Keywords: nanoscale protein motion, disordered protein, protein binding kinetics, neutron spin echo spectroscopy, protein dynamics

Effects of phosphomimic mutation on NHERF1 conformation

In order to clarify if NHERF1 has an oligomer fraction in solution, we have performed online size-exclusion chromatography small angle X-ray scattering (SEC-SAXS) to analyze the conformation of wild-type NHERF1(wt) and the phosphomimic NHERF1(S339D/S340D). In SEC-SAXS, scattering is performed immediately after the protein is eluted out of a fast protein liquid chromatography size exclusion column. SEC-SAXS is thus more able to detect the presence of oligomers or aggregates in a protein solution than a classical SAXS experiment. As shown inFig. 2A, there is only one peak eluted from the SEC column, and the radius of gyration R_g of NHERF1(wt) remains unchanged over the SEC peak, suggesting the absence of oligomerization or intermolecular interference effects. This result confirms our previous findings that NHERF1(wt) is monomeric [20,28]. R_g of NHERF1(S339D/S340D) decreases slightly at the peak tail position as compared to the peak front, seeFig. 2B, suggesting the presence of marginally attractive intermolecular interference effects in NHERF1(S339D/S340D) at high concentrations. Comparing R_g, the length distribution function P(r), and the maximum dimension D_max obtained from the SEC-SAXS data at the post-peak position indicates that NHERF1(S339D/S340D) is slightly more expanded than NHERF1(wt), seeTable 1 andFig. 2C&2D.

The small angle neutron scattering (SANS) experiments performed on deuterated^dNHERF1(wt) and^dNHERF1(S339D/S340D) in H2O buffer show similar results as SEC-SAXS, seeFig. 3 andTable 1. R_g and D_max of the^dNHERF1(S339D/S340D) are slightly larger than those of the deuterated^dNHERF1(wt), seeFig. 3B. Together, SEC-SAXS and SANS show that, although the conformational changes are detectable within experimental error, the effects of phosphomimic mutation on the ensemble averaged R_g, D_max, and P(r) function are not dramatic.

Phosphomimic mutation excites nanoscale dynamics at the tip of the disordered tail of NHERF1

We have performed neutron spin echo spectroscopy (NSE) experiments to determine the nanoscale dynamics of NHERF1(S339D/S340D) on 1–70 nanosecond time scales and in Q range 0.05–0.175 Å⁻¹. The NSE experiments were performed at the physiological salt concentration 150 mM NaCl, and 300 mM NaCl, respectively, seeFig. 4A&4B for the NSE spectra. The D_eff(Q) values, shown inFig. 4C, are obtained from the NSE spectra andEq. 1.

Notably, D_eff(Q) of NHERF1(S339D/S340D) in 150 mM NaCl is higher than that in 300 mM NaCl solution, suggesting the activation of internal motion in the phosphomimic mutant at lower buffer salt concentration. D_eff(Q) of NHERF1(S339D/S340D) in 300 mM NaCl is comparable to that of NHERF1(wt). We previously have shown that D_eff(Q) of NHERF1(wt) is of that of a rigid-body motion with no internal motion [29]. The D_eff(Q) data reveals activated nanoscale motion in the phosphomimic mutant as compared to the rigid wild-type protein, and shows that nanoscale motion in the phosphomimic mutant is highly sensitive to salt concentration.

We have developed a theoretical framework to analyze the NSE data (Eq.2), and have shown how to extract protein nanoscale dynamics in a highly sensitive way from NSE data by measuring the effective diffusion constant D_eff(Q) and comparing it with theoretical calculations [29,40]. The analysis requires structural coordinates of the full-length protein. NHERF1 is a multidomain protein with a significantly disordered C-terminal tail and two PDZ domains connected by a flexible linker. There is no high-resolution structure of the full-length NHERF1. We have determined the atomic structures of the individual PDZ1, PDZ2, as well as the disordered C-tail using high-resolution NMR [41]. With the available SANS data and atomic coordinates of the PDZ domains, structural models of the full-length NHERF1(wt) or NHERF1(S339D/S340D) are first generated using the program EOM [42]. Atoms were then added to the Cα-carbon in the disordered linker and C-terminal tail using the program SABBAC [43]. The generated structural models are then refined against the SANS data by back calculating the scattering curves, seeFig. S1. Those calculated scattering profiles that have a good fit to the experimental SANS data (χ² ≤1) were selected to compute D_eff(Q). Such a generated all-atomic structural model represents an ensemble-averaged conformation of the full-length NHERF1(wt) or NHERF1(S339D/S340D).

The center-of-mass diffusion constant D_o of NHERF1(wt) measured by pulsed field NMR in 150 mM NaCl D2O buffer is D_o=2.4 Ų/ns [29]. Dynamic light scattering shows that the center-of-mass diffusion constants of NHERF1(wt) and NHERF1(S339D/S340D) in H2O buffer are the same within experimental error [21]. The D_o=2.4 Ų/ns value was thus used in our calculation of D_eff(Q) of both NHERF1(wt) and NHERF1(S339D/S340D). With the available all-atomic structural models of NHERF1(wt) and NHERF1(S339D/S340D) and D_o, we were able to compute D_eff(Q).

Comparing our calculations with the experimental NSE data shows that D_eff(Q) of NHERF1(S339D/S340D) in 300 mM NaCl is that of rigid-body motion, similar to that of NHERF1(wt) in 150 mM NaCl, seeFig. 4C. However, in 150 mM NaCl, D_eff(Q) of NHERF1(S339D/S340D) cannot be described as rigid-body motion, seeFig. 4C. Our calculations indicate that unleashing the “tip of the whip” near the position of S339/S340 and allowing EBD to be a separate body can best describe the experimental D_eff(Q) of NHERF1(S339D/S340D) at 150 mM NaCl. Incorporating motion in other parts of the protein, such as in the linker between PDZ1 and PDZ2 or in the N-terminal portion of the C-tail, does not produce theoretical D_eff(Q) values that are in agreement with experimental value, seeFig. S2. Thus, NSE analysis via D_eff(Q) can specifically pinpoint the location of the internal motion in NHERF1 that is activated by phosphomimic mutation.

Phosphomimic mutation and salt concentration affect the kinetics of NHERF1 binding to FERM, which cannot be explained by electrostatic forces alone

Using surface plasmon resonance (SPR), we have determined the affinity and the kinetics of NHERF1 binding to FERM at various NaCl concentrations while keeping other buffer compositions unchanged.Fig. 5 shows that the equilibrium dissociation constant K_d of NHERF1(wt) is in general smaller than that of NHERF1(S339D/S340D) at different salt concentrations. For example, for NHERF1(wt) at I=0.16 (in 150 mM NaCl buffer), K_d=51±5 nM [20]. At I=0.31 (in 300 mM NaCl buffer), K_d of NHERF1(wt) increases modestly by 2.3 fold as compared to that at I=0.16. K_d of NHERF1(S339D/S340D) increases 6.3 fold at I=0.31 as compared to that at I=0.16. The result show that K_d of NHERF1(wt) is less dependent on the buffer ionic strength I than that of the NHERF1(S339D/S340D) mutant.

The kinetics of a protein-protein interaction can be obtained by analyzing the shape of the SPR sensorgrams [44–46]. The response curves of the binding of NHERF1(wt) or NHERF1(S339D/S340D) to FERM can better be fit by a two-state kinetic model (Eq.3) than a single state 1:1 binding model or any other binding models, seeFig. S4. At 150 mM NaCl (i.e. at ionic strength I=0.16M), k_a1 is in the order of 10⁶ M⁻¹ s⁻¹ for both NHERF1(wt) and NHERF1(S339D/S340D), seeFig. 6A. The k_a1 values of NHERF1 to FERM are comparable to those of other intrinsically disordered proteins measured by solution-based kinetic experimental methods [47–49]. For both NHERF1(wt) and NHERF1(S339D/S340D), the dissociation rate constant k_d1 is essentially independent of salt concentration, seeFig. S6A.

Fig. 6A shows the ionic strength dependence of k_a1 for NHERF1(wt) and NHERF1(S339D/S340D) to FERM binding, respectively. Notably, k_a1 of the NHERF1(S339D/S340D) mutant is more subject to buffer ionic strength changes than the wild-type protein. However, k_a1 of the wild-type NHERF1 is not as sensitive to the buffer ionic strengths as the S339D/S340D mutant. For instance, at I=0.31M, k_a1 of the mutant reduces 13 fold as compared to that at I=0.16M. At I=0.16M, k_a1 of NHERF1(S339D/S340D) is similar to that of NHERF1(wt). In the entire range of buffer ionic strengths measured, k_a1 of NHERF1(S339D/S340D) decreases about two order of magnitude from low to high ionic strength, while that of NHERF1(wt) decrease about 5.6 fold.

Following the analysis of Schreiber and Fersht in determining the electrostatic force dependent association rate constant of barnase with barstar [50],Fig. 6B plots logk_a1 as a function of logγ_± of NHERF1(wt) and NHERF1(S339D/S340D) to FERM binding, where γ_± is the mean activity coefficient of the buffer, and logγ_± is given by the Debye–Hückel equation, seeEq. 4. A linear dependence of logk_a1 on logγ_± suggests that the association rate constants decrease with increasing ionic strength. NHERF1(wt) and NHERF1(S339D/S340D) have different slopes and intercepts. The larger slope suggests that the k_a1 of NHERF1(S339D/S340D) is more susceptible to the buffer ionic strength changes than the wild-type protein.

If electrostatic interactions are the only factor that affects k_a1, logk_a1 vs logγ_± inFig. 6B will have different slopes but converge to the same value when extrapolating to logγ_±=−0.277 [50,51]. According to the Debye–Hückel relationship (Eq.4), logγ_± approaches the asymptotic value −0.277 when the ionic strength I is infinitely large. At infinitely large ionic strength I, all the electrostatic charges are screened. Thus, at logγ_±=−0.277, the association rate constant approaches the basal value k_a0 in the absence of electrostatic force. For the interaction of barnase and barstar in which electrostatic force is the predominant factor that influences the binding kinetics, the basal association rate constant ka0 converge at logγ_±=−0.277 [50,51].

However, for NHERF1(wt) and NHERF1(S339D/S340D), logka1 does not converge at the asymptote logγ_±=−0.277. A linear fit inFig. 6B gives log_ka10=4.70 and a basal rate constant k_a10=5.06×10⁴ 1/Ms for NHERF1(wt) at logγ_± = −0.277, whereas for NHERF1(S339D/S340D) logk_a10=1.68 and k_a10=47.9 1/MS. Thus, the Debye–Hückel analysis implies that the change in association rate constant at different ionic strengths cannot be explained by electrostatic effects alone. This results correlates with our NSE results that indicate significant changes in nanoscale dynamics of NHERF1(S339D/S340D) as we lowered the buffer salt concentration.

Analyzing the crystal structures of FERM in complex to the last 14 EBD residues of the NHERF1 C-terminal domain suggests that the complex predominantly utilizes hydrophobic interactions for high affinity binding [24,25], seeFig. S5. The structures of the complex thus explain why the binding kinetics of NHERF1(wt) to FERM is not sensitive to salt concentration. Further, in the structure of the complex, S339 and S340 are located in the loop flanking the hydrophobic binding motif. S339/S340 are also in proximity to the negatively charged surface of the FERM domain, seeFig S5. The phosphomimic mutation introduces negative electrostatic charges, which should have generated repulsive interactions with the negatively charged surface of FERM, and thus reduce k_a1 at low salt concentrations. However, such repulsive effects are likely countered by the increased nanoscale motions in the phosphomimic mutant, which allow NHERF1(S339D/S340D) to increase k_a1 at low salt concentration. At higher salt concentration of 300 mM NaCl, the electrostatic interactions are screened, and the reduced nanoscale motion in NHERF1(S339D/S340D) is largely responsible for the reduced k_a1 to bind FERM. Thus, for NHERF1(S339D/S340D), the sensitivity of k_a1 to the salt concentration correlates with the change in nanoscale motion in the mutant.

For both NHERF1(wt) and NHERF1(S339D/S340D), the general trend of k_a2 is to decrease with increasing ionic strength (Fig. S6B), whereas the effects on k_d2 of are modest (Fig. S6C). The origin of the slower kinetic binding mode k_a2 or k_d2 is likely due to the large conformational changes in NHERF1 after the engagement of NHERF1(wt) or NHERF1(S339D/S340D) with FERM. Our previous contrast variation SANS studies indicate that NHERF1(wt) undergoes significant conformational changes upon binding to FERM, with significant rearrangement of the two PDZ domains and the opening of the disordered C-terminal tail [28,29]. Alternatively, the slow binding mode may be due to a subsequent binding interaction, as implicated in the crystal structure of a 31-residue tail (a.a. 321–358) of NHERF1 bound to the Moesin FERM domain [24]. This crystal structure shows that residue 321–330 are in contact with the F2 subdomain of FERM besides the last 14 C-terminal residues that bind tightly to the F3 subdomain. The S339 and S340 phosphorylation sites are located in the loop between the two binding patches.

Correlation between binding kinetics and nanoscale motion

The kinetics binding results show that the association rate constant of NHERF1(S339D/S340D) binding to FERM is susceptible to the influence of buffer salt concentration, with a particularly enhanced association rate constant k_a1 at low salt concentration. At low buffer ionic strength, because of the activation of the nanoscale motion in the C-tip of NHERF1(S339D/S340D) as shown by NSE, the protein can overcome the negatively charged repulsive interaction with the surface of FERM, and have similar k_a1 as the wild-type protein. A high buffer ionic strength, the rigid NHERF1(S339D/S340D) has a significantly reduced k_a1 as compared to that in low salt buffer solution. Comparing the kinetics binding and the NSE results at low and high salt concentration thus suggests that the altered binding kinetics correlates with the changes in the nanoscale dynamics.

Intrinsically disordered proteins can be relatively rigid

The full-length NHERF1 and, in particular, the C-terminal tail have all the traits of an intrinsically disordered protein [1]. The largely disordered C-terminal tail can sample a variety of conformations as shown in our previous NMR study and the SAXS and SANS data [22,28]. However, the motion of the full-length wild-type protein as seen by NSE is that of a rigid-body [29,52]. The disordered C-tail of the wild-type NHERF1 as viewed by NSE, on nanometer length scale and nanosecond time scale, is a structural disorder of an ensemble of rigid and heterogeneous conformations. Only in the phosphomimic mutant of NHERF1 and at relatively low salt solution, the nanoscale protein motion at the C-terminal tip is excited. Depending on the spatial and temporal observation, a distinction between static disorder and dynamic disorder of intrinsically disordered proteins has been proposed [53]. Our results show that NSE can distinguish such static disorder from dynamic order on nanoscales.

Phosphorylation has diverse effects on the structure and function of proteins. It has been shown that phosphorylation modulates protein-protein interactions by altering the electrostatics and chemistry of the recognition site directly, or by inducing small or large structural changes that affect the recognition site allosterically [8,9,54,55]. However, a large-scale statistical study shows that the majority of phosphorylated proteins only undergo subtle conformational changes with the global root-mean-square deviation of less than 2Å [56]. Our SAXS and SANS results indicate that the effect of phosphomimic S339/S340D mutation on the overall conformation of NHERF1 is subtle and small, which corroborate the statistical study [56]. Future study should examine the effect of protein kinase phosphorylation on the dynamics and binding kinetics of full-length NHERF1.

In summary, this study reveals that phosphomimic mutation and salt concentration alter the nanoscale dynamics and target-binding kinetics in the intrinsically disordered tail of NHERF1. The altered kinetics of a phosphorylated NHERF1 binding to Ezrin could modulate the dynamic attachment of the NHERF1-assembled membrane signaling complexes to the cytoskeletal actin network, thus influencing the intracellular mobility and localization of membrane signaling complexes. Being able to reveal the nanoscale protein dynamics in the disordered tail provides a mechanistic explanation for the effects of phosphorylation on the cellular functions of NHERF1, demonstrating that controllable nanoscale protein motions inspire mesoscale function and assembly.

Protein expression and purification

The bacterial expression and purification of the full-length wild-type of human NHERF1 and the FERM domain of Ezrin (residue 1-298) are described previously [20,28]. Site-directed mutagenesis of NHERF1 was performed as described [21]. The deuterated proteins used in the SANS experiments were expressed in M9 minimum medium containing 85% D2O and purified as described previously [28,29].

Online size-exclusion chromatography small angle X-ray scattering (SEC-SAXS) experiments

The SEC-SAXS experiments were performed at SSRL (Stanford Synchrotron Radiation Light Source) Bio-SAXS beamline 4-2 in a similar manner as recently reported [57–59], seeTable S1 for SEC-SAXS setup parameters. Briefly, a 100 μl of the protein sample at 6.9 mg/ml NHERF1(wt) or 11.0 mg/ml NHERF1(S339D/S340D) was applied to a Superdex 200 PC3.2/300 column (GE Healthcare, Wisconsin, USA). The SEC running buffer and the protein buffer was 150 mM NaCl, 20 mM Tris-HCl (pH=7.5), 0.1 mM EDTA. 5mM Dithiothreitol was added to both sample and running buffer solution in order to alleviate radiation damages. Totally 600 images were recorded with 1 second exposure every 5 seconds at 0.05 ml/min flow rate. The program SasTool (http://ssrl.slac.stanford.edu/~saxs/analysis/sastool.htm) was employed for data reduction including scaling, azimuthal integration, averaging and background subtraction. The first 100 images at the early part of the void volume were averaged and used as a buffer-scattering profile for the background subtraction. After taking the first 100 images or so, the X-ray shutter was closed until main elution peak so as to keep the sample cell clean from radiation-damaged sample. The data were then presented as I(Q) vs. Q, where Q=4πsin(θ)/λ, 2θ the scattering angle, and λ the wavelength of the X-ray. The scriptfplcplots, available at SSRL beamline 4-2, was used for consecutive Guinier analysis, implemented in the programAUTORG [60], and assessing data quality (e.g. radiation damage and cleanness of sample cell) by providing Rg, I(0) and an experimental intensity at a low Q angle. Since marginal inter-particle interactions (concentration dependence) were observed over the peak, the average profiles, image number: 500^th–504^th for NHERF1(wt) and 520^th–524^th for NHERF1(S339D/S340D) were generated, scaled and merged for further analyses. The length distribution function was generated using the program GNOM [61].

Small angle neutron scattering (SANS) experiments

SANS data were collected using the EQ-SANS instrument [62], which is a time-of-flight SANS instrument located at the Spallation Neutron Source (SNS) of Oak Ridge National Laboratory. A single instrument configuration with a 4 m sample-to-detector distance was employed. The instrument choppers ran at 60 Hz and were set to provide a minimum wavelength of 2.5 Å. The beam was defined with a 25 mm diameter source aperture and a 10 mm diameter sample aperture. The configuration spans a Q-range from ~0.01 Å⁻¹ to ~0.40 Å⁻¹, which probes the length scales required to see the complex and facilitates subtraction of the solvent background from the scattering signal from the samples. Mantid [63] was used to reduce the data from the samples and from the backgrounds using standard procedures that correct for incident flux spectrum, sample transmission and detector sensitivity, as well as the detector dark current, which represents electronic noise and natural sources of radiation. Then, the data were azimuthally averaged the data to provide I(Q) vs. Q. The sample scattering was then corrected for the solvent scattering by subtracting the 1D profiles to produce the final, reduced data. Details of SANS data analysis to extract protein conformational changes are described in previous studies [27,64–66].

Neutron spin echo experiments

The NSE experiment was performed at SNS-NSE instrument, JCNS1 outstation to Spallation Neutron Source, Oak Ridge National Laboratory [67]. NSE spectra were measured at a maximum incident wavelength of 9Å, accessing a dynamical range of ≤0.1 t_max ≤70 ns (Fouriertimes) for several momentum transfers, Q, between 0.05Å⁻¹ and 0.12Å⁻¹. As an elastic reference measurement we used a Graphite powder sample loaded in an identical quartz container and measured with the same instrument setup. The samples were loaded in top loader quartz containers (Hellma cells) of 40×30×4 mm and NSE spectra were taken at 283.15K. Solvent/background measurement and transmission measurements were also necessary for proper data reduction. The data reduction was performed with the standard ECHODET software package of the NSE-SNS instrument [67]. The momentum transfer (Q) ranges between 0.052 and 0.169 Å⁻¹.

Surface plasmon resonance experiments

Surface plasmon resonance (SPR) experiments were performed on a X100 instrument (GE Healthcare Life Sciences). Before the binding experiments, the Biacore CM5 Biosensor chips was activated by N-hydroxysuccinimide and N-ethyl-N′-[3-(diethylamino) propyl] carbodimide (GE Healthcare Life Sciences). The ligand, which is the FERM domain of Ezrin dissolved at 5 μg/ml in 10mM sodium acetate pH=4.9, was injected to coat the activated sensor chip surface in one of the two flow cells. Uncrosslinked ligand was washed away, and uncoated sites were blocked by 1 M ethanolamine, pH=8.5. The control flow cell was activated and blocked without ligand injection. The analytes (NHERF1 wild-type protein or mutant) were dissolved in HBS-EP buffer containing 10 mM HEPES buffer, pH=7.4, 3 mM EDTA and 0.005% surfactant polysorbate 20 and different NaCl concentrations, respectively. The analytes, of increasing concentrations, were injected over the FERM coated surfaces at 30 μl/min for 180 seconds. The dissociation time is 800 seconds. At the end of injection-dissociation cycle, the sensor chip was regenerated with 4.0 M MgCl_2, 50 mM triethylamine (pH=9.15), and HBS-EP buffer. SPR experiments were performed at three different ligand immobilization levels and in HBS-EP buffers containing 10, 25, 50, 100, 150, 200, 250, 300, 400 mM NaCl. All SPR experiments were performed at 15 °C.

The SPR response curves were obtained using the BIA Evaluation Software. The response curves of NHERF1(wt) or NHERF1(S339D/S340D) were fit by a two-state kinetic binding model. In the two-state model, the analyte A binds to the ligand L to form an initial complex (AL), and then undergoes subsequent conformational change or binding to form the final complex (A^*L):

where A is the analyte NHERF1(wt) or NHERF1(S339D/S340D) and L is the ligand FERM immobilized on the CM5 sensor chip.

The association rate constant k_a1 was plotted against logγ_± that is calculate by the Debye–Hückel equation:

where γ_± is mean activity coefficient, I the ionic strength of NaCl in 10 mM Hepes buffer (pH=7.4), 3 mM EDTA, 0.5 mM DTT, a≈5.6Å the effective diameter of the ion, and z₊ and z₋ are the number of positive and negative charges of the ion, respectively. The value of constants A and B inEq. 4 are 0.5085 and 0.3281, respectively.

The binding responses in the steady-state region of the sensorgrams (R_eq) were also plotted against analyte concentration (C) to determine the overall equilibrium binding affinity. The data were fit to the non-linear equation:

Where R_max is the maximum binding response, andK_d is the dissociation constant.

Research highlights.

• Nanoscale protein dynamics in an intrinsically disordered protein affects protein-protein interactions in a largely unknown fashion because it is experimentally difficult to probe protein dynamics on nanoscales.

• Employing the novel technique of neutron spin echo spectroscopy (NSE), we are able to reveal nanoscale dynamics changes in a disordered protein.

• Our theoretical analysis shows that NSE pinpoints the nanoscale dynamics changes in a highly specific manner.

• Debye–Hückel analysis indicates that changes in kinetic association rate constant at different ionic strengths cannot be explained by electrostatic effects alone.

• This study shows that nanoscale dynamics in a disordered protein significantly influences the binding kinetics of signaling partners.

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