Phosphatase and tensin homologue (PTEN) and microtubule-associated serine threonine kinase 2 (MAST2) are key negative regulators of survival pathways in neuronal cells. The two proteins interact via the PDZ (PSD-95, Dlg1, Zo-1) domain of MAST2 (MAST2–PDZ). During infection by rabies virus, the viral glycoprotein competes with PTEN for interaction with MAST2–PDZ and promotes neuronal survival. The C-terminal PDZ-binding motifs (PBMs) of the two proteins bind similarly to MAST2–PDZ through an unconventional network of connectivity involving two anchor points. Combining stopped-flow fluorescence, analytical ultracentrifugation (AUC), microcalorimetry and NMR, we document the kinetics of interaction between endogenous and viral ligands to MAST2–PDZ as well as the dynamic and structural effects of these interactions. Viral and PTEN peptide interactions to MAST2–PDZ occur via a unique kinetic step which involves both canonical C-terminal PBM binding and N-terminal anchoring. Indirect effects induced by the PBM binding include modifications to the structure and dynamics of the PDZ dimerization surface which prevent MAST2–PDZ auto-association. Such an energetic communication between binding sites and distal surfaces in PDZ domains provides interesting clues for protein regulation overall.

INTRODUCTION

Microtubule-associated serine threonine kinase 2 (MAST2) is a repressor of survival in neuronal cells [1]. We previously proposed that the function of MAST2 is mediated through its interaction with the phosphatase and tensin homologue (PTEN) [2], a major tumour suppressor acting on the Akt (protein kinase B) signalling pathway [3,4]. We showed that the complex formed by PTEN and MAST2 is disrupted upon rabies infection [5]. The viral glycoprotein competes with PTEN for interaction with MAST2, triggering neurosurvival, a key characteristic of rabies infection [6]. Both PTEN and viral proteins interact with the single PDZ domain of MAST2 (MAST2–PDZ) by their C-terminal sequences.

PDZ (PSD-95, post synaptic density 95; Dlg1, disk large homologue 1; Zo-1, zona occludens 1) domains are some of the most common protein–protein interaction domains. They are involved in a variety of cellular processes by assembling and regulating protein networks [79]. PDZ domains typically recognize C-terminal motifs of four residues called PBM (PDZ-binding motif). PDZ domains interact with PBM via a binding groove located between the PDZ strand β2 and the helix α2. Two key residues of the PBM at positions 0 and −2 are responsible for the formation of an intermolecular sheet [10,11]. Even though most studies point out the crucial role of the four last residues of the PBM, the contribution of residues upstream from the canonical motif is increasingly documented in the literature [1216].

The C-termini of PTEN and the viral glycoprotein dock on to MAST2–PDZ through a similar network of connectivity, defining an unusually large interaction surface [700 Å2 (1 Å=0.1 nm)] for a PDZ–PBM complex. This interface is composed of two distinct and non-overlapping regions (Figure 1). In addition to their canonical C-terminal motif, the peptides have an additional anchoring point involving the interaction of an aromatic residue at position −11 with a hydrophobic cluster exposed on the surface of the MAST2–PDZ β2/β3 sheet [2]. This unconventional mode of binding reinforces ligand selectivity and affinity for MAST2–PDZ. We previously showed that unliganded MAST2–PDZ domain is in self-association in solution [2]. PTEN and viral peptides exclusively interact with the monomeric form of MAST2–PDZ and prevent dimerization of the domain in solution. The molecular mechanism by which PBM binding interferes with MAST2 auto-association remains unknown.

Structures of MAST2–PDZ in complex with SWCyto13ETRL and PFPTENITKV peptides

Figure 1
Structures of MAST2–PDZ in complex with SWCyto13ETRL and PFPTENITKV peptides

Ribbon diagram of the structure of MAST2–PDZ in complex with a peptide containing the last 13 residues of the virulent form of the glycoprotein of rabies virus in (a) and of PTEN, one of MAST2's cellular partners in (b). MAST2–PDZ interacts similarly with both peptides through an unconventional network of connectivity involving two anchor points. The residues of the canonical PBM of SWCyto13ETRL and PTEN peptides are represented as blue spheres. The side chains of aromatic residues (tryptophan or phenylalanine) at position −11 are shown as purple spheres. MAST2–PDZ is represented in green (with Trp41 side chain as spheres).

Figure 1
Structures of MAST2–PDZ in complex with SWCyto13ETRL and PFPTENITKV peptides

Ribbon diagram of the structure of MAST2–PDZ in complex with a peptide containing the last 13 residues of the virulent form of the glycoprotein of rabies virus in (a) and of PTEN, one of MAST2's cellular partners in (b). MAST2–PDZ interacts similarly with both peptides through an unconventional network of connectivity involving two anchor points. The residues of the canonical PBM of SWCyto13ETRL and PTEN peptides are represented as blue spheres. The side chains of aromatic residues (tryptophan or phenylalanine) at position −11 are shown as purple spheres. MAST2–PDZ is represented in green (with Trp41 side chain as spheres).

Given the prominence of PDZ domains in biology, it is clear that understanding the molecular details of their specificity is of great significance. To decipher the role of each anchor, we dissect the interaction between MAST2–PDZ and peptides from endogenous and viral origins. Combining analytical ultracentrifugation (AUC), microcalorimetry, fluorescence and NMR, we document the dynamic and structural consequences of ligand binding to MAST2–PDZ. We show that peptide binding is driven by the canonical C-terminal PBM allowing the anchoring of a specific N-terminal determinant. As a functional consequence, our results reveal that the PBM, essential for the peptide binding, prevents MAST2 auto-association by modifying the structure and the dynamics of the PDZ dimerization interface.

EXPERIMENTAL

Sample preparation

MAST2–PDZ, unlabelled, 15N-labelled or 15N/13C-labelled (residues 1099–1193 renumbered as 2–96), is expressed and purified as previously described [17]. All peptides are synthesized on solid phase supports using the Fmoc (9-fluorenyl methoxycarbonyl) strategy (ProteoGenix; see Table 1 for sequences). All biophysics experiments were performed in Tris/HCl 50 mM, NaCl 150 mM, pH 7.5.

Table 1
Equilibrium (Kd) and intrinsic rate constants (kon, koff) of peptides with varying anchors on MAST2–PDZ

The apparent equilibrium dissociation constants Kdapp are deduced from the kinetic amplitudes. Kd values are obtained by titration monitored by static fluorescence. The Kd value marked with (*) (SWPTENITKV) has been deduced from ITC experiment.

Peptide Sequence koff (s−1kon (s−1·μM−1koff/kon (μM) Kd app (μM) Equilibrium titration Kd (μM) 
AcCyto4ETRL ———ETRL 29.7±0.2 12.7±0.1 2.3±0.1 1.5 1.9±0.1 
AcCyto4QTRL ———QTRL 34.7±0.4 9.5±0.1 3.6±0.1 4.2 3.2±0.1 
SACyto13ETRL SAESHKSGGETRL 18.4±0.7 7.7±0.1 2.4±0.1 1.2 1.5±0.1 
SACyto13QTRL SAESHKSGGQTRL 23.0±0.5 7.3±0.2 3.2±0.1 3.5 4.8±0.7 
SWCyto13ETRL SWESHKSGGETRL 9.4±0.9 14.9±0.3 0.6±0.1 0.4 0.4±0.1 
SWCyto13QTRL SWESHKSGGQTRL 16.6±0.7 8.5±0.2 1.9±0.1 0.7 1.5±0.2 
PFPTENQTRL PFDEDQHTQQTRL 6.4±1.1 8.5±0.2 0.8±0.1 0.5 0.7±0.1 
PFPTENITKV PFDEDQHTQITKV 9.7±0.5 5.6±0.1 1.7±0.1 1.4 2.1±0.1 
SWPTENITKV SWDEDQHTQITKV 7.5±0.9 8.3±0.2 0.9±0.1 1.0 1.6* 
SWCyto13Δ4 SWESHKSGG–– Not measurable Not measurable Not measurable Not measurable 
Peptide Sequence koff (s−1kon (s−1·μM−1koff/kon (μM) Kd app (μM) Equilibrium titration Kd (μM) 
AcCyto4ETRL ———ETRL 29.7±0.2 12.7±0.1 2.3±0.1 1.5 1.9±0.1 
AcCyto4QTRL ———QTRL 34.7±0.4 9.5±0.1 3.6±0.1 4.2 3.2±0.1 
SACyto13ETRL SAESHKSGGETRL 18.4±0.7 7.7±0.1 2.4±0.1 1.2 1.5±0.1 
SACyto13QTRL SAESHKSGGQTRL 23.0±0.5 7.3±0.2 3.2±0.1 3.5 4.8±0.7 
SWCyto13ETRL SWESHKSGGETRL 9.4±0.9 14.9±0.3 0.6±0.1 0.4 0.4±0.1 
SWCyto13QTRL SWESHKSGGQTRL 16.6±0.7 8.5±0.2 1.9±0.1 0.7 1.5±0.2 
PFPTENQTRL PFDEDQHTQQTRL 6.4±1.1 8.5±0.2 0.8±0.1 0.5 0.7±0.1 
PFPTENITKV PFDEDQHTQITKV 9.7±0.5 5.6±0.1 1.7±0.1 1.4 2.1±0.1 
SWPTENITKV SWDEDQHTQITKV 7.5±0.9 8.3±0.2 0.9±0.1 1.0 1.6* 
SWCyto13Δ4 SWESHKSGG–– Not measurable Not measurable Not measurable Not measurable 

Equilibrium titration by fluorescence measurements

Static measurements of fluorescence were performed at 20°C in a LS50 spectrofluorometer (Perkin Elmer). Excitation and emission wavelengths were set at 295 nm and 354 nm respectively, with a 10 nm bandwidth for both. One millilitre of a solution of MAST2–PDZ at 2 μM was titrated by successive additions of small aliquots from a highly concentrated peptide solution (140–5200 μM). After 2.5 min equilibration time, emission intensity was integrated for 30 s. All fittings of data were achieved with OriginPro 7.5 software (Origin Lab Corporation) using Lenvenberg–Marquardt minimization. Static fluorescence recorded data were fitted to the following equation:

 
formula

Where p and Fi(p) are the concentration of peptide and the fluorescence recorded after the i-th addition of peptide. Fm, Fp and Fx refer to specific fluorescence of MAST2–PDZ, peptide and MAST2–PDZ–peptide complex respectively, p0 to the peptide concentration in the stock solution. R is the ratio of initial MAST2–PDZ concentration to peptide concentration in the stock solution and K is the equilibrium dissociation constant of the MAST2–PDZ–peptide interaction. Fm, R and p0 are fixed.

Stopped-flow experiments

Kinetics of association between MAST2–PDZ and various peptides were acquired using SFM300 stopped-flow equipment with fluorescence detection (BioLogic). Typically, 150 μl of MAST2–PDZ solution were rapidly mixed with an equal volume of peptide solution. For each peptide, 3–5 concentrations were tested with a constant ratio MAST2–PDZ–peptide of 1:10 to respect pseudo first order conditions. The maximal concentration of MAST2–PDZ was 1 μM. A mixing time of 15 ms is used (total flow rate of 20 ml·s−1), resulting in a 2.5 ms dead time. Data points recorded between 0 and 3 ms were excluded from the fitting process. Temperature was maintained at 20°C by circulating water from a temperature-controlled water bath. The FC15 flow cell (0.15×0.15 cm) was illuminated by a monochromatic UV light at 295 nm (bandwidth 8 nm) and emitted fluorescence was collected perpendicularly to the incident beam through a 325-nm high pass filter. Kinetics was acquired on 0–0.5 s time scale with a 0.1-ms sampling period and a time constant of 0.1 ms (5001 data points). Series of 10 successive shots were recorded and averaged. Depending on the signal to noise ratio, 4–32 averaged traces were stored and processed independently. Controls of initial fluorescence intensity were performed under strictly identical conditions by recording fluorescence contributions of MAST2–PDZ and peptides separately upon 1:1 mixing with buffer. Averaged kinetic traces from stopped-flow experiments were fitted independently to the following mono-exponential equation: F(t)=A·ekobs·t + offset. For each peptide concentration, the mean values of amplitude, the observed rate constant and the related S.D.s were calculated from the fitted values deduced from each series of average kinetics. Amplitudes deduced from the exponential fits were normalized to the concentration of MAST2–PDZ and variation of the resulting reduced amplitude with peptide concentration was fitted to the following equation:

 
formula

Where p is the peptide concentration; F, the maximum reduced amplitude; Kapp, the apparent dissociation constant; and R, the ratio of MAST2–PDZ to peptide concentrations. R was maintained constant for a given peptide assay. For each peptide, intrinsic association and dissociation rate constants, kon and koff, were determined from the linear regression of the variation of kobs as a function of peptide concentration.

Analytical ultracentrifugation

Sedimentation coefficients were determined using a Beckman Coulter XL-I centrifuge equipped with an AN60-Ti rotor. Samples were prepared by mixing MAST2–PDZ (100 μM) and peptides (200 μM). Samples were centrifuged for 17 h at 262 000 g. Sedimentation scans were analysed by the continuous size distribution procedure c(S) with the program Sedfit 12.0 (available at analyticalultracentrifugation.com). All c(S) distributions were calculated with a fitted frictional ratio f/f0 and a maximum entropy regularization procedure with a confidence level of 0.95.

NMR spectrometry

NMR spectra were recorded at 25°C on a Varian Inova 600 MHz spectrometer (Agilent Technologies) equipped with a cryoprobe. 1H-15N HSQC spectra were recorded using 120 μM 15N-labelled MAST2–PDZ and 700 μM peptide with 12% 2H2O. Spectra of apo MAST2–PDZ were obtained at a concentration of 400 μM. The 15N relaxation times T1 and T2 and {1H}-15N heteronuclear NOE were measured by standard methods [18]. The spectra were recorded in an interleaved manner with six relaxation delays, from 10 to 1200 ms and from 10 to 110 ms for T1 and T2 experiments respectively.

RESULTS AND DISCUSSION

Both N-terminal and C-terminal extremities of peptide contribute to MAST2–PDZ binding

We previously studied the interaction of MAST2–PDZ with the C-terminus of its cellular partner PTEN and of viral rabies glycoproteins using 13-residue-long C-terminally-derived peptides. The peptides showed similar affinities in the micromolar range and shared a similar binding mode [2]. We dissect here the molecular determinants that allow ligands to bind to MAST2–PDZ. Several modified versions of the viral and endogenous sequences were used to investigate the contribution of each peptidic region to the affinity and specificity (Table 1).

The C-terminal sequences of viral glycoproteins, SWCyto13ETRL (from attenuated strain) and SWCyto13QTRL (from virulent strain) and of PTEN (PFPTENITKV) bind MAST2–PDZ with Kd of 0.4, 1.5 and 2.1 μM respectively, according to equilibrium titrations (Table 1). No binding of swCyto13Δ4 (viral sequence deprived of its PBM) to MAST2–PDZ could be detected by ITC (isothermal titration calorimetry) [2] or by static fluorescence (Table 1), which shows that the canonical PBM is essential for the peptide-binding affinity. We then characterized the binding of ‘complementary’ sequences by testing the N-acetylated forms of the short PBM peptides AcCyto4ETRL and AcCyto4QTRL. N-acetylation prevents the effects of the N-terminal positive charge [12]. Note that MAST2–PDZ binds AcCyto4ETRL 5-fold more tightly than the non-acetylated form, NH3Cyto4ETRL (result not shown), indicating that the N-terminal charge significantly disturbs the interaction between NH3Cyto4ETRL and MAST2–PDZ. The affinities displayed by MAST2–PDZ for AcCyto4ETRL (1.9 μM) and AcCyto4QTRL (3.2 μM) are close to the ones observed for two viral peptides containing a W(−11)A substitution, SACyto13ETRL (1.5 μM) and SACyto13QTRL (4.8 μM). Then, the W(−11)A substitution included in the natural viral sequences significantly decrease the overall affinities of these two peptides: SWCyto13s affinities are 2–5-fold higher than those of AcCyto4s or SACyto13s. This result corroborates the intermolecular NOE associated with the N-terminal anchor, which almost exclusively involve the tryptophan residue W(−11) (80% of the N-terminal anchor intermolecular NOE; [2]). It seems therefore safe to conclude that in the presence of an efficient C-terminal anchor (QTRL or better ETRL), the N-terminal moiety of the peptides is only safely anchored if the tryptophan residue is present at position −11. We previously observed that viral peptides (44-residues long) containing Cyto13 with an N-terminal extension have an affinity ~3-fold higher than that of shorter Cyto13s peptides for MAST2–PDZ [5]. In this case, the N-terminal extension most probably stabilizes the interaction of the aromatic residue W(−11) with MAST2–PDZ.

The canonical C-terminal anchor appears crucial for the interaction, being the main contributor to the peptide-binding affinity. An efficient N-terminal anchor most probably plays a crucial role in narrowing the specificity of interaction between MAST2 and its partners, since this type of interaction with the long β2/β3 loop of MAST2–PDZ has been reported for only one other PDZ domain [16].

The C-terminal binding drives the N-terminal anchoring in an overall one-step process

To further explore the relative contribution of the N- and C-terminal sequences of these peptides, we analyse binding kinetics at different concentrations of MAST2–PDZ and peptide using stopped-flow tryptophan fluorescence. To simplify the data processing, we apply pseudo first order conditions with a constant MAST2–PDZ–peptide ratio of 1:10. Under these conditions, interactions of all peptides, containing one or two anchor points, with MAST2–PDZ can satisfactorily be fitted to a single exponential decay on the 0–0.5 s interval. In every case, the observed rate constant and the amplitude both vary with peptide concentration. Figure 2 illustrates two examples of peptide–MAST2–PDZ association. In the case of SACyto13ETRL (Figure 2a), fluorescence decreases with time. This decrease was systematically observed with long and short peptides containing the viral ETRL or QTRL C-terminal motifs. On the other hand, a time-dependent increase in fluorescence is observed with peptides terminated by the ITKV C-terminal motif (Figure 2b). For the 10 peptides studied, the variation of the observed rate constant with the peptide concentration fits to the linear function: kobs=kon.[peptide] + koff. Therefore, the rate-limiting step detected by the fluorescence time course reflects a second order process, the binding of the peptide to MAST2–PDZ. The sign of the exponential amplitude strictly depends on the E/QTRL or ITKV motif, indicating that the binding event concerns as a priority the canonical C-terminal PBM sequence.

Kinetic and equilibrium binding of SACyto13ETRL (a) and PFPTENITKV (b) to MAST2–PDZ

Figure 2
Kinetic and equilibrium binding of SACyto13ETRL (a) and PFPTENITKV (b) to MAST2–PDZ

(a1 and b1) The averaged kinetic recordings of the interaction between MAST2–PDZ at 0.5 μM and SACyto13ETRL at 5 μM or PFPTENITKV at 5 μM (see ‘Experimental’ section for detailed experimental conditions). (a2 and b2) Variation of kobs with peptide concentration. (a3 and b3) peptide concentration dependence of the kinetic amplitude normalized to the concentration of MAST2–PDZ. (a4 and b4) Variation of fluorescence of MAST2–PDZ upon titration by SACyto13ETRL and PFPTENITKV.

Figure 2
Kinetic and equilibrium binding of SACyto13ETRL (a) and PFPTENITKV (b) to MAST2–PDZ

(a1 and b1) The averaged kinetic recordings of the interaction between MAST2–PDZ at 0.5 μM and SACyto13ETRL at 5 μM or PFPTENITKV at 5 μM (see ‘Experimental’ section for detailed experimental conditions). (a2 and b2) Variation of kobs with peptide concentration. (a3 and b3) peptide concentration dependence of the kinetic amplitude normalized to the concentration of MAST2–PDZ. (a4 and b4) Variation of fluorescence of MAST2–PDZ upon titration by SACyto13ETRL and PFPTENITKV.

There are two implications of this simple behaviour: first, the apparent dissociation constant deduced from the variation of the kinetic amplitude normalized by MAST2–PDZ concentration must coincide with the back extrapolation of the linear plot of kobs compared with peptide concentration. Second, the ratio koff/kon should match the equilibrium dissociation constant deduced independently by equilibrium titration of MAST2–PDZ with the same peptide. As shown in Figure 2, the back extrapolation to the abscissa axis of the linear plot of kobs as a function of peptide concentration agrees well with both the dissociation constant deduced from the normalized amplitude and the equilibrium dissociation constant based on equilibrium titration. This is true for any of the MAST2–PDZ–peptide pairs tested (Table 1).

This set of binding data validates a simple two-state model for the anchoring of the C- and the N-terminal moieties of the peptides tested. The C-terminal anchoring event corresponds to the majority of the fluorescence change observed between isolated and assembled partners. The participation of the N-terminal sequence manifested itself through an increase of 20%–25% in the normalized amplitude when an A(−11)W substitution is made in the case of SWCyto13ETRL and SWCyto13QTRL. Therefore, in the concentration range explored for the peptide, the fact that the N-terminal anchoring does not give rise to a distinct subsequent phase means that it proceeds much more rapidly than the bimolecular event implying the C-terminal motif E/QTRL or ITKV.

Figure 3 shows values of kon and koff, plotted against each other on a double logarithmic scale. This representation allows a simple description of the effects of all the sequence modifications for the various peptides we have studied up to now. We first look at the effects of the lengthening of the strict C-terminal PBM by addition of an N-terminal sequence devoid of an anchor by comparing the peptides AcCyto4ETRL and AcCyto4QTRL to SACyto13ETRL and SACyto13QTRL. Binding clearly proceeds without a significant change in the free energy of association. The ‘on’ and ‘off’ kinetic constants are affected by the same factor for each pair of peptide when the N-terminal sequence is added. The insertion of an aromatic residue A(−11)W at the N-terminal of the Cyto13 sequences or the Q(−3)E change at the C-terminal, have similar effects on the two kinetics constants. All the points represented for Cyto13s are on a line having a slope close to −1, implying that a modification of the peptide in either the N-terminal or the C-terminal anchor proportionally affects the residence time (1/koff) and the ‘on’ constant roughly in the same proportion. When we analyse the kinetics behaviour of the sequences derived from PTEN, the effects of modifications on both the N-terminal and the C-terminal anchors display the same trend as observed before for the sequences derived from Cyto13. They affect the residence time (1/koff) and ‘on’ constant by the same factor. However, the line representative of these PTENs points is shifted from the Cyto13s one by a significant amount: −0.3 kcal·mol−1 (1 kcal ≡ 4184 J), as displayed on the graph. We suspect that this effect is due to the differences in the linker displayed by the two families of sequences. The Cyto13s sequences contain two glycines next to the canonical PBM that are absent from PTEN sequences. These two residues most probably increase the flexibility of the linker region between the two anchors favouring the exploration of the N-terminal, hence explaining the better affinities of Cyto13s peptides compared with PTENs. Our results are consistent with the extensive work of Huang and Liu [19] on the comparison of the binding kinetics of intrinsically disordered proteins (IDPs) and folded proteins. In their analysis, IDPs show higher kon and koff than folded protein, for a similar Kd, illustrating the effect of flexibility on kinetic constants.

Log–log plot giving the variation of intrinsic association and dissociation rate constants with the sequence of peptides

Figure 3
Log–log plot giving the variation of intrinsic association and dissociation rate constants with the sequence of peptides

In this representation, isoenergetic variations correspond to lines displaying a slope of +1. If the difference in free energy of binding between various partners affects, by the same factor, both the ‘on’ constant and the residence time (1/koff), then the corresponding array of points will be aligned with a slope of −1. Such is apparently the case for the two sets of points characterizing the insertion of different C- or N-terminal sequences at each end of a linker corresponding either to the viral sequence (Cyto13s, in squares) or to the PTEN sequence (PTEN, in triangles). Points reporting the kinetic parameters of the two tetrapeptides Cyto4s (in circles) are located above the two continuous lines. Dotted lines indicate the changes observed when Cyto4s peptides are extended to tridecapeptides SACyto13s.

Figure 3
Log–log plot giving the variation of intrinsic association and dissociation rate constants with the sequence of peptides

In this representation, isoenergetic variations correspond to lines displaying a slope of +1. If the difference in free energy of binding between various partners affects, by the same factor, both the ‘on’ constant and the residence time (1/koff), then the corresponding array of points will be aligned with a slope of −1. Such is apparently the case for the two sets of points characterizing the insertion of different C- or N-terminal sequences at each end of a linker corresponding either to the viral sequence (Cyto13s, in squares) or to the PTEN sequence (PTEN, in triangles). Points reporting the kinetic parameters of the two tetrapeptides Cyto4s (in circles) are located above the two continuous lines. Dotted lines indicate the changes observed when Cyto4s peptides are extended to tridecapeptides SACyto13s.

Long-range structural and dynamic perturbations are induced by N-terminal anchoring

To investigate the structural and dynamic effects of peptide interactions with MAST2–PDZ, we recorded NMR 1H-15N HSQC spectra and measured backbone 15N relaxation data. We first compared the 1H and 15N chemical shifts of MAST2–PDZ in complex with SWCyto13ETRL or with AcCyto4ETRL. Based on chemical shift perturbations (as Δδ), we identified MAST2–PDZ structural modifications caused by the N-terminal anchor upon peptide binding (Figure 4a). Out of the 96 amino acids in the MAST2–PDZ construct, we were able to assign 85 in the MAST2–PDZ–SWCyto13ETRL spectrum and 80 on the MAST2–PDZ–AcCyto4ETRL spectrum by direct comparison of the 1H-15N HSQC spectra. Spectra of both complexes are highly similar, indicating that the global fold and secondary structures of MAST2–PDZ are conserved upon binding of the two peptides. The 1H-15N Δδ values in the GLGF conserved loop of MAST2–PDZ (residues K16–F19) are weak, indicating a similar C-terminal-binding mechanism for both complexes. As expected, the structural variations of MAST2–PDZ induced by the binding of SWCyto13ETRL compared with that of AcCyto4ETRL are mainly observed near the N-terminal anchor. Thus, the largest Δδ values (Δδ > 0.04 ppm) are observed for MAST2–PDZ residues directly contacting the N-terminal region of the peptide, on the β2 strand (residues L21–M27) and on the β3 strand (residues V33–W41) (Figure 4b). Notably, the N-terminal anchor binds directly to the β3 strand by an edge-to-face interaction of its tryptophan residue W(−11) with MAST2–PDZ W41 (PDB: 2KQL). Peaks of residues Ile24 and Arg25 are missing in the MAST2–PDZ–AcCyto4ETRL spectrum. These residues of MAST2–PDZ are in the closest proximity to W(−11) of SWCyto13ETRL. A microsecond–millisecond conformational exchange regime probably broadens the peaks of the β2 strand beyond detection due to the missing N-terminal anchor.

Δδ values and relaxation times differences between MAST2–PDZ–SWCyto13ETRL and MAST2–PDZ–AcCyto4ETRL

Figure 4
Δδ values and relaxation times differences between MAST2–PDZ–SWCyto13ETRL and MAST2–PDZ–AcCyto4ETRL

(a) Δδ values (1H,15N) computed as ∆δ=[(∆δH)2 + (∆δN*0.159)2]1/2. The red line represents the threshold used to determine perturbed region with ∆δ > 0.04 ppm; blue circles mark residues with no resonances in the MAST2–PDZ–AcCyto4ETRL spectrum. (b) MAST2–PDZ–SWCyto13ETRL structure (PDB: 2KQF) with different colours depending on Δδ values. Grey: ∆δ < 0.04 ppm, red ∆δ > 0.04 ppm, blue: resonance absent from the MAST2–PDZ–AcCyto4ETRL spectrum. Cyto13 is represented in cyan and the N-terminal anchor W(−11) is represented in purple. Side chains represented as spheres show a potential energy pathway between MAST2–PDZ β2 strand and the C-terminal end. (c) Longitudinal relaxation times (T1) of MAST2–PDZ–SWCyto13ETRL (black) and MAST2–PDZ/AcCyto4ETRL (red). (d) Transverse relaxation times (T2) of MAST2–PDZ–SWCyto13ETRL (black) and MAST2–PDZ–AcCyto4ETRL (red).

Figure 4
Δδ values and relaxation times differences between MAST2–PDZ–SWCyto13ETRL and MAST2–PDZ–AcCyto4ETRL

(a) Δδ values (1H,15N) computed as ∆δ=[(∆δH)2 + (∆δN*0.159)2]1/2. The red line represents the threshold used to determine perturbed region with ∆δ > 0.04 ppm; blue circles mark residues with no resonances in the MAST2–PDZ–AcCyto4ETRL spectrum. (b) MAST2–PDZ–SWCyto13ETRL structure (PDB: 2KQF) with different colours depending on Δδ values. Grey: ∆δ < 0.04 ppm, red ∆δ > 0.04 ppm, blue: resonance absent from the MAST2–PDZ–AcCyto4ETRL spectrum. Cyto13 is represented in cyan and the N-terminal anchor W(−11) is represented in purple. Side chains represented as spheres show a potential energy pathway between MAST2–PDZ β2 strand and the C-terminal end. (c) Longitudinal relaxation times (T1) of MAST2–PDZ–SWCyto13ETRL (black) and MAST2–PDZ/AcCyto4ETRL (red). (d) Transverse relaxation times (T2) of MAST2–PDZ–SWCyto13ETRL (black) and MAST2–PDZ–AcCyto4ETRL (red).

Interestingly, in addition to these local perturbations, residues distal from the PBM-binding site are also affected. The resonances of the residues forming the α1/β4 loop (residues R55–I60) and the C-terminal residues Leu94 and Glu95 of MAST2–PDZ are affected by the absence of the peptide N-terminal anchor in terms of either their shifts (Arg55, Gln56, Gly57, Ile60) or their intensities (Asp58, Leu59). These long-range perturbations are probably due to a re-orientation of the Trp41 side chain in the absence of the N-terminal anchor (Figure 4b). Trp41 is in direct contact with the side chain of Gln56. This perturbation is most probably transmitted to MAST2–PDZ C-terminal residues Leu94 and Glu95 via the side chain of Arg55 in a ‘domino-like’ manner. The resonances of residues Asp58 and Leu59 are missing in the MAST2–PDZ–AcCyto4ETRL spectrum indicating that the α1/β4 loop is also affected by the PBM binding at the distal binding site.

To probe and compare the local dynamics of MAST2–PDZ complexed with the SWCyto13ETRL and AcCyto4ETRL peptides, the 1H-15N longitudinal (T1) and transverse (T2) relaxation times of the domain were measured at 25°C. The backbone 1H-15N heteronuclear NOE were also recorded but no significant differences could be observed between the two complexes (result not shown). The average T1 values are 530±79 ms for MAST2–PDZ–SWCyto13ETRL and 587±44 ms for MAST2—PDZ–AcCyto4ETRL (Figure 4c). The average T2 values (Figure 4d) are 96±22 ms for MAST2–PDZ–SWCyto13ETRL and 94±24 ms for MAST2–PDZ–AcCyto4ETRL. Backbone dynamics analysed from 1H-15N relaxation measurements revealed small but significant local changes in the dynamic behaviour of the two SWCyto13ETRL and AcCyto4ETRL complexes. The most affected regions are the β2−β3 loop and the C-terminal end and to a lesser extent the α2−β5 loop and the α1−β4 loop. As observed with the largest Δδ induced by the N-terminal anchor on MAST2–PDZ, the dynamical perturbations correspond to residues in the closest proximity to the N-terminal anchor but also to distal residues. In addition, these regions of MAST2–PDZ complexed to SWCyto13ETRL have decreased 1H-15N T1 values and increased 1H-15N T2 values in comparison with the values for MAST2–PDZ in complex with AcCyto4ETRL. This observation unambiguously shows that the N-terminal anchor of the PDZ ligand triggers significant changes in the conformation and dynamics at several local and distal regions of MAST2–PDZ in the fast picosecond-to-nanosecond timescale.

Long-range perturbations are also induced by modification of the canonical PBM

To study whether perturbations in MAST2–PDZ could be induced by other regions of the peptide, we compared the chemical shifts of MAST2–PDZ in complex with two forms of the C-terminal anchor. We looked at both the acetylated form of the short C-terminal peptide (AcCyto4ETRL) that mimics the PBM in a longer peptide chain and the non-acetylated form (NH3Cyto4ETRL). In the latter, the positive charge of the unprotected N-terminal amino group decreases the affinity of the peptide for MAST2–PDZ by a factor of five.

Similar to the chemical shift differences between SWCyto13ETRL and AcCyto4ETRL complexes, the MAST2–PDZ β2 and β3 strands contain the most shifted resonances. All residues within the β2 strand (residues F19–Y27) either have a ∆δ > 0.04 ppm or are absent from the MAST2–PDZ–NH3Cyto4ETRL spectrum (Figure 5). The microsecond–millisecond intermediate time-scale dynamics of β2 strand observed for MAST2–PDZ–AcCyto4ETRL is exacerbated in the MAST2–PDZ–NH3Cyto4ETRL complex with an additional missing resonance, namely that of residue Ala23. Larger Δδ values on the β2 strand are observed compared with those induced by the binding of the N-terminal anchor. The residues His37 to Val43 of the β3 strand are also affected by the amino-charge of AcCyto4ETRL (∆δ > 0.04 ppm) but to a lesser extent compared with the effect of the N-terminal binding. In addition, perturbations in the α1/β4 loop and MAST2–PDZ C-terminus are clearly observed. Gly57 from the α1/β4 loop is even absent from the MAST2–PDZ–NH3Cyto4ETRL spectrum, a consequence of microsecond-millisecond conformational exchange.

Δδ between MAST2—PDZ–AcCyto4ETRL and MAST2—PDZ–NH3Cyto4ETRL

Figure 5
Δδ between MAST2—PDZ–AcCyto4ETRL and MAST2—PDZ–NH3Cyto4ETRL

(a) Δδ values (1H,15N) computed as ∆δ=[(∆δH)2 + (∆δN*0.159)2]1/2. The red line represents the threshold used to determine the perturbed region with ∆δ > 0.04 ppm; blue circles mark residues with no resonance in MAST2—PDZ-NH3Cyto4ETRL. (b) MAST2—PDZ–SWCyto13ETRL structure (PDB: 2KQF) with different colours depending on Δδ values. Grey: ∆δ < 0.04 ppm, red ∆δ > 0.04 ppm, blue: resonances absent from the MAST2–PDZ–NH3Cyto4ETRL spectrum. AcCyto4ETRL equivalent is represented in cyan. Side chains represented in spheres show a potential energy pathway between MAST2–PDZ β2 strand and the C-terminal end.

Figure 5
Δδ between MAST2—PDZ–AcCyto4ETRL and MAST2—PDZ–NH3Cyto4ETRL

(a) Δδ values (1H,15N) computed as ∆δ=[(∆δH)2 + (∆δN*0.159)2]1/2. The red line represents the threshold used to determine the perturbed region with ∆δ > 0.04 ppm; blue circles mark residues with no resonance in MAST2—PDZ-NH3Cyto4ETRL. (b) MAST2—PDZ–SWCyto13ETRL structure (PDB: 2KQF) with different colours depending on Δδ values. Grey: ∆δ < 0.04 ppm, red ∆δ > 0.04 ppm, blue: resonances absent from the MAST2–PDZ–NH3Cyto4ETRL spectrum. AcCyto4ETRL equivalent is represented in cyan. Side chains represented in spheres show a potential energy pathway between MAST2–PDZ β2 strand and the C-terminal end.

Overall, we observe that modification of the charge at peptide position (−3) induces long-range perturbations in the MAST2–PDZ similar to the effects of N-terminal anchoring. This result is consistent with the 1H-Δδ we previously observed between the complexes of MAST2–PDZ with either SWCyto13ETRL or SWCyto13QTRL [2]. The conservative mutation E/Q is sufficient to induce perturbations that spread from the binding groove toward the α1/β4 loop of MAST2–PDZ.

Both anchors contribute to the prevention of MAST2–PDZ dimerization

We previously observed that at high concentration in solution, both monomeric and multimeric forms of MAST2–PDZ coexist [2]. To characterize this self-association, we performed ITC dilution experiments of MAST2–PDZ. We obtained a Kd of 15±7 μM (result not shown), consistent with the equilibrium dissociation constant of 29±3 μM determined by ultracentrifugation [2]. The Gibbs free energy change associated with MAST2–PDZ dissociation is mainly driven by a large enthalpy contribution (13 kcal·mol−1). We also demonstrated that the peptides PFPTENITKV and SWCyto13ETRL promote full dissociation of MAST2–PDZ by binding exclusively to its monomeric form. We initially proposed that the N-terminal anchor of the PDZ ligand precludes the self-association of MAST2–PDZ by occluding the dimerization area of MAST2–PDZ. This hypothesis was supported by the fact that the N-terminal anchor of the peptides interacts with a hydrophobic cluster that is conserved in two closely related proteins, MAST1–PDZ and MAST3–PDZ. This patch located at the surface of the β2 and β3 strands is involved in the dimerization surface of MAST1–PDZ and MAST3–PDZ X-ray structures (PDB: 3PS4 and 3KHF respectively).

To assess whether this hypothesis is valid for all regions of the peptides, we monitored the oligomeric state of MAST2–PDZ in the presence of various peptides, with or without the N-terminal anchor. Our ultracentrifugation results (Table 2) show that, regardless of the N-terminal anchor, each peptide is equally capable of maintaining MAST2–PDZ in its monomeric form with sedimentation coefficients (S20w) between 1.59 and 1.66 S ± 0.1 (2.0 S ± 0.2 for the dimeric form of MAST2–PDZ in the absence of PDZ ligand). Thus, the canonical PBM is sufficient to prevent MAST2–PDZ from dimerizing. Compared with the dimer structures of others PDZ domains of the MAST family, MAST1, MAST3 and MAST4, the short AcCyto4ETRL peptide does not mask the corresponding dimerization surfaces on MAST2–PDZ. The dimerization of MAST2–PDZ is thus probably hampered by distal structural and dynamical changes induced by AcCyto4ETRL.

Table 2
Sedimentation coefficients of MAST2–PDZ domain alone or in complex with various peptides

Sedimentation coefficients of MAST2–PDZ (100 μM) in the presence of peptides (200 μM) containing either one or two anchors are similar whereas the sedimentation coefficient of MAST2–PDZ alone is higher and consistent with a dimeric state.

Complex Peptide sequence Sedimentation coefficient (S) 
MAST2–PDZ/– – 2.07 
MAST2–PDZ/SWCyto13ETRL SWESHKSGGETRL 1.59 
MAST2–PDZ/SACyto13ETRL SAESHKSGGETRL 1.66 
MAST2–PDZ/AcCyto4ETRL ———ETRL 1.66 
Complex Peptide sequence Sedimentation coefficient (S) 
MAST2–PDZ/– – 2.07 
MAST2–PDZ/SWCyto13ETRL SWESHKSGGETRL 1.59 
MAST2–PDZ/SACyto13ETRL SAESHKSGGETRL 1.66 
MAST2–PDZ/AcCyto4ETRL ———ETRL 1.66 

1H-15N HSQC spectrum of uncomplexed MAST2–PDZ at high concentration is consistent with MAST1–PDZ and MAST3–PDZ dimeric structures

To determine the PDZ/PDZ interface of MAST2, we recorded 1H-15N HSQC spectra of the uncomplexed MAST2–PDZ at high concentration (400 μM). On the basis of the monomer–dimer dissociation constant set at 15 μM, the dimeric form represents 96% of the total population at this concentration. The resonances were broad and weak; a total of 57 out of the 91 expected resonances (for non-proline residues) were observed. We were able to assign 36 of them by direct comparison with the 1H-15N HSQC spectrum of the monomeric MAST2–PDZ in complex with AcCyto4ETRL. To determine the regions affected by dimerization, we compared the chemical shifts collected on the two spectra (Figure 6a). Sixteen have no chemical shift changes (∆δ < 0.08 ppm) and 20 resonances are shifted (∆δ > 0.08 ppm) in the spectrum. These Δδ could be due to dimerization and/or ligand binding. In addition, we identified 17 of the resonances that are absent from the dimer spectrum but are present in the MAST2—PDZ–AcCyto4ETRL spectrum. The 16 assigned residues with ∆δ < 0.08 ppm are essentially located on the surface opposite to the PBM-binding groove (Figure 6a) and correspond to residues unaffected by both peptide binding and dimerization. This surface has been reported as the dimer interface for other PDZ [20,21], involving the formation of an intermolecular sheet via the β1 strand. Our data rule out this type of auto-association for MAST2–PDZ. We propose that the 17 missing resonances are due to the monomer–dimer exchange and could help us to delimit the interface of MAST2/PDZ auto-association. Out of 17 missing resonances, nine are located in the β2 strand and the β2/β3 loop between residues Arg22 and Ser31. Out of those residues, only the Arg22 is in direct contact with the peptide [17]. Three residues of the α1/β4 loop and the β4 strand are also missing, Gly57, Asp58, Leu59. Five residues (Gly14, Tyr17, Gly18, Asp45 and Ser50) in close contact are affected by exchange on the top part of the peptide-binding groove.

Δδ values between MAST2–PDZ–AcCyto4ETRL and MAST2–PDZ in dimeric form, compared with MAST3–PDZ dimer structure

Figure 6
Δδ values between MAST2–PDZ–AcCyto4ETRL and MAST2–PDZ in dimeric form, compared with MAST3–PDZ dimer structure

(a) MAST2–PDZ–SWCyto13ETRL structure coloured according to Δδ values between MAST2–PDZ–AcCyto4ETRL and MAST2–PDZ dimer. White sphere: residues without assignment; grey sphere: the 16 residues with ∆δ < 0.08 ppm; red sphere: the 20 residues with ∆δ > 0.08 ppm; blue sphere: the 17 residues identified as absent from MAST2–PDZ dimer spectrum. (b) MAST3–PDZ dimer structure (PDB: 3KHF). Protomers interact mainly by their β2/β3 sheet, their α1/β4 loop and their C-terminus.

Figure 6
Δδ values between MAST2–PDZ–AcCyto4ETRL and MAST2–PDZ in dimeric form, compared with MAST3–PDZ dimer structure

(a) MAST2–PDZ–SWCyto13ETRL structure coloured according to Δδ values between MAST2–PDZ–AcCyto4ETRL and MAST2–PDZ dimer. White sphere: residues without assignment; grey sphere: the 16 residues with ∆δ < 0.08 ppm; red sphere: the 20 residues with ∆δ > 0.08 ppm; blue sphere: the 17 residues identified as absent from MAST2–PDZ dimer spectrum. (b) MAST3–PDZ dimer structure (PDB: 3KHF). Protomers interact mainly by their β2/β3 sheet, their α1/β4 loop and their C-terminus.

The dimeric structures of MAST1–PDZ (PDB: 3PS4) and MAST3–PDZ (PDB: 3KHF) were stabilized through the cis addition of a PBM at the C-terminal end of the PDZ domain, with each protomer binding to the PBM of the other. The surface of auto-association deduced from our NMR data overlaps the ones of liganded MAST1–PDZ, MAST3–PDZ and NHERF4 (Na+/H+ exchange regulatory factor 4)–PDZ3 (PDB: 2V90) as well as of unliganded GRASP (GRP-1-associated scaffold protein)–PDZ (PDB: 2EGK). All these proteins display large β2-β3 strands sharing an exposed hydrophobic patch. We conclude that these PDZ domains could auto-associate following a similar mechanism.

CONCLUSIONS

In the present study, we report that the unconventional binding of peptide ligands to MAST2–PDZ controls the auto-association of this domain. Both viral and endogenous PDZ ligands bind to MAST2–PDZ in a similar manner, through two distinct anchoring points. By thoroughly analysing a set of peptides with different anchors, we showed that specific peptide binding necessarily implies their canonical C-terminal sequence, the major contributor to peptide affinities. The N-terminal sequence participates in the same binding event when it carries a specific aromatic side chain interacting with the β2-β3 hydrophobic cluster of the domain. However, our results differ from those found by Chi et al. [22] on the binding of a nine residue viral peptide on to SAP97 (synapse-associated protein 97)–PDZ2 [22]. They described a two-step process initiated by an unspecific interaction between the peptide and the PDZ domain surface leading to an isomerization connecting the PBM to the binding groove. Our combined results demonstrate the variety of mechanisms of interaction that domains such as PDZ can have. In the case of MAST2–PDZ it seems likely that the N-terminal anchor is mainly beneficial to peptide specificity to MAST2–PDZ, whereas kinetics and affinity are mostly driven by the C-terminal PBM.

Furthermore, we have addressed the question of how the unconventional mode of binding of peptides to MAST2–PDZ affects the dynamics and conformation of the domain. We previously showed that PDZ ligand binding precludes the self-association of MAST2–PDZ [2]. At the time, we proposed that the N-anchoring of the peptide prevents dimerization by steric hindrance. Using NMR, we show in the present study that the dimerization surface involves residues from the β2-β3 strands, the α1/β4 loop and the C-terminal of MAST2–PDZ. The interface of the dimer is consistent with the X-ray structure of MAST isoforms and with the dimeric structure of GRASP. In the case of GRASP, PBM binding and oligomerization modulate the cellular activity of the protein [23]. Contrary to our initial hypothesis, we show in the present study that the strict canonical PBM is by itself able to prevent dimerization of MAST2–PDZ although being solely confined within the binding groove. Thus, PBM binding must transmit effects through an interconnected network of residues on MAST2–PDZ. This network of side chains involves the β2 and β3 strands, the α1/β4 loop and the C-terminus that are perturbed by the PBM binding. We propose that MAST2–PDZ auto-association is prevented by PBM binding through this network of energetically linked changes in the conformation of key residues.

The experimental evidence that we have collected over the years indicates that MAST2–PDZ sustains fine-tuned structural and dynamical changes upon ligand binding. A connected pathway signals the binding event to regions that are distal to the binding cleft, on the protein surface. These findings are compatible with a wealth of prior data on PDZ domains. Statistical coupling analysis and double mutant cycles suggested that energetic pathways within PDZ domains may support allostery [2430]. A model of protein regulation controlled by the equilibrium between PBM binding and dimerization is particularly attractive since we now know that over 30% of PDZ domains form dimers in solution [31]. Several types of heterologous complexes involving MASTs [32,33] have been documented; however, the auto-association of MAST2 in cells has not been studied yet. Allosteric perturbation of the dimeric interface of MAST2–PDZ upon peptide binding may provide interesting clues for MAST2 regulation overall. Valiente et al. [34] showed that binding of PTEN to MAST2–PDZ increases the rate of PTEN phosphorylation by MAST2 kinase. Thus, the regulation by PBM interaction might be involved at several levels in this system: recruiting kinase substrate and affecting protein auto-association. More specifically, dissociation of MAST2–PDZ might modify the catalytic activity of its kinase module. Such a mechanism acting on the substrate PTEN might lead to a modulation of neuronal proliferation and survival.

AUTHOR CONTRIBUTION

Florent Delhommel, Alain Chaffotte, Florence Cordier, Elouan Terrien and Nicolas Wolff planned the experiments. Florent Delhommel, Alain Chaffotte, Florence Cordier, Bertrand Reynal and Elouan Terrien performed the experiments. Florent Delhommel, Alain Chaffotte, Florence Cordier, Bertrand Reynal, Henri Buc and Nicolas Wolff analysed the data. Florent Delhommel, Alain Chaffotte, Florence Cordier, Muriel Delepierre, Henri Buc and Nicolas Wolff wrote the paper.

ACKOWLEDMENTS

We thank Jane Hardy and Terence Strick for careful proofreading of the manuscript and Patrick England for valuable discussions. The authors declare that they have no conflict of interest.

FUNDING

This work was supported by the Institut Pasteur and Institut Carnot Pasteur Maladies Infectieuses [grant number 13/10/120]; and the Ministère de l’Enseignement Supérieur [grant number 883/2013 (to F.D.)].

Abbreviations

     
  • Akt

    protein kinase B

  •  
  • AUC

    analytical ultracentrifugation

  •  
  • Δδ

    chemical shift perturbation

  •  
  • Dlg1

    disk large homologue 1

  •  
  • GRASP

    GRP-1-associated scaffold protein

  •  
  • GRP-1

    general receptor of phosphoinositides 1

  •  
  • IDP

    intrinsically disordered protein

  •  
  • ITC

    isothermal titration calorimetry

  •  
  • MAST

    microtubule-associated serine threonine kinase

  •  
  • PBM

    PDZ-binding motif

  •  
  • PDZ

    PSD-95, Dlg1, Zo-1

  •  
  • PSD-95

    post synaptic density 95

  •  
  • PTEN

    phosphatase and tensin homologue

  •  
  • Zo-1

    zona occludens 1

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