Autoinhibitory interactions between the PDZ2 and C-terminal domains in the scaffolding protein NHERF1

Abstract

Na(+)/H(+) exchanger regulatory factor (NHERF1) is a signaling adaptor protein comprising two PDZ domains and a C-terminal ezrin-binding (EB) motif. To understand the role of intramolecular interactions in regulating its binding properties, we characterized the complex between the second PDZ domain PDZ2 and the C-terminal 242-358 fragment of NHERF1 using NMR and fluorescence methods. NMR chemical shift and relaxation data implicate 11 C-terminal residues in binding and, together with a thermodynamic analysis of mutant proteins, indicate that the EB region becomes helical when bound to PDZ2. Both specific contacts between PDZ2 and EB as well as nonspecific interactions involving a 100-residue flexible linker contribute to stabilizing two structurally distinct closed conformations of NHERF1. The affinity of mutant proteins for an extrinsic ligand is inversely related to the helix-forming propensity of the EB motif. The findings provide a structural framework for understanding how autoinhibitory interactions modulated the binding properties of NHERF1.

Figure 1. Domain structure of NHERF1 and 3D structure of PDZ domains

(A) A linear representation of the domain structure of NHERF1. (B) Backbone overlay of NMR solution structures of NHERF1 PDZ2 (12 structures with lowest number of restraint violations). Ramachandran plot analysis of the final structures showed 64%, 30%, 6% and 0% for most favorable, additionally allowed, generously allowed and disallowed regions, respectively (excluding Gly). (C) Ribbon representation of NMR structure of PDZ2. (D) Comparison of the backbone structures of NHERF1 PDZ1 (blue) and NHERF1 PDZ2 (magenta).

Figure 2. NMR evidence for inter- and intra-molecular interactions between PDZ2 and CT

(A) HSQC at 15 °C of ¹⁵N-labeled PDZ2 in 20 mM HEPES and 150 mM NaCl, pH 7.5. (B) Transverse relaxation-optimized NMR spectrum (TROSY) at 30 °C of PDZ2-CT in 20 mM HEPES and 150 mM NaCl, pH 7.5. (C–F) Expanded regions from HSQC spectra at 20 °C of ¹⁵N-labeled PDZ2 (red), equimolar complex of ¹⁵N-labeled PDZ2 and unlabeled CT (orange), and PDZ2-CT (cyan) in 20 mM HEPES and 150 mM NaCl, pH 7.5. Assignments for PDZ2 are indicated.

Figure 3. Secondary structure and stability of PDZ2, CT and PDZ2-CT

CD spectra of (A) PDZ2 at 15 °C (solid line) and PDZ2-CT at 20 °C (dashed line); and (B) CT at 15 °C. (C) Urea unfolding of PDZ2 and PDZ2-CT monitored by using CD at 222 nm at 15 °C. The lines represent a two-state fit of the data for PDZ2 (C_m=1.25±0.33 M, m = 1.41±0.13 kcal mol⁻¹ M⁻¹; ΔG₀=1.76±0.46 kcal mol⁻¹) and PDZ2-CT (C_m=4.18±0.03 M, m = 1.6±0.11 kcal mol⁻¹ M⁻¹; ΔG₀=6.69±0.15 kcal mol⁻¹), respectively.

Figure 4. Residues on PDZ2 interacting with the EB region

(A) Fractional intensity change in percent (complex/free) from the ¹⁵N relaxation dispersion reference spectra (without CPMG sequences) recorded on free PDZ2 (1.51 mM) and PDZ2 (1.46 mM) in the presence of CT (0.032 mM). Unassigned residues (Y164, S170, and K172) and those with overlapping peaks in the spectrum of the complex (A190, I199, V202, and M207) are not shown. (B) Ribbon diagram of the NMR structure of PDZ2 with a surface plot in the background. Residues with more than 50% intensity change are shown as ball-and-stick in red. S162, the phosphorylation site in the PDZ2 alone, is shown as ball-and-stick in yellow. (C) Ribbon diagram surface plot of the NMR structure of PDZ2 showing homologous side chains in PDZ1 involved in interactions with the peptide DEQL [19].

Figure 5. NMR evidence that PDZ2 binds the C-terminal EB region in a helical conformation

¹H-¹⁵N HSQC spectra of ¹⁵N-labeled CT (242–358). (A) The superimposed spectra of HSQC of ¹⁵N-labeled CT (red) and HSQC of ¹⁵N-labeled CT (0.53 mM)/unlabeled PDZ2 (0.53 mM) equimolar complex (black); (B) The difference spectrum obtained by subtracting HSQC of ¹⁵N CT from that of equimolar ¹⁵N-labeled CT/unlabeled PDZ2 complex (red: positive contours; black: negative contours). The spectra of free and complex were normalized based on the intensities of the peak with an arrow in (A). (C) Chemical shift changes due to binding of ¹⁵N-labeled CT to unlabeled PDZ2 derived from relaxation dispersion measurements (Figure S3), including 10 of the last 11 residues at the C-terminal of NHERF1. (D) C-terminal helix of the NHERF1 peptide (346–358) as observed in the crystal structure of the FERM-NHERF1 complex [25]. The side chains are shown in ball-and-stick representation. F355 and L358 (red) are involved in binding to PDZ2 and FERM. M346, W348, and L354 (green) are involved in binding to FERM only. K350, K351, and S356 (blue) are important in the binding to PDZ2 only.

Figure 6. Effect of mutations in the EB region of NHERF1 on the three-state unfolding equilibrium of PDZ2-CT

observed by global analysis of the fluorescence emission spectra vs. urea concentration (Supplemental Data, Figure S4). In panels (A) and (B) the populations of the three predominant equilibrium states, N (solid), I (dashed) and U (dash-dot), are plotted vs. urea concentrations for PDZ-CT and three variants. (C) Intrinsic tyrosine fluorescence spectra of the I-state (normalized relative to the emission maximum of the N-state) for WT PDZ2-CT and three variants. (D) Bar graph showing the effect of mutations on the free energy of the N ⇔ I transition (Δ ΔG₁, hatched) and the I ⇔ U unfolding transition (Δ ΔG₂). Free energy changes were calculated at the C_m1/2 values of WT PDZ2-CT, using the transition parameters in Table 1.

Figure 7. Schematic diagram of the various conformational states populated in the unfolding equilibrium of PDZ2-CT

, including the native state (N) populated in the absence of denaturant, a state with the C-terminal EB helix interacting with PDZ2 (I_EB), an open state in which only the PDZ domain is folded (I_open), and the fully unfolded ensemble (U).