NHERF1 interacts with ion channels and receptors through its tandem PDZ domains, which recognize specific PDZ-binding motifs with the consensus sequence D(S/T)X(V/I/L) (X denoting any amino acid residue). A mechanism involving competing intramolecular interactions is consistent with the fact that the same motif is also found at the C-terminus of NHERF1 (FSNL). The dynamic nature and low affinity of the PDZ2-CT interaction make this system not only unsuitable for X-ray crystallographic analysis, but also pose major challenges for structure determination by conventional NMR methods. Nevertheless, we were able to resolve and assign a majority of the peaks due to PDZ2 in the presence of covalently or non-covalently bound CT (). The lack of major changes in chemical shift indicates that the overall structure of PDZ2 is preserved in the complex. By measuring the changes in peak intensity for individual residues in 15
N-labeled PDZ2 due to line broadening upon addition of unlabeled CT, we were able to identified key residues involved in recognition of the C-terminus (). These residues, located mainly on the β2 strand and adjacent loops, form a contiguous binding surface (). The interface is similar, but not identical to that observed in the complex of other PDZ domains with extended peptides, which often make more extensive contacts with the α2 helix [13
Our NMR and CD spectra (, and ) indicate that the CT domain, expressed either as an isolated fragment or covalent linked to PDZ2, is highly flexible and largely disordered in solution, as predicted on the basis of its sequence, using a disorder prediction algorithm [9
]. The following observations provide evidence for specific intramolecular interactions between PDZ2 and the C-terminal EB region of NHERF1: (1) the PDZ2-CT fragment is much more stable than the isolated PDZ2 domain (); (2) adding a small amount of unlabeled CT to an 15
N-labeled sample of PDZ2 causes a major decrease in intensity for selected residues on β2 and adjacent loops; (3) addition of unlabeled PDZ2 to 15
N-labeled CT causes major changes in peak intensity and relaxation dispersion profiles (); (4) a 12-residue peptide with a sequence corresponding to the EB region of NHERF1 exhibits changes in line-width and chemical shifts on addition of unlabeled PDZ2 (Supplemental Figure S3
); (5) mutation of residues in the EB at or near the C-terminus cause major shifts in the conformational equilibrium between alternative folded states and modulate the fluorescence of Tyr164 in the PDZ2 peptide binding groove ().
In all other PDZ-peptide complexes reported previously, the peptide ligand assumes an extended conformation and inserts itself between the α2 helix and β2 strand in an antiparallel arrangement with respect to β2 [10
]. The binding groove accommodates a maximum of five residues for an extended ligand peptide with the last three residues at the C-terminus playing the principal role in PDZ-ligand recognition. In contrast, our NMR studies () show that PDZ2 interacts with a much longer segment spanning 11 residues at the C-terminus of NHERF1, including L358, F355, S356, K351, K350 and W348 (listed in decreasing order with respect to relaxation effects). The fact that L358 experiences particularly large spectral perturbations confirms that this C-terminal residue plays a key role in PDZ2-peptide recognition, as previously found in the case of the complex between NHERF1 PDZ1 and CFTR [13
]. Our results are clearly inconsistent with binding in an extended conformation, which would extend far beyond the length of the binding groove on PDZ2. On the other hand, since the axial distance between adjacent amino acids is shorter in an α-helix (1.5 Å) compared to β-sheet or extended conformation (~3.5 Å), an α-helix of 11–12 residues covers the same length as a 5-residue extended peptide, and can thus be accommodated in the peptide binding pocket of PDZ2. Moreover, the perturbations show a periodic pattern with larger changes in relaxation parameters for every 3rd
residue (). To our knowledge, this is the first direct structural evidence for a peptide interacting with a PDZ domain in an α-helical conformation. The distribution of contact residues on PDZ2 in its complex with CT () suggests that the helical EB domain is accommodated in the groove between β2 and α2. A previous attempt to model this complex resulted in a different orientation for the C-terminal helix and a more exposed N-terminal end [18
]. This preliminary docking model has to be revised in light of the NMR data obtained in the present study.
In the crystal structure of a C-terminal fragment of NHERF1 (339–358) bound to the radixin FERM domain (structurally unrelated to PDZ), the C-terminal 11 residues of NHERF1 form a 3-turn amphipathic α-helix [25
]. Based on our NMR results, the majority of residues strongly affected by binding to PDZ2 (L358, F355, K351, and K350) are located on the hydrophobic side of the helix. By measuring the effect of mutations on affinity, Terawaki, et al. found that residue M346 also contributes to binding in the case of the FERM domain [25
]. In contrast, our data indicate that only the helical part of the EB region (residues 348–358) is involved in the binding to PDZ2. Thus, while both PDZ2 and the FERM domain recognize the EB region in an α-helical conformation, the binding interface with these structurally unrelated binding partners is not identical.
Morales, et al. published biochemical and cell biological evidence for intramolecular interactions between the PDZ domains and C-terminus of NHERF1 [8
]. They reported that mutations near the C-terminus (F355P, F355R and a C-terminal deletion) abolished the intramolecular contact between PDZ2 and CT, which they attribute to loss of α-helical structure at the C-terminus. They also showed that intramolecular interactions were only moderately perturbed by another set of mutations (S356A, L358F, and L354I/358F), which are expected to disrupt some of the canonical PDZ-peptide contacts, but not a putative α-helix. Although highly suggestive, the results from this mutagenesis study are inconclusive with respect to the exact conformation of the C-terminal peptide when bound to PDZ2, since proline substitution of an amino acid is expected to disrupt both α-helical as well as β-sheet structure. Furthermore, Pro or Arg substitutions at position 355 involve drastic changes in the size and shape of a side chain that is also predicted to contact the PDZ domain in the canonical extended binding motif.
Our NMR and mutational studies offer more detailed structural insights and provide direct support for the hypothesis that the EB region of NHERF1 adopts a helical conformation when engaged in intramolecular contacts with PDZ2. Especially compelling is our finding that amino acid changes at an exposed site of the putative helix cause significant shifts in the conformational transition preceding the main unfolding transition (, ). Mutations that destabilize the C-terminal helix (N352G, E353P and E353G) or remove a key contact with PDZ2 (L358D) shift the midpoint of the first transition (Cm1) toward higher urea concentrations and are associated with positive ΔΔG1 (). In contrast, the mutations cause only small shifts in the second (major) unfolding transition and give rise to small or negative ΔΔG2. As a result, the maximum population of the I-state () is considerably lower for destabilizing mutations (~40%) compared to wild-type and the helix-stabilizing E353A variant (~75%). Thus, contrary to expectations, disruption of the C-terminal helix or its contacts with the PDZ2 domain stabilizes the native state relative to the I-state populated at intermediate urea concentrations. Interestingly, the variation in population of the I-state correlates with its fluorescence properties; the proteins with well-populated I-states (wild-type and E353A) have substantially enhanced tyrosine emission spectra ().
Our equilibrium denaturation experiments () show that unfolding of PDZ2-CT occurs in several steps involving at least two alternative folded states in addition to fully unfolded conformations. For the wild-type protein and all mutants studied, a three-state unfolding model fully accounts for all of the fluorescence data vs. urea concentration and yields a robust set of fitting parameters characterizing the thermodynamic and spectral properties of the populated states (, Figure S4
). The second transition centered around 3.8 M urea is relatively insensitive to mutation of C-terminal residues, and thus represents the cooperative unfolding of the PDZ2 domain as well as any parts of the linker segment (residues 242–347) it may be in contact with. In contrast, the conformational transition at lower urea concentrations is very sensitive to point mutations in the EB region (except for E353A, which supports helix formation). The free energy perturbations, ΔΔG1
, due to mutations that interfere with helix formation or remove critical contacts with PDZ2 (L358D) are all positive (). Thus, mutations that disrupt binding to the PDZ domain all result in a significant stabilization of the native state relative to intermediate and unfolded states. These surprising observations can be explained in terms of the mechanism schematized in , which features two partially folded states, Iopen
, in addition to the native (N) and fully unfolded (U) states. The N
U branch is the dominant unfolding pathway for the disruptive mutants, which exhibit two distinct unfolding transitions. The wild type protein and the helix-stabilizing E353A variant can assume an alternative folded state, IEB
, in which PDZ2 engages the helical EB motif (IEB
are unresolved in our equilibrium unfolding measurements). IEB
dominates the population at intermediate urea concentrations (~1.5–3.5 M), and is thus less structured (in terms of solvent-accessible surface area) than the N-state. This suggests that portions of the linker segment (240–347) are structured in N and become solvent-exposed in IEB
. Mutations that block formation of the C-terminal helix or its contact with PDZ2 shift the conformational equilibrium from IEB
and N, which accounts for the observed stabilization of the N-state at the expense of the less structured intermediates.
Schematic diagram of the various conformational states populated in the unfolding equilibrium of PDZ2-CT
The mechanism outlined in also accounts for the observed effect of mutations on the tyrosine fluorescence spectra (). For WT and E353A, the tyrosine emission band in the non-native intermediates (comprising both Iopen
) is nearly as high as that in the N-state (Figure S4
), indicating that Tyr164 is buried in both IEB
and N. On the other hand, solvent-quenching of Tyr164 fluorescence in Iopen
explains the low I-state fluorescence of the N352G, E353G, E353P, E353D and L358D variants in which IEB
is unstable and Iopen
is well populated. Mechanisms with fewer states cannot fully account for all observations. For example, if IEB
were the predominant state in the absence of denaturant, as initially proposed [8
], disruptive mutations in the C-terminal PDZ-binding motif would greatly destabilize
the native state and shift the equilibrium towards partially or fully unfolded states (Iopen
or U). In contrast, the disruptive changes in the EB motif consistently stabilize
the folded state N, indicating that PDZ2-EB interactions are unfavorable in the absence of denaturant and are replaced by energetically more favorable interactions involving the flexible linker region.
The ligand binding properties of mutant NHERF1 constructs are fully consistent with the proposed mechanism () if we postulate that the peptide binding groove of PDZ2 is accessible for interaction with extrinsic ligand peptides only in Iopen
. This explains our observation that the affinity of PDZ2 for C-CFTR increases when we disrupt its autoinhibitory interactions with the EB motif (either be mutating the important C-terminal Leu or by blocking helix formation). Further corroborating evidence comes from the fluorescence of Tyr164 (), which reports on the solvent accessibility of a key residue in the ligand binding grove of PDZ2. Our observation of strong tyrosine fluorescence bands under native conditions and those favoring formation of IEB
() indicates that Tyr164 is buried in a non-aqueous environment in both N and in IEB
, but exposed to the quenching solvent in Iopen
. Since tight binding of PDZ ligand peptides involves intimate contact with the Tyr164 side chain [13
], it is likely that not only IEB
, but also the N-state is unable to bind extrinsic peptide ligands. Whether this reflects competing interactions involving the flexible linker region or structural changes within the PDZ2 domain remains to be explored. The latter possibility is in line with recent kinetic evidence that binding a high-affinity peptide ligand to the 2nd
PDZ domain of tyrosine phosphatase PTP-BL is accompanied by structural rearrangements at the interface between the α2 helix and β2 strand [26