From the Departments of Protein Engineering,
¶ Assay and Automation Technology, and
Molecular
Oncology, Genentech, Inc.,
South San Francisco, California 94080
Received for publication, September 23, 2002, and in revised form, November 19, 2002
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ABSTRACT |
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The LAP (leucine-rich repeat
and PDZ-containing) family of proteins play a
role in maintaining epithelial and neuronal cell size, and mutation of
these proteins can have oncogenic consequences. The LAP protein Erbin
has been implicated previously in a number of cellular activities by
virtue of its PDZ domain-dependent association with the C
termini of both ERB-B2 and the p120-catenins. The present work
describes the NMR structure of Erbin PDZ in complex with a high
affinity peptide ligand and includes a comprehensive energetic analysis
of both the ligand and PDZ domain side chains responsible for binding.
C-terminal phage display has been used to identify preferred ligands,
whereas binding affinity measurements provide precise details of the
energetic importance of each ligand side chain to binding. Alanine and
homolog scanning mutagenesis (in a combinatorial phage display format)
identifies Erbin side chains that make energetically important contacts
with the ligand. The structure of a phage-optimized peptide
(Ac-TGW In the post-genomic era, identification of interactions between
proteins has become a significant challenge to achieving a comprehensive understanding of cellular biology (1, 2). Although many
such interactions have been described (3), many more remain to be
discovered (4). Research over the past decade has identified many types
of protein-protein interactions that participate in intracellular
signaling pathways (5). A common feature of such pathways is the
involvement of several types of small protein domains (<100 residues)
whose sole function is to recognize sequence motifs presented by other
members of the pathway (6, 7). Single proteins often contain multiple
copies of the same or different protein interaction modules, permitting the formation of the complex, multicomponent assemblies necessary to
transmit a specific signal. Indeed, some proteins contain only protein
interaction modules and may be considered adapters or scaffolds on
which other "active" components of a signaling pathway are brought
into proximity (8). Although these interaction domains are often
readily identified on the basis of primary sequence, identification of
the binding partner relevant to a particular signaling cascade is often difficult.
The PDZ domain, so-called because it was first recognized in the
proteins post-synaptic density-95, discs large,
and zonula occludens 1 (9-11), is a common component of
such scaffold proteins. As many as 440 PDZ domains in 259 different
proteins have been proposed to exist within the human genome (12). PDZ
domains are ~90 residues in size and adopt a common fold consisting
of a Initial studies identified a C-terminal (S/T)XV motif as
being necessary for PDZ binding (14-16). Study of ligands from
synthetically or biochemically derived peptide libraries has revealed a
more extensive and complex picture of selectivity that involves as many
as six C-terminal residues (20-24). These methods also provide an
alternative to protein ligand identification from yeast two-hybrid experiments; data base searches for proteins that have C termini that
match the optimal ligand sequence are potential protein ligands in vivo. The optimized peptide ligands themselves may also
be used to antagonize a particular PDZ domain and observe cellular phenotypes, giving further insight into function (24).
The LAP (leucine-rich repeat and
PDZ-containing) proteins are a recently described family of
scaffold proteins that are involved in the formation of membrane
complexes and the maintenance of epithelial and neuronal cell shape and
polarity (25). For example, in Drosophila, mutation of the
Scribble LAP protein results in loss of epithelial cell polarity and
morphology as well as uncontrolled, tumor-like growth (26). The LAP
proteins have a domain structure comprising 16 N-terminal leucine-rich
repeats and up to four C-terminal PDZ domains. On the basis of yeast
two-hybrid experiments, a mammalian LAP protein that recognizes the C
terminus of ERB-B2 (a member of the epidermal growth factor receptor
family) has been identified and given the name Erbin, for
ERB-interacting protein (27). More recently, we
have used C-terminal phage display (23) to identify optimal ligands for
the Erbin PDZ domain (24). These ligands are quite different in
sequence from the C terminus of ERB-B2 and also bind ~1000-fold more
tightly to Erbin PDZ than the C-terminal peptide from ERB-B2 (24).
In vivo interactions with the p120-like catenin proteins
have been proposed and tested on the basis of these results (24),
whereas yeast two-hybrid screens have also identified these same
interactions (28, 29). These data suggest that LAP proteins are
targeted to p120-catenin-localized junctional regions via a
PDZ-mediated interaction (24).
In the present work, we have explored in detail the interactions
between Erbin PDZ and the optimal phage-derived peptide ligand. A
preference for a penultimate tryptophan residue in the ligand has been
confirmed by extensive phage library selections, a feature that is also
present in the optimal ligand for the second PDZ domain of the
membrane-associated guanylate kinase Magi-3 (23). Affinity measurements
of synthetic peptide analogs of the optimal ligand have been made to
quantitate the energetic contributions of the five C-terminal ligand
residues; the penultimate tryptophan is indeed beneficial to binding
(affinity decreases by >1000-fold when replaced by alanine). An
efficient phage-based combinatorial scanning approach has also been
utilized to identify residues within Erbin PDZ that contribute
energetically to ligand binding (30). Although we have previously
described a homology-based model of a domain of Magi-3 (23), the use of
homology modeling to interpret the structure-activity data is of
limited value because the primary sequence of Erbin PDZ differs in
several key regions from that of other PDZ domains whose structures are
known (Fig. 1). Thus, we have also used
NMR spectroscopy to determine the structure of Erbin PDZ in
complex with the p120-catenin-like
phage-derived ligand.1 This structure provides a
clear view of the interactions made by the penultimate tryptophan
residue and more generally provides a framework within which to
understand the affinity and selectivity of peptide binding to Erbin
PDZ. This work provides one of the most extensive characterizations to
date of the structural and energetic (ligand and protein) components of
a PDZ domain interaction with a C-terminal peptide.
Materials--
Enzymes were from New England Biolabs. Maxisorp
immunoplates and 384-well assay plates were from Nalge NUNC
International (Naperville, IL). Escherichia coli XL1-Blue,
E. coli BL21, and M13-VCS were from Stratagene. Plasmid
pET15b was from Novagen. Thrombin was from Calbiochem. Bovine serum
albumin and Tween 20 were from Sigma. Horseradish peroxidase/anti-M13
antibody conjugate and Superdex-75 were from Amersham Biosciences.
Nickel-nitrilotriacetic acid was from Qiagen. 3,3',
5,5'-Tetramethyl-benzidine/H2O2 peroxidase substrate was from Kirkegaard and Perry Laboratories Inc.
AlphaScreenTM reagents and a plate reader were from
PerkinElmer Life Sciences.
Oligonucleotide Synthesis--
Oligonucleotides for
combinatorial scanning were designed as described previously using
equimolar DNA degeneracies (30). The particular mutagenic
oligonucleotides are listed in Supplementary Table I.
Synthetic Peptides--
The peptides were synthesized using
standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) protocols,
cleaved off the resin with 2.5% triisopropylsilane and 2.5%
H2O in trifluoroacetic acid, and purified by reversed-phase
high performance liquid chromatography. The purity and mass of each
peptide were verified by liquid chromatography/mass spectrometry.
Statistical Analysis of Erbin PDZ Binding
Specificity--
Previously described procedures were used to isolate
peptides that bound to a
GST-Erbin2 PDZ fusion, using
a library of random heptapeptides fused to the C terminus of the M13
gene-8 major coat protein (23, 24). After two rounds of selection,
individual clones were grown in a 96-well format in 500 µl of 2YT
broth supplemented with carbenicillin and M13-VCS, and the culture
supernatants were used directly in phage enzyme-linked immunosorbent
assays (31) to detect peptides that bound specifically to Erbin PDZ. A
total of 148 peptide sequences derived from positive clones were
aligned, and the occurrence of each natural amino acid at each position
was tabulated. The occurrence of each amino acid was normalized by
dividing by the number of times the amino acid was encoded by the
NNS codon. The normalized data set was used to calculate the
percentage of occurrence of each amino acid at each position (see Table
I).
Construction of Libraries for Erbin PDZ Shotgun
Scanning--
Erbin PDZ was displayed on the surface of M13
bacteriophage by modifying a previously described phagemid (pS1602)
(32). Standard molecular biology techniques were used to replace the fragment of pS1602 encoding human growth hormone with a DNA fragment encoding Erbin PDZ. The resulting phagemid (pS2202d) contained an open
reading frame that encoded the maltose-binding protein secretion
signal, followed by an epitope tag (amino acid sequence: SMADPNRFRGKDLGS), followed by Erbin PDZ and ending with the C-terminal domain of the M13 gene-3 minor coat protein. E. coli
harboring pS2202d were co-infected with M13-VCS helper phage and grown
at 37 °C without
isopropyl-1-thio-
Libraries were constructed using previously described methods (31) with
appropriately designed "stop template" versions of pS2202d. For
each library, we used a stop template that contained TAA stop codons
within each of the regions to be mutated. The stop template was used as
the template for the Kunkel mutagenesis method (33) with mutagenic
oligonucleotides (see above) designed to simultaneously repair the stop
codons and introduce mutations at the desired sites.
For shotgun scanning, wild type codons were replaced with the
corresponding degenerate codons shown in Table I of Vajdos et
al. (34). Two separate libraries were constructed with each library designed to mutate 22 Erbin PDZ residues with no overlap between the two. Libraries A1 and A2 were constructed with mutagenic oligonucleotides A1a and A1b or A2a
and A2b, respectively. Library A1 mutated residues in two
continuous stretches of sequence between positions 16-28 and 46-55,
whereas library A2 mutated residues between positions 31-43 and
78-95. For the shotgun homolog scan, libraries H1 and H2 were
constructed in an analogous fashion with mutagenic oligonucleotides
H1a and H1b or H2a and H2b,
respectively. The library diversities were as follows: A1, 4.2 × 1010; A2, 4.0 × 1010; H1, 4.4 × 1010; and H2, 4.2 × 1010.
Library Sorting and Analysis--
Phage from the libraries
described above were propagated in E. coli XL1-blue with the
addition of M13-VCS helper phage. After overnight growth at 37 °C,
phage were concentrated by precipitation with polyethylene glycol/NaCl
and resuspended in PBS, 0.5% bovine serum albumin, 0.1% Tween 20 as
described previously (31). Phage solutions (1012 phage/ml)
were added to 96-well Maxisorp immunoplates that had been coated with
capture target and blocked with bovine serum albumin. Two different
targets were used; for the display selection the target was an
immobilized antibody that recognized the epitope tag fused to the N
terminus of Erbin PDZ, whereas for the function selection a
biotinylated peptide that binds to Erbin PDZ with high affinity
(biotin-TGWETWV) (24) was immobilized on streptavidin-coated plates.
Following a 2-h incubation to allow for phage binding, the plates were
washed 10 times with PBS, 0.05% Tween 20. Bound phage were eluted with
0.1 M HCl for 10 min, and the eluent was neutralized with
1.0 M Tris base. Eluted phage were amplified in E. coli XL1-blue and used for further rounds of selection.
Individual clones from each round of selection were grown in a 96-well
format in 500 µl of 2YT broth supplemented with carbenicillin and
M13-VCS, and the culture supernatants were used directly in phage
enzyme-linked immunosorbent assays (31) to detect phage-displayed Erbin
PDZ variants that bound to either biotin-TGWETWV or anti-tag antibody.
After two rounds of selection, greater than 50% of the clones
exhibited positive phage enzyme-linked immunosorbent assay signals at
least 2-fold greater than signals on control plates coated with bovine
serum albumin. These positive clones were subjected to DNA sequence
analysis (see below).
The sequences were analyzed with the program SGCOUNT as described
previously (30). SGCOUNT aligned each DNA sequence against the wild
type DNA sequence by using a Needleman-Wunch pairwise alignment
algorithm, translated each aligned sequence of acceptable quality, and
tabulated the occurrence of each natural amino acid at each position.
For the function selection, the number of analyzed clones are indicated
in parentheses following the name of each library: A1 (185 clones), A2
(180 clones), H1 (190 clones), and H2 (170 clones). For the display
selection, the following numbers of clones were analyzed: A1 (83 clones), A2 (83 clones), H1 (94 clones), and H2 (96 clones).
DNA Sequencing--
Culture supernatants containing phage
particles were used as templates for PCRs that amplified DNA fragments
containing the Erbin PDZ gene, and these fragments were sequenced as
described previously (34).
Affinity Assays--
An Erbin PDZ construct with GST fused to
the N terminus was prepared as described (24). The binding affinities
of peptides for Erbin PDZ were determined as IC50 values
using the AlphaScreenTM, a bead-based chemiluminescence
assay with optimized concentration of reagents. The IC50
was defined as the concentration of peptide that blocked 50% of the
chemiluminescence arising from the interaction of anti-GST acceptor
beads coated with Erbin PDZ-GST and streptavidin donor beads coated
with biotinylated peptide (biotin-TGWETWV). The assays were performed
at room temperature in white opaque 384-well plates (25 µl/well)
under subdued lighting to reduce nonspecific chemiluminescence. The
assay buffer was PBS, 0.5% Tween 20, 0.1% bovine Purification of Erbin PDZ Protein for NMR Spectroscopy--
A
DNA fragment encoding residues 1273-1371 of Erbin was cloned into the
NdeI/BamHI sites of the pET15b expression vector, creating a fusion with an N-terminal His tag followed by a thrombin cleavage site. E. coli BL21 cultures harboring the
expression plasmid were grown at 37 °C to mid-log phase
(A600 = 0.7) in M9 medium with
15NH4Cl or
15NH4Cl/13C glucose supplemented
with 50 µg/ml carbenicillin. Protein expression was induced by the
addition of 1.0 mM
isopropyl-1-thio- NMR Spectroscopy and Structure Determination--
NMR samples
typically contained 1.0-1.5 mM protein, 50 mM
phosphate buffer, pH 6.5, 50 mM NaCl, and 0.1 mM
d11-2,2-dimethyl-2-silapentane-s-sulphonate (DSS) for chemical shift referencing in 93% H2O,
7% D2O. A "100% D2O" sample was prepared
by lyophilization and resuspension in 99.995% D2O. NMR
spectra were acquired at 25 °C on Bruker DRX600 and DRX800
spectrometers equipped with triple resonance, triple axis actively
shielded gradient probes. Addition of the phage-optimized peptide
(AcTGWETWV) to Erbin PDZ indicated that the free and bound resonances
were in slow exchange. Aliquots of peptide were added until
1H-15N HSQC peaks for the free protein
had disappeared and no further change of intensity or line width was
seen for the bound peaks. Backbone resonance assignments for Erbin PDZ
were obtained from the following double and triple resonance
experiments acquired at 600 MHz in H2O solution, as
described (35, 36): 1H-15N three-dimensional
NOESY-HSQC, 1H-15N three-dimensional
TOCSY-HSQC, three-dimensional HNCA, three-dimensional CBCA(CO)NH, and three-dimensional HBHA(CBCACO)NH. Erbin PDZ side chain
assignments were completed from a three-dimensional HCCH-TOCSY spectrum
acquired at 800 MHz in D2O solution. Peptide resonance assignments were obtained from 15N-filtered two-dimensional
1H NOESY and TOCSY spectra at 600 MHz in H2O
solution and 13C-filtered 1H NOESY and TOCSY
spectra at 800 MHz in D2O solution. Details of these
experiments are presented in Supplementary Table II.
Distance restraints were obtained from analysis of the following NOESY
spectra acquired at 800 MHz: three-dimensional
1H-15N NOESY-HSQC, two-dimensional
15N-filtered 1H NOESY, three-dimensional
13C-edited NOESY, and two-dimensional
13C-filtered 1H NOESY. Intermolecular
interactions were identified unambiguously in a three-dimensional
Statistical Analysis of Erbin PDZ Binding Specificity--
A
phage-displayed peptide library was used to explore Erbin PDZ binding
specificity. Instead of focusing on a small set of molecules with high
affinity, we used a statistical analysis of a large number of potential
ligands to accurately define the specificity of peptides able to bind
to Erbin PDZ. After two rounds of binding selection, approximately half
of the phage clones bound specifically to Erbin PDZ in a phage
enzyme-linked immunosorbent assay; DNA sequencing revealed that most of
these clones were unique. An alignment of 148 sequences defined the
optimal binding consensus for Erbin PDZ, which agreed with earlier
results obtained from sequencing a limited number of clones after three
rounds of selection (24). In addition, the presence of rare clones
among the large number of sequences revealed suboptimal but still
significant preferences for certain amino acids at most binding
positions (Table I). In the remainder of
the manuscript, the standardized PDZ ligand nomenclature will be used
to describe the peptide residues, wherein the C terminus is designated
residue 0 (e.g. Val0) and the remaining residues
are numbered with negative integers whose absolute value increases
toward the N terminus (42). At the peptide C terminus, valine was
overwhelmingly preferred along with a much rarer occurrence of leucine
and isoleucine, whereas tryptophan was selected exclusively at the Specificity of Peptide Binding--
To more fully understand the
energetic contributions of different residues at each ligand position,
the relative binding affinity was measured for a series of synthetic
peptides (Table II). The heptapeptide
identified in the preliminary phage analysis (24) bound with an
IC50 of 0.15 µM; the lack of consensus for
the two N-terminal residues (Table I) suggests that they are not
important for binding, and indeed removal of them actually leads to a
slight improvement in affinity (7-fold; IC50 = 0.02 µM). In the context of the pentapeptide, at most ligand
sites the relative binding affinity of a given amino acid substitution
correlates well with the preference for that residue in the phage
selection. For example, Leu0 and Ile0 bind
~90-fold less tightly then Val0 and are selected in less
than 3% of the clones. Similarly, Ser Structure of Erbin PDZ domain--
The Erbin PDZ contains six
Ligand Binding to Erbin PDZ Domain--
The interaction between
the five C-terminal residues of the phage-derived heptapeptide and
Erbin PDZ is clearly defined by more than 200 intermolecular nuclear
Overhauser effect restraints (Fig. 3A and Supplementary
Table III). The peptide extends one edge of the Shotgun Alanine Scanning and Homolog Scanning of Erbin PDZ
Domain--
The contribution to peptide binding of individual Erbin
PDZ domain residues was assessed by combinatorial alanine scanning (30). A pair of libraries were constructed in which 44 residues in and
around the peptide-binding site were represented by trinucleotides that
encoded either the wild type Erbin amino acid or alanine (note that
because of the particular codons used, some non-alanine mutants were
also possible; see Ref. 30). Two additional libraries were constructed
in which the same 44 residues were present as either the wild type or a
homolog of the wild type residue (the so-called "homolog scan";
Table III). These libraries were then selected for binding to immobilized peptide (Ac-TGWETWV), and ~180
clones positive for binding were sequenced after two rounds of
selection. The number of clones with the wild type residue at each position were compared with the number with each designed mutant (either alanine or homolog) and categorized as substitutions that reduce (ratio > 1), do not affect (ratio = ~1), or
improve (ratio < 1) binding to peptide. To control for variation
in expression or display level for different library members, the
libraries were also selected for binding to an immobilized antibody
capable of recognizing an epitope tag that was displayed at the N
terminus of all library members. The ratio of wild type to mutant in
the peptide selection was then scaled by the ratio of wild type to mutant observed in the antibody selection to give a normalized frequency of occurrence (F; Table III).
The results of the alanine and homolog substitutions on peptide binding
are mapped onto the structure of Erbin PDZ in Fig. 4. The majority of alanine mutations that
have a significant effect on peptide binding (F > 20)
are proximal to only three of the peptide side chains
(Val0, Thr
The homolog scan data (Fig. 4B and Table III) reiterates the
view seen from the alanine scan, with the high F values
occurring proximal to Val0, Thr The ligand binding and structural studies described herein for
Erbin PDZ recapitulate earlier findings on the importance of a ligand
C-terminal aliphatic and a Earlier studies of PDZ ligand interactions usually failed to find any
preference for residues at the 4ETW
1V; IC50 = ~0.15 µM) in complex with Erbin PDZ provides a
structural context to understand the binding energetics. In particular,
the very favorable interactions with Trp
1 are not
Erbin side chain-mediated (and therefore may be generally applicable to
many PDZ domains), whereas the
2-
3 loop provides a binding site
for the Trp
4 side chain (specific to Erbin because it has
an unusually long loop). These results contribute to a growing
appreciation for the importance of at least five ligand C-terminal side
chains in determining PDZ domain binding energy and highlight the
mechanisms of ligand discrimination among the several hundred
PDZ domains present in the human genome.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-barrel capped by
-helices (13). The predominant function of
PDZ domains is to recognize the extreme C termini of other proteins,
thereby bringing signaling pathway components into proximity (14-16).
Numerous structural and biochemical studies have demonstrated that
C-terminal peptide ligands always bind in a groove between a
-strand
(
2; see Figs. 1 and 2 below) and an
-helix (
2) (reviewed in
Refs. 17 and 18). The ligand is arranged in an antiparallel fashion
with respect to the PDZ domain strand, and the ligand carboxylate is
hydrogen-bonded to backbone amide nitrogen groups in a conserved GLGF
motif located prior to strand
2 (19).
View larger version (32K):
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Fig. 1.
Structure-based sequence alignment of Erbin
PDZ domain (Erbin; Protein Data Bank code 1N7T) with
the third PDZ domain of PSD-95 (PSD95.3; Protein Data
Bank code 1BE9), -syntrophin
(aSyn; Protein Data Bank code 2PDZ), and
H+/Na+ exchange
regulatory factor PDZ domain 1 (HNERF; Protein Data
Bank code 1I92). Regular secondary structure elements (strands and
helices denoted by arrows and ellipses above the
sequence) were superimposed onto those of Erbin PDZ, and the
corresponding sequence alignments were extracted. Additional sequences
for Magi-3 PDZ2 (Magi3.2), Densin-180 (Densin),
and human Scribble PDZ domains 1-4 (Scrib1-Scrib4) were
manually aligned to the other sequences. The numbers in
bold type correspond to the residue position within the
Erbin PDZ domain, whereas the numbers in regular
type correspond to the position within each element of secondary
structure (see text). The asterisks under the sequence
indicate those residues in Erbin PDZ that contact the ligand.
Underlined Erbin PDZ residues correspond to sites that were
varied in the combinatorial scanning mutagenesis; alanine mutations
with F > 10 are in bold type and colored
red.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside induction, resulting in the production of phage particles that encapsulated pS2202d DNA and displayed Erbin PDZ in a monovalent format.
-globulin,
1 ppm proclin. The reaction mixture contained fixed concentrations of
anti-GST acceptor beads (16 µg/ml), biotin-TGWETWV (36 nM), and Erbin PDZ-GST (3 nM). Serial dilutions
of peptide were added, followed by addition of streptavidin donor beads
(20 µg/ml). The mixture was incubated at room temperature for 1 h and read on an AlphaQuest plate reader set at 1 s/well.
-D-galactopyranoside, and the cells
were harvested after 2 h further growth by centrifugation at
4,000 × g for 15 min and stored at
80 °C. The
pellet was resuspended in 50 mM Tris, pH 8.0, 500 mM NaCl, 1 µM phenylmethylsulfonyl fluoride
and sonicated for 3 min on ice. The suspension was centrifuged for 30 min at 10,000 × g, and the supernatant was loaded onto a nickel-nitrilotriacetic acid-agarose column. The column was washed
with 50 mM Tris, pH 8.0, 500 mM NaCl, 10 mM imidazole, and then the protein was eluted with 250 mM imidazole in the same buffer. Fractions containing the
protein of interest were pooled, thrombin was added (1 unit/mg of
protein), and the sample was dialyzed overnight against PBS at 4 °C.
The protein sample was then concentrated and further purified over a
Superdex-75 column in PBS to remove thrombin and the cleaved His tag.
The identity of the purified protein was verified by N-terminal
sequencing and mass spectrometry. In addition to Erbin PDZ domain
(residues 1273-1371), the construct also contains an N-terminal GSHM
tail from the expression vector (i.e. Gly5 of
the present construct corresponds to Gly1273 of full-length Erbin).
1-13C-filtered,
2-13C-edited NOESY spectrum. Initial NOESY
peak assignments were made on the basis of the assigned resonance
positions and a homology model of Erbin PDZ as described previously
(37), followed by several rounds of structure calculation and manual
restraint checking and peak assignment. The homology model was based on
the second and third PDZ domain of PSD-95 (1QLC (38); 1BE9 (19)) and the location of secondary structure elements identified in a
preliminary analysis of the NMR data. Dihedral angle restraints were
obtained from analysis of three-dimensional
15N-1H HNHA (
), three-dimensional
15N-1H HNHB, and three-dimensional
15N-1H TOCSY-HSQC (
1) spectra.
Additional loose backbone dihedral angle restraints were obtained from
analysis of backbone chemical shifts with the program TALOS (39).
Restraints were applied for good fits to the chemical shifts (as
defined by the program) with the allowed range being the TALOS-defined
mean ± the larger of 30° or three times the TALOS-calculated
standard deviation. Finally, residual dipolar coupling restraints were
measured from a solution of Erbin PDZ with peptide in the presence of
15 mg/ml of Pf1phage (ASLA Ltd.) (40) using the in-phase
anti-phase method of Tjandra and Bax (41). The values for the axial
component of the molecular alignment tensor (Da)
and the rhombicity (R) were obtained by fitting calculated
to experimental residual dipolar coupling values using a simple Powell
minimization procedure (41). Coordinates from initial structures
calculated on the basis of nuclear Overhauser effect and dihedral angle
restraints only were used for this purpose and yielded values of
19
Hz and 0.4 for Da and R,
respectively. The structures were calculated using the program CNX
(v2000.1; Accelrys, San Diego, CA). 100 structures were calculated
using torsion angle dynamics followed by Cartesian dynamics and
minimization. The 20 structures of lowest restraint violation energy
were chosen to represent the solution structure of the Erbin PDZ
domain-peptide complex. Details of the input restraints and structural
statistics are presented in Supplementary Table III.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
position. Threonine was the predominant selection at the
2 position,
although serine and, to a lesser extent, valine or acidic residues were
also observed. Acidic residues were selected almost exclusively at the
3 site with a 2-fold preference for aspartate over glutamate.
Although all amino acid types were observed at the
4 and
5 sites,
aromatic residues, especially tryptophan, did predominant at
4, and
there was a slight preference for Trp
5 (Table I).
Statistical analysis of Erbin PDZ binding specificity
2 and
Val
2 are ~10-fold reduced in affinity compared with
Thr
2 and are also selected 6- and 25-fold less often than
threonine, respectively. The one point of disagreement between the
selection data and the binding data is in the relative affinity of
Asp
3 versus Glu
3 because
aspartate is selected more often yet binds 20-fold less tightly.
Replacement of Trp
4 with other aromatic residues leads to
an 8-fold loss in affinity, as expected from the presence of all three
aromatic residues at this site; replacement with alanine was more
detrimental to binding (~50-fold) as expected by selection of alanine
in <2% of ligands sequenced. Tryptophan is the only amino acid
selected at the
1 position, and even replacement with phenylalanine
caused a dramatic loss of affinity (165-fold). Interestingly, replacing
Trp
1 with Pro, as found in the C-terminal peptide of
ERB-B2, which has been implicated in Erbin binding (27), reduces the
affinity of the pentapeptide by >3300-fold. The alanine scan data in
Table II give an indication of the relative contribution of the peptide side chains to the binding energy. Generally, the closer a residue to
the C terminus, the more important it is for binding, with Val0
Ala being most deleterious (3700-fold). As found
in previous studies, the C-terminal carboxylate is also critical for
high affinity binding (2400-fold decrease in affinity when amidated). The importance of the C-terminal residues is also seen in the N-terminal truncation analogs in Table II; even the acetylated dipeptide Ac-WV binds with an affinity of ~300 µM.
IC50 values for Erbin PDZ-binding synthetic peptides
-strands arranged into two antiparallel
-sheets (Fig.
2), as has been observed in other PDZ
domain structures (e.g. see Ref. 17 and the references
therein). The majority of the Erbin PDZ domain structure is well
defined by and agrees well with the 1991 experimental NMR restraints
(Fig. 3A and Supplementary
Table III). Residues within Erbin PDZ domain will be referred to by
their location within each of the secondary structure elements or loops
(42) as defined in the canonical third PDZ domain of PSD-95 (19) (Figs.
1 and 2). Thus,
2,
3, and
4 form the "top" sheet, and
1,
6,
4, and
5 form the lower sheet, whereas helix
2
caps the
2-
5 edge of the sandwich (Fig. 2). The extreme termini
of the domain (Gly1-Lys10 and
Ser102-Ser103) and the N terminus of the ligand
(Thr
6-Gly
5) are disordered (Fig.
3A); the longer loops (
2:
3 and
3:
4) are defined
slightly less well than residues in regular elements of secondary
structure (see Supplementary Table III). At the sequence level, several
differences are apparent between Erbin PDZ and the canonical third
domain from PSD-95, namely one residue fewer in the
1:
2 loop, an
extra nine residues in the
2:
3 loop, and two fewer residues in
the
3:
4 loop (Fig. 1). The structure of Erbin PDZ reveals that
these sequence differences may be readily accommodated without
distortion of the fold (Fig. 2). The
1:
2 loop is able to make a
more direct connection between the strands without perturbing the
important side chain- and backbone-mediated interactions with the
ligand (see below). The
2:
3 loop is highly variable in length and
character among PDZ domains, and in the case of Erbin it initially
turns toward the
5:
2 loop before a reverse turn at
Val31-Gly32 (type II) redirects the chain back
across the top of strand
2 into two final reverse turns
(Pro37-Phe38, distorted type I;
Pro40-Asp41, type I) that lead the chain into
the start of strand
3 (Fig. 2). The shorter
3:
4 loop in Erbin
PDZ precludes the formation of the
1 helix that normally caps this
edge of the
-sandwich; instead a series of nested reverse turns are
present (Glu53-Gly54,
Pro55-Ala56,
Ser57-Lys58, and
Lys58-Leu59).
View larger version (56K):
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Fig. 2.
Ribbon view of representative Erbin PDZ
structure bound to phage-derived peptide. Elements of regular
secondary structure are labeled, as are the side chains of the
phage-derived peptide ligand.
View larger version (55K):
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Fig. 3.
A, ensemble of structures for Erbin PDZ
domain (gray) bound to the phage optimized peptide
(green). Only the backbone N, C , and C atoms are shown.
Selected peptide side chain heavy atoms are included. Root mean square
deviation to the mean structure = 0.40 ± 0.05 Å for
backbone heavy atoms of residues 10-100; no distance or dihedral
angle restraints violated by more than 0.17 Å or 2.5°, respectively.
B, representative structure showing specific side chains
around the peptide binding groove.
-sandwich via
contacts with strand
2 (Figs. 2 and 3), with several backbone
hydrogen bonds observed between Erbin PDZ (
2-1(Phe25)
and
2-3(Ile27)) and ligand (Val0 and
Thr
2). The backbone amide protons of
1:
2-4
(Leu23),
1:
2-5(Gly24) and
2-1(Phe25) are all directed toward the carboxylate
oxygen atoms of Val0 but at distances slightly longer than
that usually considered for a hydrogen bond. The Ne amino
groups of
1-7(Lys19) and
2-9(Lys87) are
in the vicinity of the Val0 carboxylate and may give rise
to favorable Coulombic contacts. The poor definition of these
interactions results from the absence of restraint-generating protons
on the carboxylate and amino groups; in addition, we cannot rule out
the presence of a bound water molecule in the carboxylate-binding
pocket, as seen in other PDZ domain complexes (19). Turning to the
ligand side chains, Val0 pokes into the core of the protein
and is surrounded by residues from
1:
2,
2, and
2, whereas
Thr
2 abuts helix
2, is in van der Waals' contact with
2-5(Val83), and participates in a hydrogen bond from
its hydroxyl group to N
2 of
2-1(His79)
(Fig. 3B). Although in a number of other studies the peptide side chain at position
1 does not make specific structural or energetically favorable intermolecular contacts (19, 20), this is not
the case for Erbin PDZ domain: Trp
1 reaches across strand
2 and inserts between the side chains of
3-5(Arg49)
and
3:
4-1(Gln51) (Fig. 3B).
Glu
3 also reaches across strand
2 toward strand
3;
although a lack of restraints precludes the definition of a precise
orientation for this side chain, an ionic interaction with
3-5(Arg49) is likely. Finally, Trp
4
also lies toward the
2 side of the binding cleft and has a number of
interactions with Glu
3 and residues at the C terminus of
the
2:
3 loop (Fig. 3B).
Erbin PDZ shotgun scan
2, and Trp
4),
emphasizing the importance of these interactions for peptide binding
(Fig. 4A). Non-alanine mutations of many of these residues are also highly detrimental to peptide binding, even in the case of
subtle substitutions of isoleucine or leucine for valine
(e.g.
1:
2-4(L23V),
2-3(I27V),
3-1(I45V), and
2-8(L86V)). Moreover, several of these mutations also cause
a drop in display level, indicating that the wild type residue is
necessary for efficient folding of the domain. Non-alanine mutations at
a number of additional sites are also detrimental to peptide binding.
Although some of these may indicate the loss of direct contacts with
the peptide (e.g.
1-7(K19E)), many of the mutations are
to proline and hence may decrease peptide binding by an indirect
structural perturbation. Curiously, alanine substitutions of residues
that contact Trp
1 and Glu
3 did not decrease
peptide binding and in some cases actually improved it
(F(S28A) = 0.44; F(E51A) = 0.25).
However, alanine substitutions elsewhere in
3 were detrimental to
binding, suggesting that
3-1(Ile45),
3-2(Phe46),
3-4(Val47), and
3-6(Val50) are important for maintaining the
3
conformation necessary for tight ligand binding.
View larger version (46K):
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Fig. 4.
Results of the shotgun alanine scan
(A) and homolog scan (B) of Erbin PDZ
domain. The PDZ domain is represented with a Connolly surface, and
the peptide ligand is shown in tube form. The protein side chains
varied in the libraries are colored red (F > 100), orange (100 > F > 20),
yellow (20 > F > 5), light
blue (5 > F > 0.5), and dark
blue (F < 0.5); residues not varied in the libraries are
left white. All residues with F > 5 or < 0.5 are
labeled; in cases where the side chain is not visible, the labels are
in parentheses and colored according to F
value.
1, and
Trp
4 with the caveat that the homologs are generally less
disruptive to peptide binding, perhaps as expected from the less
dramatic nature of many of the substitutions. One exception to this
trend is for Val83; substitution with isoleucine
(F = 31) disfavors binding much more than substitution
with alanine (F = 11), indicating that the recognition
of Thr
2 is less tolerant of larger side chains at the
2:4 position. Despite the homolog mutations being conservative
substitutions, a number of them do lead to a significant decrease in
display level. The loss of display often involves substituting leucine or valine with isoleucine, implying that even subtle changes of some
hydrophobic core positions can perturb the ability of the domain to
fold correctly.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 position hydroxyl-containing residue for
binding to type I PDZ domains (14, 15, 20-22). However, in contrast to
these earlier studies, we have also investigated the relative
importance of PDZ domain residues in a systematic fashion. The current
data show that the hydrophobic core residues surrounding the C-terminal
side chain cannot be substituted even conservatively without loss of
binding to the phage-derived peptide, thereby providing selectivity.
High display levels on phage indicate that these mutants are well
folded, suggesting that hydrophobic pockets of varying shape and size
can be generated that recognize a variety of C-terminal ligand
residues. Thus, libraries similar to those described herein may be used
to select PDZ domain sequences that recognize ligands with particular
sequences, including C-terminal residues other than valine. Indeed, a
computational approach that achieves the same goal has recently been
described (43).
1 site (20-22), a finding rationalized by early structural studies in which this side was often
found to be oriented away from the PDZ domain (Fig.
5A) (19, 44, 45). More
recently, structures have been published for PDZ domains from
-syntrophin (22) and the H+/Na+ exchange
regulatory factor (HNERF PDZ1) (48, 49), in which specific hydrophobic
or hydrogen bond interactions are observed to the
1 position side
chain (Fig. 5). The energetic benefit of this contact in
-syntrophin
is unclear because almost all amino acids are selected at the
1 site
from libraries of potential ligands (22). Likewise, the effect of the
1 site of HNERF on affinity is also uncertain because peptide library
selection experiments and Western blot overlay studies have shown that
it has a preference for ligands with Arg
1,
Leu
1, Phe
1, or Tyr
1 (46, 47),
and ligands with Ala
1 also appear to bind well (47).
Thus, although these studies point to a compelling structural rationale
for recognition of a
1 position ligand side chain, the absence of
precise affinity measurements for mutant proteins or peptide analogs
makes the absolute contribution of these interactions hard to
gauge.
View larger version (53K):
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Fig. 5.
Comparison of intermolecular contacts
observed with the peptide 1 side chain. Note that for
B-D, the exact energetic contribution of the interaction
has not been evaluated experimentally. All of the structures are shown
in the same orientation after superposition of the common elements of
the regular secondary structure. A, third PDZ domain of
PSD95 with the C-terminal peptide KQTSV from Crypt (Protein Data Bank
code 1BE9). B,
3:
4-1(Phe) of
-syntrophin PDZ
domain contacts Leu
1 of the peptide ligand GVKESLV (the C
terminus of the vertebrate voltage-gated sodium channel; Protein Data
Bank code 2PDZ). C, hydrogen bond formation from the
Arg
1 side chain of the peptide QDTRL (C terminus of the
cystic fibrosis transmembrane conductance regulator) to HNERF side
chain
3:
4-1(Glu) and
1:
2-3(Asn) backbone carbonyl (via an
intervening water molecule; not shown) (Protein Data Bank code 1I92).
D, HNERF side chains
1:
2-3(Asn) and
3:
4-1(Glu)
HNERF reorientate to accommodate the Leu
1 side chains of
the peptide ligand NDSLL (C terminus of the
2-adenergic receptor;
Protein Data Bank code 1GQ4). E, homology model of
Magi3-PDZ2 in which Trp
1 of the phage-optimized ligand
interacts with
2-2(Ala),
3-5(Met) and
3:
4-1(Leu) (23).
F, Trp
1 of the phage-derived peptide TGWETWV
reaches over
2-2(Ser26) and inserts between
3-5(Arg49) and
3:
4-1(Gln51) (Protein
Data Bank code 1N7T).
In contrast to these earlier studies, we have shown a distinct
energetic preference for Trp1 in ligands that bind to
Magi-3 PDZ2 (23) and Erbin PDZ (Table I). An homology model constructed
for Magi-3 PDZ2 suggested that interactions with residues in
2 and
3 might be the source of the favorable contribution to binding by
Trp
1 (Fig. 5E) (23). Although these contacts
have been confirmed in the present study of Erbin PDZ (Fig.
5F), shotgun alanine scan data indicate that these side
chains do not confer any specific energetic contribution to binding
(Fig. 4A). Thus, the benefit to binding conferred by
Trp
1 derives from Erbin PDZ domain side chain-independent
interactions with the backbone of strands
2 and
3. The
orientation of the Trp
1 side chain with respect to strand
2 in the Erbin PDZ complex is reminiscent of the interstrand
tryptophan contacts observed in recent studies of peptide
-hairpin
stability (50-52). These studies indicated that tryptophan is best
able to stabilize antiparallel interactions between two
-strands
regardless of the residue type on the opposite strand (50). The
similarity in backbone and tryptophan conformation in these two cases
(Fig. 6) suggests a common mechanism for
stabilization (either tight ligand binding in the PDZ case or
-hairpin stability in the peptide case) based on the side chain to
backbone contacts. Thus, a Trp
1 residue may be a general
and somewhat nonselective means to increase the affinity of C-terminal
peptides for PDZ domains. A further degree of positive selection may be
garnered by making the site around Trp
1 more hydrophobic,
as observed in the case of Magi-3 PDZ2 (Fig. 5E) (23), in
PDZ domains engineered to recognize hydrophobic peptides (43), and in
the alanine scan results of Erbin PDZ (Fig. 4). Conversely, selection
against Trp
1 ligands may be achieved by the inclusion of
large bulky residues in the vicinity of the
1 site that might
obstruct tryptophan-strand interactions.
|
The C-terminal phage display process selected only acidic residues at
the 3 site (Table I). Structural analysis indicated the presence of a
proximal basic residue at position
3-5(Arg49) (Fig. 3B),
suggesting that a favorable Coulombic interaction is the cause of the
140-fold decrease in affinity for the Glu
3 to
Ala
3 substitution. Curiously, mutagenesis of Erbin PDZ
residues close to the Glu
3 location had very little
effect on peptide binding (Fig. 4). One possible explanation is that
given the interdigitation of side chains from the ligand and
3 (Fig.
3B) and the contacts between Glu
3 and
Trp
4, replacement of the Glu
3 side chain
may decrease the ability of other ligand side chains to make optimal
contacts with Erbin PDZ. In contrast, contacts between Erbin PDZ and
Trp
4 (Fig. 3B) suggest a structural rationale
for the selection of aromatic residues at this site that is born out by
the alanine scanning mutagenesis results (Fig. 4). The opportunity for
these interactions to arise is made possible by the much longer, but still structured,
2:
3 loop in Erbin (15 residues) compared with other PDZ domains (usually 4-6 residues; Fig. 1). Utilization of an
enlarged
2:
3 loop to provide additional ligand selectivity has
been noted previously (38, 53). In addition to the
2:
3 residues
that contact ligand, the alanine and homolog scan data reveal that
several more are necessary for tight ligand binding (e.g.
Asn36 and Asp42; Fig. 4). The
2:
3 loop
wraps around the side chain of the Asn36 (Fig.
3B) with potential hydrogen bonds to the backbone of
residues Arg39, Asp42, and Gly44,
suggesting an important role for Asn36 in stabilizing the
2:
3 loop in a conformation competent for favorable interaction
with the ligand.
On the basis of the optimal ligand identified by phage display, we have
previously suggested that the interaction between Erbin and the family
of p120-like catenins may be more physiologically relevant than the
earlier postulated interactions with ERB-B2 (24, 27). The present, more
expansive investigations yielded the same optimal ligand and
substantiate our original hypothesis of relevant protein ligands for
Erbin, and yeast two hybrid studies have also identified such an
interaction (28, 29). The detailed structure-activity relationships
discussed above allow us to make hypotheses about potential ligands for
other mammalian LAP protein PDZ domains. Densin-180 is the most similar
in primary sequence to Erbin PDZ (61% identity), with conservation of
all of the side chains that contribute to ligand binding/selectivity
described above. In accord with this, we have previously shown that the optimal ligand for Erbin PDZ is very similar to that for Densin-180 PDZ
(24), and Densin-180 has been shown to co-localize with p120-catenins
at neuronal synaptic junctions (54). The other mammalian LAP protein,
Scribble, contains four PDZ domains and is also involved in maintenance
of epithelial cell polarity, the formation of multi-protein membrane
complexes, and the control of epithelial cell growth (26). All four
domains have 30-40% sequence identity to Erbin PDZ (Fig. 1). The
pocket at the 0 site is hydrophobic in all four cases, although
2-1(Phe25) is conserved only in PDZ2 of Scribble, and
the smaller aliphatic side chains present in Scribble PDZ1, PDZ3, and
PDZ4 suggest that these domains may accommodate larger C-terminal
ligand residues. Variability at
3:
4-1(Gln51)
suggests that the preference for Trp
1 may differ from
that observed in Erbin PDZ. The residues at
2-1(His79),
2-5(Val83), and
3-5(Arg49) are
identical or very similar in all cases, suggesting a conserved preference for Thr
2 and either Glu
3 or
Asp
3. The
2-
3 loop is long in all cases, suggesting
that favorable contacts with the
4 ligand residue will be possible,
although sequence variability in the loop makes it difficult to predict the preferred ligand residue. Thus, the consensus ligands for the human
Scribble domains are likely to be X(D/E)(T/S)XV
for domain 2 and XDETSX
for the other three
domains (where X indicates specificity of an as yet
undefined nature and
is a large hydrophobic residue). Further
studies are in progress with these and other PDZ domains to identify
optimal ligands via C-terminal phage display so that potential binding
partners may be ascertained.
In summary, we have used an expanded C-terminal phage display library
to confirm and extend our earlier proposal of an optimal and
biologically significant ligand for the Erbin PDZ domain (24). Importantly, the energetic contributions to binding of the side chains
within this optimal ligand have been ascertained by binding affinity
measurements with a large number of synthetic peptide analogs. All five
C-terminal ligand residues are found to make a beneficial contribution
to binding. Structural studies have identified the subset of Erbin PDZ
residues that contact ligand side chains, and an efficient
combinatorial scanning approach has been used to investigate the
origins of affinity and selectivity within PDZ domains. A novel
interaction with Trp1 has been observed that is likely to
be a generic method of stabilizing the interaction between C-terminal
peptides and many PDZ domains. The particular conformation adopted by
the long
2:
3 loop of Erbin PDZ also allows additional contacts
with ligand residues at the
4 site. The combination of these
investigations thus gives deep insight into the manner by which PDZ
domains can recruit their particular cellular targets with both high
affinity and selectivity. These results have been extended to make
hypotheses about ligands that other LAP PDZ domains will recognize.
Further applications of the experimental techniques described herein
will be used to confirm or refute these hypotheses and to add to our growing knowledge of the manner by which PDZ domains participate in
interactions of biological significance.
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge Drs. Kurt Deshayes, Heike Held, Yan Wu, Dean R. Artis, and Rich Laura for many for helpful discussions during the course of this work. We are grateful to Drs. Melissa Starovasnik, Wayne Fairbrother, and Borlan Pan for assistance with NMR data collection and preliminary analysis of the NMR data. We also thank the Genentech DNA synthesis group for oligonucleotides, Alan Zhong for DNA sequencing, and Cliff Quan for help with peptide synthesis.
![]() |
FOOTNOTES |
---|
* 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.
The atomic coordinates and the structure factors (code 1N7T) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
The on-line version of this article (available at
http://www.jbc.org) contains supplemental tables.
§ To whom correspondence may be addressed: Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080. E-mail: skelly@gene.com.
** To whom correspondence may be addressed: Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080. E-mail: sidhu@gene.com.
Published, JBC Papers in Press, November 20, 2002, DOI 10.1074/jbc.M209751200
1 Model 2 in this ensemble is closest to the geometric mean and has been used as a representative single structure for the figures in this manuscript.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GST, glutathione S-transferase; HNERF, H+/Na+ exchange regulatory factor; NOESY, nuclear Overhauser effect spectroscopy; wt, wild type; PBS, phosphate-buffered saline; TOCSY, total correlation spectroscopy; HSQC, heteronuclear single quantum coherence.
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REFERENCES |
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---|
1. | Eisenberg, D., Marcotte, E. M., Xenarios, I., and Yeates, T. O. (2000) Nature 405, 823-826[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Hazbun, T. R.,
and Fields, S.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
4277-4278 |
3. |
Xenarios, I.,
Salwinski, L.,
Duan, X. J.,
Higney, P.,
Kim, S. M.,
and Eisenberg, D.
(2002)
Nucleic Acids Res.
30,
303-305 |
4. | Xenarios, I., and Eisenberg, D. (2001) Curr. Opin. Biotechnol. 12, 334-339[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Pawson, T.,
and Scott, J. D.
(1987)
Science
278,
2075-2080 |
6. | Kuriyan, J., and Cowburn, D. (1997) Annu. Rev. Biophys. Biomol. Struct. 26, 259-288[CrossRef][Medline] [Order article via Infotrieve] |
7. | Lee, C. H., Cowburn, D., and Kuriyan, J. (1998) Methods Mol. Biol. 84, 3-31[Medline] [Order article via Infotrieve] |
8. | Cowburn, D. (1996) Structure 4, 1005-1008[Medline] [Order article via Infotrieve] |
9. | Cho, K. O., Hunt, C. A., and Kennedy, M. B. (1992) Neuron 9, 929-942[Medline] [Order article via Infotrieve] |
10. | Woods, D. F., and Bryant, P. J. (1993) Mech. Dev. 44, 889 |
11. | Kim, E., Niethammer, M., Rothschild, A., Jan, Y. N., and Sheng, M. (1995) Nature 378, 85-88[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Schultz, J.,
Copley, R. R.,
Doerks, T.,
Ponting, C. P.,
and Bork, P.
(2000)
Nucleic Acids Res.
28,
231-234 |
13. | Morais Cabral, J. H., Petosa, C., Sutcliffe, M. J., Raza, S., Byron, O., Poy, F., Marfatia, S. M., Chishti, A. H., and Liddington, R. C. (1996) Nature 382, 649-652[CrossRef][Medline] [Order article via Infotrieve] |
14. | Kornau, H. C., Schenker, L. T., Kennedy, M. B., and Seeburg, P. H. (1995) Science 269, 1737-1740[Medline] [Order article via Infotrieve] |
15. | Matsumine, A., Ogai, A., Senda, T., Okumura, N., Satoh, K., Baeg, G. H., Kawahara, T., Kobayashi, S., Okada, M., Toyoshima, K., and Akiyama, T. (1996) Science 272, 1020-1023[Abstract] |
16. | Niethammer, M., Kim, E., and Sheng, M. (1996) J. Neurosci. 16, 2157-2163[Abstract] |
17. | Sheng, M., and Sala, C. (2001) Annu. Rev. Neurosci. 24, 1-29[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Harris, B. Z.,
and Lim, W. A.
(2001)
J. Cell Sci.
114,
3219-3231 |
19. | Doyle, D. A., Lee, A., Lewis, J., Kim, E., Sheng, M., and MacKinnon, R. (1996) Cell 85, 1067-1076[Medline] [Order article via Infotrieve] |
20. |
Songyang, Z.,
Fanning, A. S., Fu, C., Xu, J.,
Marfatia, S. M.,
Chishti, A. H.,
Crompton, A.,
Chan, A. C.,
Anderson, J. M.,
and Cantley, L. C.
(1997)
Science
275,
73-77 |
21. | Stricker, N. L., Christopherson, K. S., Yi, B. A., Schatz, P. J., Raab, R. W., Dawes, G., Bassett, D. E., Jr., Bredt, D. S., and Li, M. (1997) Nat. Biotech. 15, 336-342[Medline] [Order article via Infotrieve] |
22. | Schultz, J., Hoffmuller, U., Krause, G., Ashurst, J., Macias, M. J., Schmieder, P., Schneider-Mergener, J., and Oschkinat, H. (1998) Nat. Struct. Biol. 5, 19-24[Medline] [Order article via Infotrieve] |
23. |
Fuh, G.,
Pisabarro, M. T., Li, Y.,
Quan, C.,
Lasky, L. A.,
and Sidhu, S. S.
(2000)
J. Biol. Chem.
275,
21486-21491 |
24. |
Laura, R. P.,
Witt, A. S.,
Held, H. A.,
Gerstner, R.,
Deshayes, K.,
Koehler, M. F.,
Kosik, K. S.,
Sidhu, S. S.,
and Lasky, L. A.
(2002)
J. Biol. Chem.
277,
12906-12914 |
25. | Bryant, P. J., and Huwe, A. (2000) Nat. Cell Biol. 2, E141-E143[CrossRef][Medline] [Order article via Infotrieve] |
26. | Bilder, D., and Perrimon, N. (2000) Nature 403, 676-680[CrossRef][Medline] [Order article via Infotrieve] |
27. | Borg, J. P., Marchetto, S., Le, Bivic, A., Ollendorff, V., Jaulin-Bastard, F., Saito, H., Fournier, E., Adelaide, J., Margolis, B., and Birnbaum, D. (2000) Nat. Cell Biol. 2, 407-414[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Jaulin-Bastard, F.,
Arsanto, J. P., Le,
Bivic, A.,
Navarro, C.,
Vely, F.,
Saito, H.,
Marchetto, S.,
Hatzfeld, M.,
Santoni, M. J.,
Birnbaum, D.,
and Borg, J. P.
(2002)
J. Biol. Chem.
277,
2869-2875 |
29. |
Izawa, I.,
Nishizawa, M.,
Tomono, Y.,
Ohtakara, K.,
Takahashi, T.,
and Inagaki, M.
(2002)
Genes Cells
7,
475-485 |
30. |
Weiss, G. A.,
Watanabe, C. K.,
Zhong, A.,
Goddard, A.,
and Sidhu, S. S.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
8950-8954 |
31. | Sidhu, S. S., Lowman, H. B., Cunningham, B. C., and Wells, J. A. (2000) Methods Enzymol. 328, 333-363[Medline] [Order article via Infotrieve] |
32. | Sidhu, S. S., Weiss, G. A., and Wells, J. A. (2000) J. Mol. Biol. 296, 487-495[CrossRef][Medline] [Order article via Infotrieve] |
33. | Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol. 154, 367-382[Medline] [Order article via Infotrieve] |
34. | Vajdos, F. F., Adams, C. W., Breece, T. N., Presta, L. G., de Vos, A. M., and Sidhu, S. S. (2002) J. Mol. Biol. 302, 415-428 |
35. | Cavanagh, J., Fairbrother, W. J., Palmer, A. G., and Skelton, N. J. (1995) Protein NMR Spectroscopy: Principles and Practice , Academic Press, New York |
36. | Pan, B., Li, B., Russell, S. J., Tom, J. K., Cochran, A. G., and Fairbrother, W. J. (2002) J. Mol. Biol. 316, 769-787[CrossRef][Medline] [Order article via Infotrieve] |
37. | Starovasnik, M. A., Christinger, H. W., Wiesmann, C., Champe, M. A., de Vos, A. M., and Skelton, N. J. (1999) J. Mol. Biol. 293, 531-544[CrossRef][Medline] [Order article via Infotrieve] |
38. | Tochio, H., Hung, F., Li, M., Bredt, D. S., and Zhang, M. (2000) J. Mol. Biol. 295, 225-237[CrossRef][Medline] [Order article via Infotrieve] |
39. | Cornilescu, G., Delaglio, F., and Bax, A. (1999) J. Biomol. NMR 13, 289-302[CrossRef][Medline] [Order article via Infotrieve] |
40. | Hansen, M. R., Mueller, L., and Pardi, A. (1998) Nat. Struct. Biol. 5, 1065-1074[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Tjandra, N.,
and Bax, A.
(1997)
Science
278,
1111-1114 |
42. | Aasland, R., Abrams, C., Ampe, C., Ball, L. J., Bedford, M. T., Cesareni, G., Gimona, M., Hurley, J. H., Jarchau, T., Lehto, V. P., Lemmon, M. A., Linding, R., Mayer, B. J., Nagai, M., Sudol, M., Walter, U., and Winder, S. J. (2002) FEBS Lett. 513, 141-144[CrossRef][Medline] [Order article via Infotrieve] |
43. | Reina, J., Lacroix, E., Hobson, S. D., Fernandez-Ballester, G., Rybin, V., Schwab, M. S., Serrano, L., and Gonzalez, C. (2002) Nat. Struct. Biol. 9, 621-627[Medline] [Order article via Infotrieve] |
44. | Daniels, D. L., Cohen, A. R., Anderson, J. M., and Brunger, A. T. (1998) Nat. Struct. Biol. 5, 317-325[Medline] [Order article via Infotrieve] |
45. | Tochio, H., Zhang, Q., Mandal, P., Li, M., and Zhang, M. (1999) Nat. Struct. Biol. 6, 417-421[CrossRef][Medline] [Order article via Infotrieve] |
46. | Wang, S., Raab, R. W., Schatz, P. J., Guggino, W. B., and Li, M. (1998) FEBS Lett. 427, 103-108[CrossRef][Medline] [Order article via Infotrieve] |
47. |
Hall, R. A.,
Ostedgaard, L. S.,
Premont, R. T.,
Blitzer, J. T.,
Rahman, N.,
Welsh, M. J.,
and Lefkowitz, R. J.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
8496-8501 |
48. |
Karthikeyan, S.,
Leung, T.,
and Ladias, J. A.
(2001)
J. Biol. Chem.
276,
19683-19686 |
49. |
Karthikeyan, S.,
Leung, T.,
and Ladias, J. A.
(2002)
J. Biol. Chem.
277,
18973-18978 |
50. | Russell, S. J., and Cochran, A. G. (2000) J. Am. Chem. Soc. 122, 12600-12601[CrossRef] |
51. |
Cochran, A. G.,
Skelton, N. J.,
and Starovasnik, M. A.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
9081 |
52. | Cochran, A. G., Tong, R. T., Starovasnik, M. A., Park, E. J., McDowell, R. S., Theaker, J. E., and Skelton, N. J. (2001) J. Am. Chem. Soc. 123, 625-632[CrossRef][Medline] [Order article via Infotrieve] |
53. | Kozlov, G., Banville, D., Gehring, K., and Ekiel, I. (2002) J. Mol. Biol. 320, 813-820[CrossRef][Medline] [Order article via Infotrieve] |
54. |
Izawa, I.,
Nishizawa, M.,
Kazuhiro, K.,
and Inagaki, M.
(2002)
J. Biol. Chem.
277,
5345-5350 |