From the Molecular Medicine Laboratory and Macromolecular Crystallography Unit, Division of Experimental Medicine, Harvard Institutes of Medicine, Harvard Medical School, Boston, Massachusetts 02115
Received for publication, March 28, 2001, and in revised form, April 9, 2001
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ABSTRACT |
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The PDZ1 domain of the
Na+/H+ exchanger regulatory factor
(NHERF) binds with nanomolar affinity to the carboxyl-terminal sequence QDTRL of the cystic fibrosis transmembrane conductance regulator (CFTR)
and plays a central role in the cellular localization and physiological
regulation of this chloride channel. The crystal structure of human
NHERF PDZ1 bound to the carboxyl-terminal peptide QDTRL has been
determined at 1.7-Å resolution. The structure reveals the specificity
and affinity determinants of the PDZ1-CFTR interaction and provides
insights into carboxyl-terminal leucine recognition by class I PDZ
domains. The peptide ligand inserts into the PDZ1 binding pocket
forming an additional antiparallel The cystic fibrosis transmembrane conductance regulator
(CFTR)1 is an ATP-regulated
chloride channel that determines the rate of electrolyte and fluid
transport in the apical membrane of epithelial cells (1-3). Abnormal
CFTR function is associated with the pathogenesis of cystic fibrosis
and secretory diarrhea (1-3). The CFTR activity is modulated through
interactions with other proteins; however the regulatory mechanisms
remain unknown. One protein that interacts with the carboxyl terminus
of CFTR is the Na+/H+ exchanger regulatory
factor (NHERF), a cytoplasmic protein originally cloned as an essential
cofactor for the cAMP-dependent protein kinase-mediated inhibition of the Na+/H+
exchanger 3 (4-6). NHERF is also known as EBP50
(ezrin-radixin-moesin-binding phosphoprotein-50), a
membrane-cytoskeleton linking protein that binds to membrane proteins
through its two PDZ (PSD-95/Discs-large/ZO-1) domains and to the
cortical actin cytoskeleton through its carboxyl-terminal domain (7).
The NHERF PDZ1 and PDZ2 domains (Fig. 1A) bind with
nanomolar affinity to the CFTR carboxyl-terminal sequence QDTRL and
play a critical role in the regulation of channel gating (8-10). In
addition, the NHERF-related protein, NHERF2, also binds to the
carboxyl-terminal tail of CFTR through its two PDZ domains (11) (Fig.
1A). Interestingly, CAP70, the murine homolog of the PDZK1
protein (12, 13), also interacts with the CFTR carboxyl terminus
through its PDZ3 domain and modulates the channel activity (12). These
findings corroborate previous studies in establishing the essential
role of the CFTR carboxyl-terminal motif DTRL for the functional
expression of this channel in the apical plasma membrane (14-16).
PDZ domains are protein modules that mediate specific interactions
between proteins and participate in the assembly of membrane receptors,
ion channels, and other signaling molecules into specific signal
transduction complexes (17, 18). PDZ domains bind to short
carboxyl-terminal peptides and have been categorized into two classes
based on target sequence specificity. Class I domains bind to peptides
with the consensus sequence (S/T)X(V/I/L) (X denoting any amino acid), whereas class II domains recognize the motif
(F/Y)X(F/V/A) (19). The PDZ fold comprises a six-stranded antiparallel To elucidate the structural determinants of the NHERF PDZ1-CFTR
interaction, we solved the crystal structure of the PDZ1 domain in
complex with the CFTR carboxyl-terminal peptide QDTRL. The structure
reveals for the first time that the arginine at position Protein Purification and Crystallization--
A DNA fragment
encoding the human NHERF PDZ1 (residues 11-94), and having the
carboxyl-terminal extension Q95DTRL99 that
corresponds to residues 1476-1480 of human CFTR (1), was amplified
using the polymerase chain reaction and cloned in the vector pGEX-2TJL
(27). PDZ1 was expressed in Escherichia coli BL21 (DE3)
cells as a glutathione S-transferase fusion protein, purified using glutathione-Sepharose 4B resin, and the PDZ1 was released by digestion with thrombin, as described previously (27). PDZ1
protein (18 mg/ml) was crystallized using the sitting drop vapor
diffusion method in 0.1 M sodium acetate, pH 4.6, 2 M sodium chloride, at 20 °C. Diffraction data were
collected at room temperature using an R-AXIS IV detector and
CuK Structure Determination and Refinement--
The structure was
solved by molecular replacement using the program MOLREP (30) and the
human NHERF PDZ1 (Protein Data Bank code 1g9o) as the search model. The
rotation function search in the 20-3 Å resolution range produced a
clear solution with a peak height of 5.9 sigma. The translation
function indicated that the correct space group was P3121
with a correlation coefficient of 0.36 and an Rcryst
of 0.50, compared with its enantiomorphic mate P3221, which
had a correlation coefficient of 0.23 and an Rcryst
of 0.56. The model was built using the program O (31) and was
refined by the maximum likelihood method using REFMAC5 (32). The
structure is well ordered except for the loop regions 31-35 and
81-85, which are disordered. The PDZ1 also contains at its amino
terminus the vector-derived residues GSSRM, from which only methionine
is ordered and included in the final model.
Structure Determination--
We recently determined the crystal
structure of the human NHERF PDZ1 domain (residues 11-99) at 1.5-Å
resolution (27). The crystal structure produced a dimeric arrangement
of PDZ1 domains with the carboxyl-terminal region
T95DEQL99 of one PDZ1 molecule bound to a
neighboring PDZ1 because of its resemblance to the PDZ1 ligand
consensus (27). We exploited this intermolecular association of NHERF
PDZ1 in the crystalline state to facilitate the co-crystallization of
this domain with the CFTR ligand by converting the PDZ1 sequence
T95DEQL99 to Q95DTRL99,
which corresponds to the CFTR carboxyl-terminal tail. Recombinant NHERF
PDZ1 was crystallized, and its structure was determined by molecular
replacement. The model was refined to an Rcryst
of 18.7% and an Rfree of 21.7% (Table
I), and the evaluation of its
stereochemistry using PROCHECK (33) showed that 89.2% of the residues
are in the most favored, 8.1% in the additional allowed, and 2.7% in
the generously allowed regions.
Overview of the Structure--
The present NHERF PDZ1 crystal
structure produces infinite head-to-tail polymers of PDZ1 molecules
along the z axis, with the carboxyl-terminal extension
Q95DTRL99 of one PDZ1 molecule serving as a
ligand for a neighboring PDZ1 (Fig.
1B). The overall topology of
NHERF PDZ1 is similar to other PDZ structures (20-27), consisting of
six Structural Basis for the Specificity of the NHERF PDZ1-CFTR
Interaction--
The peptide ligand Q95DTRL99
inserts into the PDZ1 binding pocket antiparallel to the
The side chain and carboxylate group of Leu 0 enter into a deep cavity
formed by Tyr24, Gly25, Phe26,
Leu28, Val76, and Ile79 residues
(Fig. 1D). The C The Importance of Arg Perspective--
The present work reveals the specificity and
affinity determinants of the NHERF PDZ1-CFTR interaction and provides
insights into carboxyl-terminal leucine recognition by class I PDZ
domains, particularly those of NHERF, NHERF2, and PDZK1/CAP70. The
sequence similarity shared among the aforementioned PDZ domains (Fig.
1A) suggests similar modes of interactions with CFTR.
Elucidation of the molecular mechanisms underlying the interaction
between these proteins and CFTR may facilitate the design of potent and specific modulators of CFTR activity with important clinical
applications in the treatment of secretory diarrhea and cystic fibrosis.
-strand to the PDZ1
-sheet, and an extensive network of hydrogen bonds and hydrophobic
interactions stabilize the complex. Remarkably, the guanido group of
arginine at position
1 of the CFTR peptide forms two salt bridges and
two hydrogen bonds with PDZ1 residues Glu43 and
Asn22, respectively, providing the structural basis for the
contribution of the penultimate amino acid of the peptide ligand to the
affinity of the interaction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-barrel capped by two
-helices (20-27). Peptide ligands interact with PDZ domains by a
-sheet augmentation process, in which the peptide forms an additional antiparallel
-strand in the
PDZ
-sheet (28). It is believed that the specificity and affinity of
the PDZ-peptide interaction is achieved by the residues at positions
3,
2, and 0 of the peptide (position 0 referring to the
carboxyl-terminal residue), whereas residue
1 does not play an
important role in the interaction.
1 of the
peptide ligand interacts with PDZ1 residues, thus contributing to the
affinity of the NHERF PDZ1-CFTR interaction.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
radiation. The data were processed using
the programs DENZO and SCALEPACK (29) (Table I). Crystals belong to
space group P3121 with unit cell parameters a = b = 51.7 Å, c = 67.0 Å, and one molecule in the asymmetric unit.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Statistics of structure determination and refinement
|(I
I
)|/
(I), where I is the
observed integrated intensity,
I
is the average
integrated intensity obtained from multiple measurements, and the
summation is over all observed reflections.
Rcryst =
||Fobs|
k|Fcalc||/
|Fobs|.
-strands (
1-
6) and two
-helices (
1 and
2) (Fig.
1, A and C).
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Fig. 1.
Structure of the NHERF PDZ1 domain bound to
the CFTR sequence QDTRL. A, sequence comparison
of PDZ domains that bind to CFTR. The indicated PDZ domains from human
NHERF (5), human NHERF2 (8), and murine PDZK1/CAP70 (12) were aligned
using MACAW (36). Absolutely conserved residues are shown as
white letters on blue background. Identical
residues in four domains are shaded in cyan. The secondary
structure of NHERF PDZ1 is indicated at the top. Conserved
acidic residues proposed to interact with Arg 1 of the CFTR ligand
are denoted by an asterisk. B, stereo view of the NHERF PDZ1
crystal packing. Each carboxyl terminus serves as a ligand for a
neighboring PDZ1 molecule. C, ribbon diagram of the NHERF
PDZ1 domain bound to the QDTRL peptide. The strands
1-
6 are
shown in yellow, and the helices
1 and
2 are shown in
green. The peptide ligand QDTRL is shown in pink.
The figure was made using MOLSCRIPT (37) and Raster3D (38).
D, surface topology of the NHERF PDZ1 bound to the peptide
QDTRL. The figure was generated using GRASP (39).
2
strand and extends the
-sheet of PDZ1 (Fig. 1, C
and D). In this arrangement, the invading pentapeptide is
highly ordered, as indicated by the high quality electron density map
(Fig. 2A). The carbonyl oxygen
of Gln
4 hydrogen bonds with the amide nitrogen of Gly30
(Fig. 2, B and C), indicating that Gln
4 does
not contribute to the specificity of the interaction. By contrast, Asp
3, Thr
2, and Leu 0 are engaged in numerous interactions with PDZ1, consistent with biochemical evidence on the central role of these residues in the specificity and affinity of the NHERF PDZ1-CFTR interaction (8-10). Specifically, the O
1 atom of Asp
3 hydrogen bonds with N
1 of
His27, and the O
2 atom of Asp
3 forms a salt bridge with N
1 of Arg40
(Fig. 2, B and C). Similarly, the amide nitrogen
and carbonyl oxygen of Thr
2 hydrogen bond with the carbonyl oxygen
and amide nitrogen of Leu28, respectively, while the
O
1 atom of Thr
2 hydrogen bonds with the
N
2 atom of the conserved His72
(Fig. 2, A-C).
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Fig. 2.
NHERF PDZ1 interactions with the peptide
QDTRL. A, stereo view of a
2Fobs Fcalc electron
density map calculated at 1.7-Å resolution and contoured at 1
at
the peptide-binding site. B, stereo image of the NHERF PDZ1
binding pocket bound to the carboxyl-terminal peptide ligand
(gray). Carbon, oxygen, and nitrogen atoms are shown in
black, red, and blue, respectively.
Water molecules are shown as red spheres and hydrogen bonds
as dashed lines. C, two-dimensional representation of the
interactions observed between the NHERF PDZ1 residues
(orange) and the peptide ligand (purple).
Dashed lines denote hydrogen bonds, and numbers
indicate hydrogen bond lengths in Å. Hydrophobic interactions are
shown as arcs with radial spokes. The figure was
made using LIGPLOT (40).
1 atom of Leu 0 makes
hydrophobic contacts with the atoms C
2 and
C
of Phe26 and C
1 of
Ile79 (Fig. 2C). In addition, the carboxyl
oxygen of Leu 0 hydrogen bonds with the amide nitrogens of
Gly25 and Phe26, whereas the carbonyl oxygen of
Leu 0 hydrogen bonds directly with the amide nitrogen of
Tyr24 and indirectly with the N
atom of
Arg80 in the
2 helix through two ordered water molecules
(Fig. 2, B and C). The involvement of
Arg80 in carboxylate binding through ordered water
molecules represents a novel feature of the PDZ-ligand interaction and
differs from other PDZ structures where this function is mediated by an
arginine residue in the
1-
2 loop (20), corresponding to NHERF
PDZ1 Lys19. In the present structure, Lys19
does not appear to be involved in hydrogen bonding with the terminal carboxylate group. The isobutyl side chain of Leu 0 fits tightly in the
hydrophobic cavity of PDZ1, suggesting that this stereochemical complementarity may underlie the strict requirement for
carboxyl-terminal leucine in all the high affinity ligands of NHERF
PDZ1 (8-10). Conceivably, smaller side chains would leave vacated
spaces within this hydrophobic cavity that would be energetically
unfavorable (34), whereas bulkier side chains would not readily fit
within this pocket. Moreover, the hydrophobic character of the cavity would likely exclude polar and charged side chains. It therefore appears that the volume, shape, and hydrophobicity of the PDZ pocket
provide the structural determinants for the selection of stereochemically complementary hydrophobic carboxyl-terminal side chains for high affinity binding.
1 for the Affinity of the NHERF
PDZ1-CFTR Interaction--
Strikingly, the guanido group of Arg
1
forms two salt bridges with O
2 of
Glu43 and two hydrogen bonds with the carbonyl oxygen of
Asn22 (Fig. 2, A-C). This finding
was unexpected because the residue
1 of the peptide has been
considered to be unimportant for the PDZ-ligand interaction. Indeed, in
other PDZ structures the side chain of the penultimate residue is
oriented toward the solution and does not interact with PDZ residues
(20, 22). Nevertheless, previous biochemical studies demonstrated that
arginine is the preferred residue at position
1 for optimal binding
to NHERF PDZ1 (8, 9). Affinity selection experiments showed that NHERF
PDZ1 selected almost exclusively ligands with arginine at position
1
from random peptides (9). Furthermore, point mutagenesis of the
penultimate arginine to alanine, phenylalanine, leucine, or glutamic
acid decreased the affinity of the PDZ1-ligand interaction by
2-10-fold (8). The multiple interactions between the Arg
1 guanido
group and PDZ1 residues Glu43 and Asn22
observed in our structure explain the remarkable preference for a
penultimate arginine by NHERF PDZ1. Taken together, these observations indicate that although the peptidic residue
1 is not important for
specificity, it may contribute to the affinity of the PDZ-ligand interaction. Consequently, PDZ domains have a preference for specific side chains at position
1 and interact optimally with peptide ligands
having the corresponding penultimate residues. In support of this
conclusion, it was found that the MAGI3 PDZ2 domain exclusively selected ligands with Trp
1 from random sequences, and it was predicted that tryptophan at this position interacts with
Leu40 for high affinity binding (35). Furthermore, the
solution structure of the
-syntrophin PDZ domain showed that Leu
1
of the peptide ligand makes hydrophobic contacts with Phe34
(23). Remarkably, both MAGI3 Leu40 and
-syntrophin
Phe34 residues correspond to NHERF Glu43,
suggesting that the amino acid at this position may play a critical role in the PDZ-ligand affinity through interaction with residue
1 of
the peptide. Interestingly, the NHERF, NHERF2, and PDZK1/CAP70 PDZ
domains that bind to the CFTR tail (8-13) have either glutamate or
aspartate at the position corresponding to NHERF Glu43
(Fig. 1A), suggesting that Arg
1 of the CFTR tail may form
similar salt bridges with these residues.
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FOOTNOTES |
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* 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 1i92) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
Established Investigator of the American Heart Association. To
whom correspondence should be addressed: Molecular Medicine Laboratory
and Macromolecular Crystallography Unit, Harvard Institutes of
Medicine, Rm. 354, 4 Blackfan Circle, Boston, MA 02115. Tel.: 617-667-0064; Fax: 617-975-5241; E-mail:
John_Ladias@caregroup.harvard.edu.
Published, JBC Papers in Press, April 13, 2001, DOI 10.1074/jbc.C100154200
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ABBREVIATIONS |
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The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; NHERF, Na+/H+ exchanger regulatory factor; PDZ, PSD-95/Discs-large/ZO-1 homology.
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