Crystal structure of the PDZ1 domain of human Na⁺/H⁺ exchanger regulatory factor provides insights into the mechanism of carboxyl-terminal leucine recognition by class I PDZ domains¹

Abstract

The Na⁺/H⁺ exchanger regulatory factor (NHERF; also known as EBP50) contains two PDZ domains that mediate the assembly of transmembrane and cytosolic proteins into functional signal transduction complexes. The NHERF PDZ1 domain interacts specifically with the motifs DSLL, DSFL, and DTRL present at the carboxyl termini of the β₂ adrenergic receptor (β₂AR), the platelet-derived growth factor receptor (PDGFR), and the cystic fibrosis transmembrane conductance regulator (CFTR), respectively, and plays a central role in the physiological regulation of these proteins. The crystal structure of the human NHERF PDZ1 has been determined at 1.5 Å resolution using multiwavelength anomalous diffraction phasing. The overall structure is similar to known PDZ structures, with notable differences in the NHERF PDZ1 carboxylate-binding loop that contains the GYGF motif, and the variable loop between the β2 and β3 strands. In the crystalline state, the carboxyl-terminal sequence DEQL of PDZ1 occupies the peptide-binding pocket of a neighboring PDZ1 molecule related by 2-fold crystallographic symmetry. This structure reveals the molecular mechanism of carboxyl-terminal leucine recognition by class I PDZ domains, and provides insights into the specificity of NHERF interaction with the carboxyl termini of several membrane receptors and ion channels, including the β₂AR, PDGFR, and CFTR.

Introduction

PDZ (PSD-95/discs-large/ZO-1 homology) domains are structurally conserved protein modules that mediate specific interactions between proteins1, 2, 3. A large number of PDZ-containing proteins function as scaffolds for assembling membrane receptors, ion channels, and other signaling molecules in the vicinity of their substrates. By organizing such signal transduction pathways at specific intracellular locations, PDZ proteins play fundamental roles in the specificity and efficiency of signal transduction1, 2, 3.

Previous structural studies have demonstrated that PDZ domains share a common fold, consisting of a six-stranded antiparallel β-barrel capped by two α-helices4, 5, 6, 7, 8, 9, 10. These protein modules bind to short carboxyl-terminal peptides and have been categorized into two classes on the basis of 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).11 In addition, PDZ domains can interact with internal protein sequences that adopt β-hairpin structures10. Peptidic ligands interact with PDZ domains by a process of β-sheet augmentation, in which the peptide forms an additional, antiparallel β-strand in the PDZ β-sheet4, 6, 7, 8, 9, 10. The loop between the first two β-strands plays an important role in recognizing and binding the terminal carboxylate group of the target peptide and is referred to as the carboxylate-binding loop. The specificity of the PDZ-peptide interaction is achieved by the residues at positions −3, −2, and 0 of the peptide ligand (position 0 referring to the carboxyl-terminal residue), whereas the residue at position −1 does not play an important role. The mechanism for selecting a valine residue at position 0 by class I PDZ domains has been determined4, 7, 8, 9. However, the structural basis for carboxyl-terminal leucine recognition has not been elucidated to date.

The Na⁺/H⁺ exchanger regulatory factor (NHERF) was originally cloned as an essential cofactor for the inhibition of the Na⁺/H⁺ exchanger NHE-3 by the cAMP-dependent protein kinase A in the renal brush-border membrane12. The human NHERF was also identified independently as EBP50 (ezrin-radixin-moesin-binding phosphoprotein-50), a membrane-cytoskeleton linking protein that binds to the carboxyl termini of integral membrane proteins through its two PDZ domains and to the cortical actin cytoskeleton through its carboxyl-terminal domain13. The emerging theme from recent studies of NHERF and its close relative NHERF214 is that these proteins play central roles in the membrane targeting, trafficking, and sorting of several ion channels, transmembrane receptors, and signaling proteins in many other tissues15, 16. The PDZ domains of human NHERF17 and NHERF2 share a high degree of sequence similarity (Figure 1) and similar ligand-binding specificities15, 16. Interestingly, PDZ1 homodimerization leads to NHERF self-association in solution18, 19; however, the PDZ1-PDZ1 interaction interface(s) has not been mapped.

The NHERF PDZ1 is a class I domain that interacts specifically with carboxyl-terminal sequences present in many membrane proteins, including the β₂ adrenergic receptor (β₂AR), the platelet-derived growth factor receptor (PDGFR), and the cystic fibrosis transmembrane conductance regulator (CFTR)14, 20, 21, 22, 23. Through high-affinity binding of PDZ1 to the β₂AR carboxyl-terminal motif DSLL, NHERF plays an important role in β₂AR-mediated regulation of Na⁺/H⁺ exchange20, and mediates the sorting of internalized β₂AR between degradative endocytic pathways and plasma membrane recycling24. Likewise, NHERF potentiates PDGFR activity through PDZ1 interaction with the carboxyl-terminal sequence DSFL of the receptor23. Remarkably, a single mutation of the carboxyl-terminal leucine to alanine residue in both β₂AR and PDGFR abolishes their interaction with NHERF PDZ1 in vitro and markedly impairs the function of these receptors in vivo20, 23. Furthermore, the NHERF PDZ1-binding carboxyl-terminal motif DTRL of CFTR is essential for the functional expression of CFTR in the apical plasma membrane and mutations that delete or destroy this motif result in abnormal apical polarization of CFTR and defective vectorial chloride transport25, 26.

In order to elucidate the structural determinants of the human NHERF PDZ1 ligand-binding specificity, we solved the crystal structure of this domain at 1.5 Å resolution. The structure of NHERF PDZ1 was determined in complex with a ligand provided by the carboxyl terminus of a neighboring PDZ1 molecule related by 2-fold crystallographic symmetry. The structure reveals the molecular mechanism of carboxyl-terminal leucine recognition by class I PDZ domains and provides a starting point for understanding the structural basis of the NHERF interaction with several membrane receptors and ion channels, including the β₂AR, PDGFR, and CFTR.