, it is a
globular domain formed by six strands (designated
A-
F) and
two
helices (designated
A and
B) arranged into an up-and-down
-barrel (14, 15). Analysis of the crystal structure of the third PDZ
domain of Dlg revealed a groove on the surface that ends in a conserved
hydrophobic pocket created by the
B strand, the
B helix, and a
loop connecting the
A and
B strands (15). The crystal structure
of a S/T-X-V-COOH peptide ligand complexed with the third
PDZ domain of PSD-95 revealed that the peptide binds within this groove
and that the terminal carboxylate group of the peptide inserts into the
hydrophobic pocket (14). The specificity of binding is determined by
the interaction of certain residues in the PDZ domain with side chains
of the last three or four residues of the C-terminal peptide.
The specificities of several different PDZ domains have been defined
using combinatorial peptide libraries (16, 19, 20). The peptide ligand
preferences of one family of nine PDZ domains was shown to be
X-S/T-X-V/I-COOH, where X was either
any, or a preferred amino acid, depending on the PDZ domain (19).
Recently, the PDZ domain of 1-syntrophin was shown to be
selective for peptides with a similar motif,
R/K/Q-E-S/T-X-V-COOH (16). The C termini of two skeletal
muscle sodium channels (NaChs) conform to this consensus and bind
directly to the PDZ domains of
1-,
1-,
and
2-syntrophins (21). This interaction mediates the association of NaChs with the DAPC and links them to the cortical actin
cytoskeleton and the extracellular matrix.
In this report, we utilized phage-displayed combinatorial peptide
libraries to define the specificities of the PDZ domains of
1-,
1-, and
2-syntrophin.
We have isolated novel peptide ligands for syntrophin PDZ domains which
conform to the consensus sequence
K/R-E-(S/T)-X-(V/L/I/M)-COOH previously determined to bind
syntrophin PDZ domains, but lack a C-terminal carboxylate group.
Instead, these peptides are able to interact with syntrophin PDZ
domains when fused to either the N terminus of a bacteriophage M13
capsid protein or bacterial alkaline phosphatase (AP). Cyclization of
these peptides through intramolecular disulfide bond formation was
found to be essential for binding to syntrophin PDZ domains since
reduction with DTT, or replacement of cysteines with alanine, eliminated this interaction. These cyclic peptide ligands appear to be
specific for syntrophin PDZ domains since several other closely related
PDZ domains do not bind them. Additional experiments showed that these
peptides bind to the same site on syntrophin PDZ domains as C-terminal
peptides and suggest that they act as C-terminal peptide mimetics. Our
results define a novel mode of binding of peptide ligands to PDZ
domains and suggest that proteins containing similar conformationally
constrained peptide sequences may be capable of interacting with other
PDZ domain-containing proteins.
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EXPERIMENTAL PROCEDURES |
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Isolation and Characterization of Syntrophin PDZ Domain-binding
Phage--
Glutathione S-transferase (GST) fusion proteins
of the PDZ domain of mouse 1-,
1-, and
2-syntrophins were prepared in the following manner.
Oligonucleotides were designed to flank the PDZ domains and the regions
were selectively amplified from cDNA clones by polymerase chain
reaction (22). The amplified segments were subcloned between the
BamHI and EcoRI sites of pGEX (Amersham Pharmacia
Biotech) and recombinants were verified by DNA sequencing. GST fusion
proteins were expressed and purified according to the manufacturer's
instructions.
Construction and Testing of AP Fusion Proteins--
The insert
of the phage isolate F was amplified by the polymerase chain reaction
with flanking oligonucleotides, digested with SalI and
XbaI, and ligated into pMY101 (24). Bacteria, harboring the
resulting recombinant plasmid, secreted a chimeric protein with the
peptide fused to the N terminus of Escherichia coli AP into
the culture medium, upon induction with
isopropyl--D-thiogalactopyranoside. The conditioned
medium was then added to microtiter plate wells coated with various
proteins, with or without soluble competitor peptides, and the amount
of enzyme retained in the wells after 1 h incubation was
quantitated with the colorimetric substrate, p-nitrophenyl
phosphate. Optical absorbance of wells was measured at 405 nm
wavelength with a plate reader (Molecular Devices, Sunnyvale, CA).
Alanine Scan of the Syntrophin PDZ Domain Ligand--
Each amino
acid of one ligand sequence, AKETCLAGYYC, was independently substituted
by alanine and fused to the N terminus of AP using standard molecular
biological methods. A peptide ligand-AP fusion, in which the two
cysteines were substituted with serines, was generated in the same
manner. All 12 chimeric proteins were tested for their ability to bind
GST fusion proteins of the 1-,
1-, and
2-syntrophin PDZ
domains immobilized on microtiter plates. Wells were coated with 0.5 µg of the purified syntrophin PDZ domain fusion proteins or with
anti-FLAG M1 antibody (Boehringer Mannheim), and blocked with 1%
bovine serum albumin in 0.1 M NaHCO3, pH 8.3, for 1 h at room temperature. Wells were incubated for 2 h at
room temperature with bacterial culture media conditioned with each of
the peptide ligand-AP fusions (24). After extensive washing with
phosphate-buffered saline, pH 7.4, and 0.1% Tween 20, the amount of
the AP retained in the wells was quantitated after addition of
p-nitrophenyl phosphate by measuring optical absorbance at 405-nm wavelength.
Blot Overlay and Affinity Purification Experiments-- N-terminal biotinylated peptides corresponding to the phage F insert (AKETCLAGYYCS-COOH), the C terminus of the SkM1 skeletal muscle sodium channel (PGQTVRPGVKESLV-COOH) and the C terminus of Fas (ENSNFRNEIQSLV-COOH) were synthesized. All synthetic peptides contained an N-terminal three amino acid (SGS) linker sequence. The identities of all peptides were determined by mass spectroscopy. Peptides were dissolved either in water, or in Me2SO, then diluted with water until the organic solvent was less than 1%. The binding of the peptides to various proteins was followed in two ways. Overlay assays with biotinylated peptides (used at a final concentration of 1 µM) were performed as described previously (21). Isolation of syntrophins from skeletal muscle extracts with peptides coupled to streptavidin-linked beads (Sigma) was carried out as described previously (21).
Mass Spectrometry-- Measurements of peptide F mass were performed on a Micromass Quattro II triple quadrapole electrospray ionization mass spectrometer. Peptide F was dissolved in neat methanol and infused directly into the electrospray source. The m/z range for data collection was set from 700 to 2000.
Surface Plasmon Resonance Measurements-- All experiments were performed on a BIAcore 2000 instrument (BIAcore Inc., Piscataway, NJ) at the University of North Carolina Macromolecular Interactions Facility. Biotinylated peptides were immobilized on Neutravidin (Pierce)-coated sensor chips as described previously (21). Increasing concentrations of syntrophin PDZ domains were injected onto the peptide surfaces at a flow rate of 20 µl/min for 2 min in 20 mM HEPES, pH 7.5, 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P-20. Between successive measurements the surfaces were regenerated with 10 mM HCl (2-min contact time). Binding to Neutravidin alone was also measured and was used to subtract nonspecific interactions. Blank sensorgrams recorded for injection of running buffer were also subtracted. Background corrected sensorgrams were fitted to a single site binding model using the numerical integration functions of the BIAevaluation 3.0 software (BIAcore AB, Uppsala, Sweden). The steady state binding value, Req, for each PDZ analyte concentration was then plotted as a function of analyte concentration. To derive binding constants, data from these plots were analyzed by the steady state affinity binding model provided in the BIAevaluation software (25-27). The reported values are the average of four separate experiments.
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RESULTS |
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Isolation of Syntrophin PDZ Domain-binding Phage--
Three
syntrophin isoforms, 1,
1, and
2, are encoded by separate genes but have an identical
domain organization (28, 29). Each syntrophin contains a single PDZ
domain, which is 78-84% identical in primary structure among the
three isoforms (28). To identify peptide ligands for syntrophin PDZ
domains, we screened a bacteriophage M13 library displaying
combinatorial peptides of the type X12 (where
X is any amino acid encoded by NN(G/T) codons) with a
recombinant GST fusion protein of the
1-syntrophin PDZ
domain. After three rounds of affinity selection, individual phage
isolates were tested for binding. No positive isolates were selected
using this library. We next screened a library of the type
X10C, where every peptide had 10 random residues
followed by a fixed cysteine residue. Seven independent phage were
isolated from a library of 109 different peptides that
encoded three different, but related peptide sequences (Fig.
1). The peptides share the consensus
sequence X-(R/K)-E-T-C-(L/M)-A-G-X-
-C-COOH,
where
represents a hydrophobic residue.
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Specificity of Phage Peptide Isolates--
Next, we determined the
specificity of three phage isolates, each representing a unique peptide
sequence, for different PDZ domains. Microtiter wells coated with
equivalent amounts of GST fusion proteins of 1-,
1-, and
2-syntrophin PDZ domains, as well
as a fusion protein containing the PDZ domain of nNOS (amino acids
1-150), were incubated with equivalent amounts (1010
plaque-forming units) of phage particles. The binding of the phage
particles was monitored by ELISA. Positive and negative control targets
consisted of GST fused to the Src homology 3 (SH3) domain and GST
alone, respectively. Phage isolates B, D, and F bound best to the
2-syntrophin PDZ domain and nearly equivalently to the
PDZ domains of
1- and
1-syntrophin (Fig.
2A). In contrast, the three
phage isolates did not bind to either the nNOS PDZ or Src SH3 domains
above background (i.e. GST alone). Moreover, phage displaying a peptide ligand for the Src SH3 domain bound to the GST-Src
SH3 domain but not to any of the PDZ domains tested. Thus, although the
peptides were selected with the
1-syntrophin PDZ domain,
they bound better to the
2-syntrophin PDZ domain than to
either
1 or
1. These differences in
binding may reflect either the different specificities of the PDZ
domains or the ability to retain their native conformations when
immobilized on plastic.
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Isolated Peptides Require Conformational Constraint for
Binding--
Even though the location of only one cysteine was
specified within the X10C peptides, all three
isolates had a second cysteine spaced six residues from the first. To
determine whether or not these cysteine residues were disulfide-bridged
in the phage-displayed peptides, we tested the effect of the reducing
agent DTT on binding. Equivalent numbers of phage particles were
incubated in microtiter wells coated with approximately equal amounts
of the 1-syntrophin PDZ domain fusion protein and the
extent of binding was determined by phage ELISA. As shown in Fig.
3, binding of three phage isolates (B, E,
and F) to the target decreased with increasing DTT concentration. Fifty
percent inhibition (IC50) was observed at approximately 2-5 mM DTT. Since PDZ domains lack disulfide bonds, this
decreased binding can be attributed to the reduction of disulfide bonds in the displayed peptides by DTT. In contrast, the binding of a Src SH3
domain-binding phage (30) to the Src SH3 domain was unaffected over the
same range of DTT concentrations. In this case neither the SH3 domain
nor the peptide ligand have cysteines. The binding of the peptide F-AP
fusion protein to syntrophin PDZ domains was also DTT sensitive (not
shown) suggesting that it forms disulfide bonds as well. These data
suggest that conformational constraint imparted by the disulfide bond
in these peptides is required for binding to syntrophin PDZ
domains.
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Isolated Cyclic Peptides are Structural Mimics of C-terminal
Peptides with an S/T-X-V Motif--
We recently reported that peptides
corresponding to the C terminus of two skeletal muscle sodium channels
(designated SkM1 and SkM2) with the motif
R/K-E-S/T-X-V-COOH, bound strongly to syntrophin PDZ domains
(21). Schultz et al. (16) obtained a similar consensus
(R/K/Q-E-S/T-X-V-COOH) for 1-syntrophin PDZ domain ligands using combinatorial peptide libraries. Together, our
results define the ligand preferences of syntrophin PDZ domains. Close
inspection of the consensus sequence in Fig. 1
(X-R/K-E-T-C-L/M-A-G-X-
-C) revealed a remarkable likeness (residues underlined) to the C-terminal consensus for binding to syntrophin PDZ domains. To test the hypothesis that the phage-displayed peptides isolated here bind syntrophin PDZ
domains at the same site as C-terminal peptides, peptide F-AP and Src
SH3 ligand-AP fusion proteins were added to microtiter wells coated
with the
1-syntrophin PDZ domain fusion protein in the
presence of increasing concentrations of soluble C-terminal peptides.
Binding of peptide F-AP to the
1- syntrophin PDZ domain was blocked by the SkM1 peptide (K-E-S-L-V-COOH) but not by a peptide
corresponding to the C terminus of the Fas antigen (I-Q-S-L-V-COOH), a
ligand for the PDZ domain of the Fas-associated protein tyrosine phosphatase PTPL1/Fas-associated protein-1 (31). The IC50
value of the SkM1 peptide was ~30 µM. Neither of the
two peptides blocked, at any concentration tested, the binding of the
Src SH3 ligand-AP to the Src SH3 domain fusion protein.
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Peptide F Binds Specifically and with High Affinity to Syntrophin
PDZ Domains--
We further defined the specificity of the peptide F
in overlay experiments using several different PDZ domains,
1-,
1-, and
2-syntrophin
PDZ domain fusion proteins, two overlapping nNOS fusion proteins
containing the PDZ domain, and fusion proteins of PSD-93 and PSD-95
containing two and three PDZ domains, respectively. When fusion
proteins, that had been resolved by SDS-polyacrylamide gel
electrophoresis and transferred to nitrocellulose membranes, were
incubated with a peptide corresponding to the C terminus of the
N-methyl-D-aspartate receptor 2B subunit (NR2B),
a strong signal corresponding to the position of the intact PSD-93 and PSD-95 fusion proteins was observed, as expected (8, 9, 32) (Fig.
6, panels CBB and
NR2B). In comparison, binding of the NR2B peptide to
syntrophin PDZ domains was observed only with longer exposures (not
shown), consistent with previous data showing that NR2B peptide binds
weakly to the PDZ domain of syntrophins. As shown in Fig. 6
(panels SkM2 and Peptide F), a significant fraction of all three syntrophin PDZ domain fusion proteins
consistently migrated in SDS-polyacrylamide gels as ~60-kDa bands,
twice the size expected. The basis for this apparent dimerization is
not known. As reported previously, a peptide corresponding to the SkM2
NaCh C terminus bound to both the monomeric and dimeric forms of all
three syntrophin PDZ domain fusion proteins (Fig. 6, panel SkM2) (21). SkM2 also bound to fusion proteins of both PSD-93 and
PSD-95. In contrast, peptide F bound to all three syntrophin PDZ
domains, but not to the PDZ domains of nNOS, PSD-93, or PSD-95 (Fig. 6,
panel Peptide F). None of the peptides tested bound to either of the nNOS PDZ domain fusion proteins or to GST alone. These
results further demonstrate the specificity of peptide F for syntrophin
PDZ domains and that the peptide F sequence can bind syntrophin PDZ
domains when removed from the context of a fusion protein.
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Syntrophin PDZ Domains Cannot Accommodate Additional Residues
following the Hydrophobic Residue in Linear
Peptides--
Collectively, our results suggest that peptide F binds
to the same site on syntrophin PDZ domains as C-terminal peptide
ligands. However, previous studies have shown that PDZ domains cannot
accommodate additional residues following the C-terminal hydrophobic
residue. For example, the addition of an alanine to the C terminus of
Fas abolished binding to the PDZ domains of the protein-tyrosine
phosphatase PTPL1/Fas-associated protein-1 (33). One possibility is
that the peptide binding groove in syntrophin PDZ domains is long or deep enough to contain additional residues. To test this possibility, we synthesized a peptide corresponding to the C terminus of SkM2 with
an additional alanine residue at the C terminus. As shown in Fig.
9, this peptide did not bind to the PDZ
domain of 1-syntrophin. Thus, syntrophin PDZ domains,
like other PDZ domains, cannot accommodate additional residues
following the hydrophobic amino acid at the C terminus.
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DISCUSSION |
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We have used an N-terminal phage-displayed peptide library to identify cyclic peptides with the motif X-R/K-E-T-C-L/M-A-G-X-W/Y-C that bind specifically to syntrophin PDZ domains. These conformationally constrained peptide ligands are similar to the carboxyl-terminal sequence R/K/Q-E-S/T-X-V-COOH previously demonstrated to bind syntrophin PDZ domains (16, 21), except that, in these peptides, this sequence lacks a free carboxylate group which has been shown to contribute significantly to the peptide-PDZ domain interaction (14). The binding of the phage-displayed peptides is dependent on cyclization of this sequence by a pair of disulfide-bridged cysteines. Our data suggest that these cyclic peptides mimic C-terminal peptides and may be useful as specific inhibitors of syntrophin PDZ domain interactions.
Phage-displayed libraries have been used to isolate high affinity cyclic ligands for other proteins including integrins (34), the erythropoietin receptor (35), the Grb2 SH2 domain (36), and calmodulin (37). In most cases these peptide ligands do not resemble the primary sequence of the natural interacting proteins. Instead, they act as structural mimics. The intrachain disulfide bonds of these peptides constrain them into a conformation that is favorable for strong binding. Accordingly, we found that substitution of both cysteines with serines, or of either cysteine with alanine, completely eliminated the syntrophin PDZ-binding activity of one cyclic peptide. Consistent with the idea that the cyclic conformation of these peptides is important, we also found that syntrophin PDZ domains did not bind any peptides from a library (X12) encoding linear peptides. This negative result is in agreement with Songyang et al. (19), who found that none of the nine PDZ domains they investigated retained specific peptides from a library encoding non-C-terminal linear peptide sequences.
Although the affinity of peptide F for syntrophin PDZ domains was 2-3-fold lower than the SkM2 peptide, the interaction was of sufficient strength to isolate native syntrophins from detergent extracts (Fig. 8). Alanine-substitution of the tyrosine at position 9 of the peptide produced a peptide that bound stronger to all three recombinant syntrophin PDZ domains than the parental sequence. These data suggest that it may be possible to isolate higher affinity peptide ligands for syntrophin PDZ domains by varying the identity of the amino acid at this position. Moreover, these peptides should serve as an excellent starting point for the design of compounds which mimic C-terminal ligands of syntrophin PDZ domains.
Our results are entirely consistent with the idea that the cyclic peptides isolated here bind to syntrophin PDZ domains in the same manner as homologous C-terminal peptides, even though they lack a free carboxylate group. This binding is contingent upon a constrained conformation provided by an intramolecular disulfide bond which may allow residues following the hydrophobic residue (L/M; which is normally at the C terminus) to bend away from the peptide binding groove. This is likely to be an essential feature of these cyclic peptides since additional residues following the hydrophobic residue in linear peptides cannot be accommodated (Fig. 9). Syntrophin PDZ domains also bind to regions of nNOS that contain a PDZ domain (21). Since the PDZ domain of nNOS is not at the C terminus, this interaction cannot occur via a C-terminal peptide sequence. One possibility is that a constrained sequence in nNOS mediates the binding of the nNOS PDZ domain to syntrophin PDZ domains. However, nNOS does not appear to contain any obvious R/K-E-S/T-X-L/M/V-related sequence within its PDZ domain, or in sequences upstream of the PDZ domain, which are necessary for strong binding to syntrophin PDZ domains (38),2 nor does nNOS contain any cysteines in this region which could potentially form intramolecular disulfide bonds. Nevertheless, it should be kept in mind that syntrophin PDZ domains may bind to sequences where flanking amino acids other than cysteines are capable of inducing a secondary structure conducive to binding.
Collectively, our results suggest that the peptide F sequence is
specific for syntrophin PDZ domains. Rational explanations for this
specificity can be deduced by comparing the solution structure of the
1-syntrophin PDZ domain complexed with a C-terminal peptide (G-V-K-E-S-L-V-COOH) (16) to the crystal structure of the third
PDZ domain of PSD-95 complexed with its peptide ligand (14). Consistent
with the idea that syntrophin PDZ domains bind C-terminal peptide
ligands in a manner similar to PSD-95, amino acids whose side chains
are known to be involved in binding to the general S/T-X-V
motif in PSD-95 are well conserved in syntrophin PDZ domains (14).
Indeed, Schultz et al. (16) have shown that the syntrophin
PDZ domain binding site is structurally conserved. In contrast,
residues that are proposed to determine ligand specificity are strongly
conserved in syntrophin PDZ domains, but vary considerably among PDZ
domains from different families (16). These findings account for the
identical ligand preferences of the three syntrophin PDZ domains (21).
Moreover, small differences in these key residues are likely to explain
the unique ability of syntrophin PDZ domains to bind peptide F.
An unexpected finding of this study was that the SkM2 NaCh C-terminal peptide bound to the PSD-93 and PSD-95 fusion proteins (Fig. 6, SkM2). Since these fusion proteins contain two and three PDZ domains, respectively, we cannot conclude at this time which PDZ domain binds the SkM2 peptide. Interestingly though, of the three PDZ domains in PSD-93 and PSD-95, the second PDZ domain in each most closely resembles syntrophin PDZ domains at the residues described by Schultz et al. (16). These similarities may explain why syntrophin PDZ domains bind to some extent to many of the same C-terminal peptide sequences as the second PDZ domain of PSD-93 and PSD-95, including NR2B, Kv1.4, and SkM2 (21)2 and to the nNOS PDZ domain (11). This observation raises the possibility that the PDZ domains of syntrophins, PSD-93, and PSD-95 compete for similar ligands.
In summary, our results suggest that syntrophin PDZ domains can potentially interact with target proteins via three different modes of binding. (i) Syntrophins bind skeletal muscle NaChs and target them to the DAPC through the interaction of the PDZ domain with C-terminal S/T-X-V-COOH sequences (21). (ii) Syntrophins also mediate the membrane localization of nNOS in skeletal muscle by forming presumptive heterodimers with a N-terminal PDZ domain in nNOS (11). (iii) Syntrophin PDZ domains may also be capable of interacting with internal, conformationally constrained, sequences in other proteins. Such proteins may play a role in modulating the localization or function of nNOS and NaChs in skeletal muscle or brain.
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ACKNOWLEDGEMENTS |
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We thank Drs. Jay Brenman, Karen Christopherson, and David Bredt for PSD-95, PSD-93, and nNOS constructs.
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FOOTNOTES |
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* This work was supported in part by grants from the National Institutes of Health (to S. C. F.), the Muscular Dystrophy Association (to B. K. K. and S. C. F.), and Novalon Pharmaceutical Corporation (to B. K. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a Human Frontier Science Program Organization Postdoctoral Fellowship.
To whom correspondence should be addressed: Dept. of
Pharmacology, University of Wisconsin-Madison, 1300 University Ave., Madison, WI 53706-1532. Tel.: 608-265-5218; Fax: 608-262-1257; E-mail:
bkkay{at}facstaff.wisc.edu.
The abbreviations used are: PDZ, PSD-95/Dlg/ZO-1; PSD-95, postsynaptic density protein of 95 kDa; Dlg, Drosophila discs-large tumor suppressor proteinZO-1, zona occludens-1DTT, dithiothreitolELISA, enzyme linked immunoabsorbent assayGST glutathione S-transferase, NR2B, N-methyl-D-aspartate receptor 2B subunitnNOS, neuronal nitric oxide synthaseSH3, Src homology 3NaCh, sodium channelAP, alkaline phosphataseDAPC, dystrophin-associated protein complex.
2 S. Gee and S. Froehner, unpublished observations.
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REFERENCES |
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