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Structural Basis of the Na+/H+ Exchanger Regulatory Factor PDZ1 Interaction with the Carboxyl-terminal Region of the Cystic Fibrosis Transmembrane Conductance Regulator*

Subramanian Karthikeyan, Teli Leung, and John A. A. LadiasDagger

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

    ABSTRACT
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 beta -strand to the PDZ1 beta -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
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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 beta -barrel capped by two alpha -helices (20-27). Peptide ligands interact with PDZ domains by a beta -sheet augmentation process, in which the peptide forms an additional antiparallel beta -strand in the PDZ beta -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.

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 -1 of the peptide ligand interacts with PDZ1 residues, thus contributing to the affinity of the NHERF PDZ1-CFTR interaction.

    EXPERIMENTAL PROCEDURES
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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 CuKalpha 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.

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.

    RESULTS AND DISCUSSION
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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.

                              
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Table I
Statistics of structure determination and refinement
Rsym = Sigma |(I - < I> )|/Sigma (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 = Sigma ||Fobs- k|Fcalc||/Sigma |Fobs|.

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 beta -strands (beta 1-beta 6) and two alpha -helices (alpha 1 and alpha 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 beta 1-beta 6 are shown in yellow, and the helices alpha 1 and alpha 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).

Structural Basis for the Specificity of the NHERF PDZ1-CFTR Interaction-- The peptide ligand Q95DTRL99 inserts into the PDZ1 binding pocket antiparallel to the beta 2 strand and extends the beta -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 Odelta 1 atom of Asp -3 hydrogen bonds with Ndelta 1 of His27, and the Odelta 2 atom of Asp -3 forms a salt bridge with Neta 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 Ogamma 1 atom of Thr -2 hydrogen bonds with the Nepsilon 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 1sigma 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).

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 Cdelta 1 atom of Leu 0 makes hydrophobic contacts with the atoms Cepsilon 2 and Czeta of Phe26 and Cdelta 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 Nzeta atom of Arg80 in the alpha 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 beta 1-beta 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.

The Importance of Arg -1 for the Affinity of the NHERF PDZ1-CFTR Interaction-- Strikingly, the guanido group of Arg -1 forms two salt bridges with Oepsilon 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 alpha -syntrophin PDZ domain showed that Leu -1 of the peptide ligand makes hydrophobic contacts with Phe34 (23). Remarkably, both MAGI3 Leu40 and alpha -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.

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.

    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 1i92) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

Dagger 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

    ABBREVIATIONS

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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