From the Department of Life Science, Kwangju
Institute of Science and Technology, Gwangju 500-712, South Korea, the
Department of Biological Sciences, Korea Advanced Institute of
Science and Technology, Daejeon 305-701, South Korea, and the
¶ Picower Center for Learning and Memory and the Howard Hughes
Medical Institute, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139
Received for publication, December 3, 2002, and in revised form, December 18, 2002
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ABSTRACT |
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PDZ domains bind to short segments within target
proteins in a sequence-specific fashion. Glutamate receptor-interacting
protein (GRIP)/ABP family proteins contain six to seven PDZ
domains and interact via the sixth PDZ domain (class II) with the C
termini of various proteins including liprin- Synaptic localization and clustering of ion channels and receptors
is often mediated by scaffolding molecules containing the protein-protein interaction motifs called PDZ (Postsynaptic
density-95/Discs large/Zona occludens-1)
domains (1). One of the most abundant molecular recognition elements,
these globular domains each contain two Members of the GRIP1 family
proteins (GRIP1 and GRIP2/ABP) contain six to seven PDZ domains (9, 10,
11). GRIP PDZ45, which is classified as a class II PDZ domain (1),
binds to the C terminus of the GluR2/3 subunit of AMPA glutamate
receptors (9, 10, 12), while GRIP PDZ6, also a class II PDZ domain, interacts with the C terminus of ephrin-B1 ligand and EphB2/EphA7 receptor tyrosine kinases (13, 14, 15). GRIP PDZ6 also interacts with
the C terminus of the liprin- The first reported crystal structure of a class II PDZ domain (from
hCASK) revealed the presence of a second hydrophobic pocket not seen in
class I PDZ domain (19). In addition, this study showed that hCASK PDZ
forms a homotetramer by binding to the truncated C-terminal tail of a
neighboring PDZ domain that partially mimics the specific peptide
ligand, though the self-associated structure does not faithfully
represent the actual binding mode of class II PDZ domains due to
differences in the amino acid sequences of the true target peptides and
the truncated C-terminal tail (19). Another structure of a class II PDZ
domain in InaD revealed its association with the To better understand the structural basis of peptide recognition by
class II PDZ domains and the mechanisms underlying PDZ-mediated GRIP
multimerization, we determined the crystal structures of the GRIP1 PDZ6
domain alone and in complex with a synthetic C-terminal octapeptide of
liprin- Protein Purification and Crystallization--
Recombinant GRIP1
PDZ6 (residues 665-761) from Rattus novergicus with a
cleavable glutathione S-transferase tag was expressed in
BL21(DE3) Escherichia coli, cleaved, purified, and
crystallized as previously described (21). The C-terminal octapeptide
(ATVRTYSC) of the human liprin- Site-directed Mutagenesis and Mutant Protein
Preparation--
The mutants Y671D and R718D were obtained by
site-directed mutagenesis of the plasmid carrying the
GRIP-PDZ6 gene using the QuikChange mutagenesis kit from
Invitrogen. The expression and purification of the mutants were done by
same procedures with the wild type protein.
Data Collection--
A native data set was collected from a
peptide-free frozen crystal to 1.5 Å resolution using an ADSC Quantum
4R CCD detector at beamline X8C in the National Synchrotron Light
Source. Since bromine is a convenient anomalous scatterer for MAD
phasing, a bromine MAD data set was collected from a single crystal
soaked for 30 s in cryoprotection solution containing 1 M NaBr (22). A data set was collected from a PDZ-peptide
complex to 1.8 Å Bragg spacing using an ADSC Quantum 4R CCD detector
at beamline BL-18B at Photon Factory, Tsukuba, Japan.
Structure Determination and Refinement--
The structure of the
peptide-free PDZ6 domain was determined by MAD using bromine as an
anomalous scatterer. The positions of three bromines in the asymmetric
unit were located and refined using the program SOLVE (23). The initial
phases (overall figure of merit, 0.51) were improved by solvent
flattening using the program DM (24). The resultant map was readily
interpretable, and model building proceeded using the program O (25),
after which the initial model was refined using the program CNS (26). When the crystallographic R value for the model was 28.3%,
the coordinates were used in the refinement procedures against the 1.5-Å native data set. The final crystallographic R value
for the peptide-free PDZ6 model using data from 15 to 1.5 Å was 25.4% (Rfree = 27.8%).
The structure of the PDZ6-octapeptide complex was determined using
standard molecular replacement methods using the structure of
peptide-free PDZ6 as a starting model. There were two PDZ6 domains
related by NCS in the asymmetric unit. After applying a simulated
annealing procedure using data to 1.8 Å, the location of the two bound
peptides was determined from a
fo Overall Structure of the GRIP PDZ6 Domain--
GRIP1 PDZ6 is a
compact, globular domain containing eight segments of secondary
structure: six
GRIP1 PDZ6 binds to the liprin- Molecular Basis of Peptide Recognition--
Because the amino acid
sequence of the rat GRIP1 PDZ6 domain used in this study is identical
to human GRIP1 PDZ6, we used a synthetic octapeptide (ATVRTYSC) that
mimics the C terminus of human liprin-
Despite variations in the sequences of many PDZ domains, their
carboxylate-binding loops are highly conserved in terms of overall
structure and the hydrogen-bonding pattern to the ligand carboxylate
group. The C-terminal carboxylate group of our octapeptide formed
hydrogen bonds with the backbone amide groups of Leu-682, Gly-683, and
Ile-684 in the carboxylate-binding loop, and was within
hydrogen-bonding distance of two fully occupied water molecules (Fig.
3A).
The amino acid residues at ligand positions 0 and
The second hydrophobic pocket, which accommodates the Tyr residue at
ligand position
Within the structure of the GRIP1 PDZ6-peptide complex, by contrast,
the side chain of Tyr-2 did not make a direct contact with the
conserved Leu-732 at
Interestingly, superposition of peptide-free and peptide-bound
structures showed that a slight reorientation of the
Within the structure of the PDZ6-peptide complex, ligand residues at
the Dimerization of PDZ6 Domains--
PDZ domain-mediated
multimerization is a common feature for a number of PDZ domain
proteins. Such multimerization of Drosophila InaD, which is
mediated by its PDZ3 or PDZ4 domain, does not disturb the binding of
target proteins (30) nor does oligomerization of NHERF/EBP50, which is
mediated by its two PDZ domains (31). Unfortunately, these studies
provide no structural evidence for the mechanism of multimerization or
why it does not affect ligand binding. At present, the structures of
InaD PDZ3 or PDZ4 remain unknown, and the crystal structures of the
NHERF PDZ1 domains provide no clues (28, 29).
GRIP/ABP proteins reportedly form homo- or heteromultimers through
their PDZ456 domains (11). Although it is not known how these three PDZ
domains mediate multimer formation, our preliminary results from size
exclusion chromatography, and dynamic light-scattering experiments
indicate that GRIP1 PDZ6 forms a dimer whether free or complexed, which
suggests that GRIP1 PDZ6, itself, has the ability to mediate dimer
formation. Consistent with that idea, the crystal structure of
peptide-bound GRIP1 PDZ6 showed formation of a tightly associated PDZ6
dimer related by a non-crystallographic 2-fold axis in the asymmetric
unit (Fig. 4A). The dimeric
interface between the two PDZ domains involves a
In the peptide-free crystal structure with the space group
P6522, there was one molecule in the asymmetric unit.
However, the same dimeric interaction described above was observed via a crystallographic 2-fold axis. It is possible that the dimer in the
solution can arrange into a crystallographic symmetry axis in a
different crystallographic environment because it shows little difference of the r.m.s.d. for all C
To further confirm the presence of PDZ6 dimer in solution, which is
shown in Fig. 4A, we did mutational analysis on the residues at the dimeric interface and measured the molecular weights of the
mutants and wild type PDZ domains in solution using size exclusion chromatography (Fig. 4). The Y671D mutant, which was expected to
disrupt the hydrophobic interaction in the dimeric interface, was
eluted as a monomer, while the wild type PDZ6 was a dimer in solution
(Fig. 4C). It indicates that Tyr-671 is located at the
dimeric interface and plays an important role in hydrophobic interaction of the interface. However, the disruption of salt-bridge by
the mutation R718D on the residue participating in self-association of
the domains in the crystalline state did not affect the oligomeric state of the PDZ domains. These results together with the dimerization of peptide-bound PDZ6 in both the crystalline state and in solution strongly suggests that the dimeric structure of PDZ6 shown in Fig.
4A is representative of dimeric PDZ6 in vivo.
We therefore propose that the GRIP PDZ6 domain plays a crucial role in
heteromultimerization of GRIPs. GRIP2, a homolog of GRIP1, has 58%
overall amino acid sequence identity with GRIP1, and the two isoforms
share 84% identity in the region containing the PDZ456 domains and
91% identity (95% similarity) in the PDZ6 domain. Notably, the
residues in the region of the dimeric interface (
In summary, we observed a novel mode of peptide recognition by the
class II GRIP PDZ6 domain. Ile-736 at the . In addition the
PDZ456 domain mediates the formation of homo- and heteromultimers of GRIP proteins. To better understand the structural basis of peptide recognition by a class II PDZ domain and PDZ-mediated multimerization, we determined the crystal structures of the GRIP1 PDZ6 domain alone and
in complex with a synthetic C-terminal octapeptide of human liprin-
at resolutions of 1.5 and 1.8 Å, respectively. Remarkably, unlike
other class II PDZ domains, Ile-736 at
B5 rather than conserved
Leu-732 at
B1 makes a direct hydrophobic contact with the side chain
of the Tyr at the
2 position of the ligand. Moreover, the
peptide-bound structure of PDZ6 shows a slight reorientation of helix
B, indicating that the second hydrophobic pocket undergoes a
conformational adaptation to accommodate the bulkiness of the Tyr side
chain, and forms an antiparallel dimer through an interface located at
a site distal to the peptide-binding groove. This configuration may
enable formation of GRIP multimers and efficient clustering of
GRIP-binding proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-helices and six
-strands. They usually bind selectively to the C terminus or a short
internal segment of interacting proteins (1) and are categorized into
four classes according to their specificity for the C-terminal target
sequences (2). Class I PDZ domains bind to a C-terminal motif with the
sequence X-Ser/Thr-X-Val/Leu-COOH, where
X represents any residue, while class II PDZ domains prefer X-
-X-
-COOH, where
is usually a large
hydrophobic residue. Both class I and II domains have a preference for
a hydrophobic residue at the 0 position of the ligand. Class III PDZ
domains prefer the sequence
X-Asp-X-Val-COOH in which a negatively charged amino acid is at the
2 position (3), while class IV domains prefer
the sequence X-
-Asp/Glu-COOH in which an acidic residue is at the C-terminal position and where
represents an aromatic residue (4). In addition, there are other classes of PDZ domains that
do not fall into any of the aforementioned classes (5, 6), and there
are minor discrepancies in the proposed classifications of PDZ domains
(7, 8).
family of multidomain proteins (16),
which interact with the leukocyte antigen-related protein family of
receptor tyrosine phosphatases (17, 18). Interestingly, the PDZ456
region also reportedly mediates homo- and heteromultimerization of
GRIPs (11), suggesting that the PDZ domain is a module mediating multimerization as well as peptide recognition.
1 position of the
ligand via a distinctive intermolecular disulfide bond (20), which is
not a canonical non-covalent peptide-PDZ interaction.
1 at resolutions of 1.8 and 1.5 Å, respectively. This is the
first description of the crystal structure of a class II PDZ domain
non-covalently complexed with its specific peptide ligand, showing an
additional role of PDZ domains in the multimerization of PDZ-containing proteins.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1 used for PDZ-peptide
cocrystallization was chemically synthesized.
fc difference electron density map. Densities of all eight residues of the peptide were obvious, indicating they were well ordered within the structure. The octapeptide was modeled using the program O, and the peptide-bound PDZ6 domain was refined to a final crystallographic R value
of 20.0% and a free R value of 22.2%. The refined model of
the peptide-bound PDZ6 domain consisted of a PDZ domain dimer
(Ala-668-Gln-753) related by 2-fold NCS, two octapeptides, and 240 water molecules. Because of disorder, the N-terminal three and
C-terminal eight residues were not modeled. The stereochemistry of the
model was analyzed with PROCHECK (27); no residues were found in the
disallowed regions of the Ramachandran plot. Data collection and
refinement statistics are summarized in Table
I. The coordinates and structure factors
of PDZ6 and peptide-PDZ6 complex have been deposited with the Protein
Data Bank accession numbers 1N7E and 1N7F, respectively.
Data collection and refinement statistics
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-strands that form an antiparallel
-barrel and two
-helices (Fig. 1A). Within
the crystal structure of the PDZ6-peptide complex, all eight amino acid
residues of the peptide ligand were well defined, as shown by the
difference electron density map calculated without inclusion of the
peptide (Fig. 1B), and the strong electron density indicates
that the octapeptide was highly ordered. As is typical of most PDZ
complexes, the ligand was positioned in the groove between the
B
strand and the
B helix, oriented anti-parallel to
B as an
additional strand. In addition, the carboxylate-binding loop contained
a PLGI sequence (residues 681-684), often referred to as the "GLGF motif."
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Fig. 1.
Structure of the GRIP1 PDZ6
domain. A, ribbon diagram showing the overall structure
of the GRIP1 PDZ6-peptide complex. The -strands are labeled as
A-
F, and the
-helices are labeled
A and
B. The peptide
(orange) inserts between the
B strand and the
B helix
and forms an antiparallel
-sheet with
B. This picture was
drawn using MolMol (33). B, quality of the
peptide electron density map. An omit map was calculated using
coefficients of
Fo
Fc at 1.8 Å resolution, where Fc is the calculated structure
factor from the PDZ6-peptide complex with the peptide removed. The map,
contoured at 3
, defines the peptide carboxylate group and all eight
peptide residues. This figure was made using PyMOL (available on the
web at www.pymol.org).
C-terminal TYSC sequence
(X-
-X-
-COOH) via a class II hydrophobic PDZ
interaction; it contains two hydrophobic pockets that can accommodate
bulky hydrophobic residues at the 0 and
2 positions of the ligand. In
the peptide-bound state, PDZ6 domain forms an antiparallel dimer in an
asymmetric unit of the crystallized complex, whereas peptide-free PDZ6
forms a similar dimer through a crystallographic 2-fold axis in its crystal. As was seen in the crystal structures of the hCASK and NHERF
PDZ domains, the peptide binding pocket of peptide-free PDZ6 is
occupied by the C-terminal end (PASS-COOH) of a neighboring molecule
mimicking the recognition of the peptide ligand (19, 28). Superposition
of the crystal structures of the peptide-free and peptide-bound PDZ6
domains shows a slight shift in the
B helix, which widens the
peptide binding pocket and enables accommodation of the bulky side
chain of tyrosine from the ligand. The free and peptide-bound
structures of the PDZ6 domain showed an
-carbon root-mean square
deviation (r.m.s.d.) of 1.1 Å when the six structurally conserved
-strands were used for the superposition. With an r.m.s.d. of 1.57 Å for the residues spanning positions 732-742, helix
B showed the
highest structural variance among secondary elements.
1 as a ligand. The last five
residues of liprin-
are identical among known isoforms, and the
residues at the 0 and
2 positions are well conserved as compared with
those from other known PDZ6 binding partners, e.g. EphB2
receptor (Fig. 2B). The C
terminus of the ligand binds as an additional strand to the
anti-parallel
-sheet of the PDZ domain and makes hydrophobic contacts with helix
B, while the peptide backbone of the C-terminal four residues is anchored to strand
B by hydrogen bonds.
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Fig. 2.
Sequence alignment of selected PDZ
domains. A, the amino acid sequences of the indicated
PDZ domains from rat GRIP1, rat GRIP2, Drosophila InaD,
human CASK, and rat PSD95 were aligned using the program ClustalX (32).
Highly conserved residues are shaded in black and
gray. The secondary structure elements of GRIP1 PDZ6 are
shown as arrows ( -sheet), bars (
helix),
and lines (connecting loops). The variant residues within
the PDZ6 domains of GRIP1 and GRIP2 are indicated with black
dots. B, several known ligands of the GRIP PDZ6 domain.
Only the C-terminal eight residues of each ligands are shown. The
sequence of the octapeptide used for the PDZ6-peptide complex examined
here is indicated with an asterisk.
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Fig. 3.
The peptide-binding site.
A, ball and stick model of the peptide binding pocket and
its specific ligand (ATVRTYSC). Only the last four residues of the
peptide are shown (green), and for clarity only side chains
of the residues involved in the peptide binding are shown. Dashed
lines represent hydrogen bonds around the carboxylate binding
site. A water molecule, shown as a red ball, forms a
hydrogen bond with one of the ligand C-terminal carboxylic oxygen
atoms. The majority of the hydrogen bonds are between the peptide
backbone and the carboxylate-binding loop or strand B. B,
molecular surfaces of GRIP PDZ6 showing the hydrophobic binding pocket
and the bound peptide. The hydrophobic residues are colored
white, and the hydrophobic side chains within the binding
pocket are colored in gray scale. The polar, acidic, and
basic residues are colored yellow, red, and
blue respectively. The two hydrophobic binding pockets are
indicated by circles. The side chains of hydrophobic ligand
residues Cys-0 and Try-1 dip into the hydrophobic binding pockets.
C, conformational changes upon peptide binding.
Superposition of the peptide-free and peptide-bound structures was done
using the six
-strands, which do not undergo conformational change
upon peptide binding. The peptide-free and peptide-bound PDZ structures
are shown in white and gray, respectively. The
segments that undergo large conformational changes upon peptide binding
are colored black. D, ball and stick model
showing a conformational change of Ile-736. Peptide-bound PDZ6 is
colored dark blue; self-associated PDZ6 is colored
green. The peptide ligand and C-terminal tail of the PDZ6
construct are colored pale blue and yellow,
respectively. The bulky hydrophobic side chain of Tyr-2 makes
hydrophobic contact with Ile-736.
2 are key to the
specific recognition by the PDZ domain. The side chain of Cys-0 is
situated at the center of the first hydrophobic cavity, which is
composed of Leu-682, Ile-684, Ile-686, and Leu-739 (Fig. 3B)
and is of sufficient size to accommodate various hydrophobic side
chains ranging from those of Ala to Phe, but not hydrophilic residues.
2, is composed of Ile-686 from the
B strand and
Ile-736 and Ile-739 from helix
B. Within the structures of
previously described class I and class II PDZ domains, the residue at
ligand position
2 usually binds to the side chain of a residue
located at the N-terminal end of helix
B (position
B1) of the PDZ
(7, 29). In class I PDZ domains, serine or threonine at the
2
position forms hydrogen bonds with the side chain of a polar residue at
the start of the
B helix. In class II PDZ domains, the corresponding
residue at the
B1 position is highly conserved (mostly hydrophobic)
and considered to be critical for determining the specificity of ligand
binding. In hCASK PDZ, for example, Val at
B1 recognizes the residue
at ligand position
2 (19). Another known class II PDZ domain, the
crystal structure of InaD, showed that the C
and C
atoms of Glu
at
B1 take part in the hydrophobic interactions with Phe at the
ligand
2 position (20).
B1. Instead, the Tyr aromatic ring was oriented
toward the first hydrophobic cavity and stacked with the side chain of
Ile-736 located at second turn of the helix
B (position
B5).
Still, Leu-732 appeared to indirectly contribute to the ligand binding
by offering the hydrophobic environment necessary accommodate a bulky
hydrophobic residue at ligand position
2. This finding suggests a
novel mode of peptide recognition in which a hydrophobic residue at the
B5 position can supersede the role of the conserved hydrophobic
residue at the
B1 position in class II PDZ domains.
B helix occurs
to accommodate the side chain of Tyr-2 (Fig. 3C). The
B-factor plots of the structures showed helix
B to have the highest
B values among the secondary structure elements in both the
peptide-free and peptide-bound structures. Gly-743, which is located in
the
F-
B loop, adjacent to Ala-742 in the C-terminal end of helix
B, exhibited the largest positional shift upon peptide binding. The
resultant shift in the C-terminal end of helix
B enlarged the second
hydrophobic pocket, suggesting that Gly-743 plays a key role in the
conformational adaptation of helix
B by providing geometrical
freedom to the preceding residues. In addition, a positional shift of
1.25 Å in the C
atom of Leu-736 at
B5, which interacts with
ligand Tyr-2 likely avoids steric hindrance due to the bulkiness of the
Tyr side chain (Fig. 3D). Supporting this view, the four
peptide backbone positions of the ligand PASS-COOH in the
self-associated structure and TYSC in the PDZ-peptide
complex were identical in both structures; however, there was a
positional difference of 1.25 Å in the C
atom of Leu-736, which
interacts with Tyr-2, due to the difference of the bulkiness of the
side chain at that position (Fig. 3D). Thus, the shift of
helix
B is determined by the difference in the size of the residue
at ligand position
2. This structural flexibility may explain the ability of GRIP1 PDZ6 to bind various target peptides with different hydrophobic amino acids at the
2 position (Fig. 2B).
1 and
3 positions also appear to contribute to its recognition.
Ser-1 hydrogen bonds with Thr-685 in the
B strand in one protomer of
the dimer, while Thr-3 hydrogen bonds with Ser-687 in the other
protomer. The residues at the
3 position of known PDZ6 ligands are
highly conserved and all contain hydroxyl groups (Fig. 2B),
implying direct contribution to the specificity and affinity for the
GRIP1 PDZ6 domain. The side chain of Arg at ligand position
4 also
participates in water-mediated hydrogen bonding with Glu-690 at the
terminus of the
B strand. Although there is no conserved pattern
of interaction at the
1,
3, and
4 positions among PDZ domains,
it likely fine tunes the specificity of PDZ-ligand recognition.
A strand and an
A-
D loop from each protomer; the
A strands form anti-parallel
-sheets around the center of the 2-fold axis, with the N and C
termini of each PDZ domain pointing in opposite directions. The peptide binding pockets were located at the distal sides of the dimeric interface, situated in anti-parallel fashion, which enabled spatially independent binding of target ligands. This dimeric interaction was
supported by six hydrogen bonds between the two anti-parallel
A
strands and hydrophobic forces between non-polar atoms in the interface. The amount of surface area buried upon dimer formation is
619.0 Å2, or 12.3% of the total surface area of each
monomer. Such independent target binding by PDZ multimers was also
detected with InaD and NHERF proteins using biochemical analyses
(30, 31).
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Fig. 4.
Dimerization of GRIP PDZ6.
A, dimeric structure of PDZ6 domain. PDZ6 domains form a
dimer via interaction of antiparallel A strands and
A-
D loops.
Peptide ligands bound to the opposite side of the PDZ6 dimer are
represented in ball-and-stick. B, self-association of two
GRIP PDZ6 domains related by a 2-fold crystallographic axis was
observed in the peptide-free PDZ6 crystal. Each C terminus serves as a
ligand for a neighboring PDZ molecule. C, effects of
mutations on dimerization. Molecular weights of mutants Y671D and R731D
and a wild type PDZ domain were estimated by size exclusion
chromatography (Superdex 75 HR 16/60 column). The mutated residues,
Y671D and R718D, were shown in ball and stick with van der Waals radius
in A and B. The elution profiles of a wild type,
Y671D and R718D mutants. This result suggests that the dimer in
solution is the form shown in A. D, the variable
residues within the PDZ6 domains of GRIP homologues are represented in
ball-and-stick. Only one variable residue, Ile-669, which is Val in
GRIP2, is located in the dimeric interface.
atoms (0.6 Å) between the two
protomers in the asymmetric unit of a peptide-bound crystal. Another
intermolecular interaction with a symmetry-related molecule in the
peptide-free crystal structure is the association through C-terminal
exchange into the ligand binding pockets between two monomers (Fig.
4B). In this case C termini serve as ligands for neighboring
PDZ molecules, which is reminiscent of the crystal structures of NHERF
PDZ1 and hCASK PDZ domains (19, 28). However, the C-terminal amino acid
sequence of the PDZ6 construct (PASS-COOH) differs from the usual class
II PDZ target peptides
(X-Phe/Tyr-X-Phe/Val/Ala-COOH). The backbone
atoms of the C-terminal four residues of the neighboring molecule,
located in the ligand-binding groove, shows little positional difference from the location C-terminal octapeptide of liprin-
1 complexed with the PDZ6. Apparently, hydrophobic pockets of GRIP1 PDZ6
accommodated smaller hydrophobic residues or the residues that partly
mimic a hydrophobic moiety. Most likely, the self-association observed
in the peptide-free crystal structure is not dimeric interaction but
molecular packing interaction within the crystal lattice. Consistent
with this idea, a PDZ6 construct in which the last seven residues
(residues 665-754 lacking DAQPASS) were deleted still formed a dimer
in solution identified by size exclusion chromatography (data not shown).
A strand and
A-
D loop) are identical except for one conserved change (Ile-669
in GRIP1 and Val-655 in GRIP2) (Fig. 4C). Collectively, these findings indicate that GRIP PDZ6 dimers use the same dimeric interface for both homo- and heterodimerization.
B5 position was involved
in specific recognition of the ligand, and the conformational adaptation of the
B helix induced by ligand binding accommodated the
hydrophobic moiety at ligand position
2. In addition, the dimeric
PDZ6 structure described in this study is the first example of a
functional role of PDZ domains in multimerization. Formation of
antiparallel PDZ6 domain dimers is mediated by an interface located at
a site distinct from the peptide-binding groove, resulting in
independent target binding by the PDZ multimer. This mechanism could
enable efficient clustering of various target proteins though multimerization of GRIP proteins.
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ACKNOWLEDGEMENTS |
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We thank Professor N. Sakabe and Drs. M. Suzuki and N. Igarashi for kind support in x-ray data collection at beamline BL-18B of Photon Factory, Tsukuba, Japan. We thank Dr. J. Berendzen and L. Flaks for data collection at beamline X8C of National Synchrotron Light Source at Brookhaven National Laboratory. We also thank Dr. H. S. Lee and G. H. Kim at the BL6B of Pohang Accelerator Laboratory, Pohang, Korea.
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FOOTNOTES |
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* This work was supported by grants from the K-JIST project, the Brain Korea 21 Project and Critical Technology 21 (Neurobiology Research Center).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 1N7E and 1N7F.) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ These authors contributed equally to this work.
** To whom correspondence should be addressed. Tel.: 82-62-970-2549; Fax: 82-62-970-2548; E-mail: eom@kjist.ac.kr.
Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M212263200
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ABBREVIATIONS |
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The abbreviations used are:
GRIP, glutamate
receptor-interacting protein;
ABP, AMPA receptor-binding
protein;
AMPA, -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid;
MAD, multiwavelength anomalous dispersion;
r.m.s.d., root-mean square
deviation.
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REFERENCES |
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