Deciphering the kinetic binding mechanism of dimeric ligands using a potent plasma-stable dimeric inhibitor of postsynaptic density protein-95 as an example

Abstract

Dimeric ligands can be potent inhibitors of protein-protein or enzyme-substrate interactions. They have increased affinity and specificity toward their targets due to their ability to bind two binding sites simultaneously and are therefore attractive in drug design. However, few studies have addressed the kinetic mechanism of interaction of such bivalent ligands. We have investigated the binding interaction of a recently identified potent plasma-stable dimeric pentapeptide and PDZ1-2 of postsynaptic density protein-95 (PSD-95) using protein engineering in combination with fluorescence polarization, isothermal titration calorimetry, and stopped-flow fluorimetry. We demonstrate that binding occurs via a two-step process, where an initial binding to either one of the two PDZ domains is followed by an intramolecular step, which produces the bidentate complex. We have determined all rate constants involved in the binding reaction and found evidence for a conformational transition of the complex. Our data demonstrate the importance of a slow dissociation for a successful dimeric ligand but also highlight the possibility of optimizing the intramolecular association rate. The results may therefore aid the design of dimeric inhibitors in general.

FIGURE 1.

Structures of dimeric ligand and PDZ1–2 tandem. A, model of the PSD-95 PDZ1–2 tandem structure in complex with cypin peptide (Protein Data Bank code 2KA9) from Wang et al. (11). B, structure of the dimeric ligand, made by dimerization of two pentapeptides with a polyethylene glycol (PEG) linker (8).

FIGURE 2.

Equilibrium binding experiments. A, K_i values for PDZ-peptide interactions measured from fluorescence polarization displacement experiments. Error bars were calculated from the average of three independent measurements. B, ITC data for PDZ1–2* with monomeric peptide inhibitor and dimeric inhibitor at 10 °C. Top, raw data. Bottom, integrated titration curves. Fitting of a single site model (solid line) yielded the parameters reported in Table 1.

FIGURE 3.

Displacement experiments for wild type PDZs and peptides. Observed rate constants for single PDZ2 WT/monomeric peptide displacement (A), tandem PDZ1–2 WT/monomeric and dimeric peptide displacement (B), by a Trp-containing PDZ. In B, data for the PDZ1–2 R70A/K98A mutant also is shown. Observed rate constants were obtained by fitting of Equation 1 to experimental traces (not shown).

FIGURE 4.

Binding kinetics of Trp PDZs, PDZ1*, PDZ2*, PDZ1*–2, and PDZ1–2* to monomeric peptide. A, binding trace for the interaction between PDZ2* (3 μm) and monomeric peptide (10 μm) along with a fit to a single exponential (solid line, Equation 1). B, residuals from the single exponential fit in A. C, binding trace for the interaction between PDZ1–2* (3 μm) and monomeric peptide (10 μm) along with a fit to a double exponential (solid line, Equation 2). D, residuals from the double exponential fit in C. E, observed rate constants for PDZ/monomeric peptide interactions plotted against increasing concentration of monomeric peptide and fitted to Equation 3 to obtain on-rate constants (Table 2).

FIGURE 5.

Binding reaction of Trp PDZs, PDZ1*, PDZ2*, PDZ1*–2, and PDZ1–2* to dimeric peptide. A, binding trace for PDZ1–2*/dimeric peptide interaction with [PDZ1–2*]>[peptide], fitted to a double exponential (solid line, Equation 2). B, binding trace for PDZ1–2*/dimeric peptide interaction with [peptide]>[PDZ1–2*], fitted to a double exponential (solid line, Equation 2). C, residuals from the double exponential fit in A (upper panel). For comparison, the residuals of a single exponential fit (Equation 1) is shown in the lower panel. D, residuals from double (upper panel) and single (lower panel) exponential fits for the experiment in B. E and F show plots of observed rate constants for the PDZ1–2/dimeric peptide interaction. Observed rate constants were plotted against increasing concentrations of PDZ1–2* E or dimeric peptide F (four data sets reported for the slow phase; in data set 1 the concentration of PDZ1–2* was 1.5 μm and in the other three 3.0 μm). The fast phases in E and F were fitted to Equation 3 (thin solid line), whereas the slow phases in F were fitted to Equation 4 to estimate k_obs^max for each set (at 15 μm dimeric peptide, fit not shown), the rate constant of the intramolecular association, which corresponds to k₃ in Fig. 6 (62 ± 6 s⁻¹). Simulated data using the rate constants shown in Fig. 6 and a concentration of the non-varied species of 3 μm are in gray. In E, the slow phase is divided into two, since the amplitudes of the phase changed sign around 4 μm dimeric peptide, in agreement with experimental data. Furthermore, in E, a scheme with accumulation of the ternary complexes between two PDZ1–2 and one dimeric peptide was used in the simulation, whereas, in F, a square according to Fig. 6 was simulated.

FIGURE 6.

Kinetic reaction scheme for the interaction between PSD-95 PDZ1–2 and a dimeric peptide. The initial association and dissociation rate constants (k₁, k₂, k₋₁, k₋₂) in the scheme were obtained from experiments with monomeric peptide, but are fully consistent with the dimeric peptide binding data. The reverse rate constants (k₋₃, k₋₄) were measured from displacement kinetics of the PDZ tandem constructs and dimeric peptide. In the displacement reaction from wild type PDZ1–2 tandem, only the slowest intramolecular k_off can be determined (0.9 s⁻¹); the rate constant of 1.8 s⁻¹ is inferred from, first, the intermolecular k_off values between monomeric peptide and single PDZ domains and, second, from the ratio of intramolecular k_off values from dimeric peptide and Trp mutant tandems PDZ1*–2 and PDZ1–2* (not shown). In both cases the off-rate constant from PDZ1 is roughly two times that of PDZ2. The intramolecular forward rate constant k₃ was determined from the dependence of the slow step for PDZ1–2* and dimeric peptide (Fig. 5F, k_obs^max ≈ k₃ = 62 s⁻¹), as described in the Results section. Finally, k₄ was calculated as 31 s⁻¹, given that the product of rate constants on one half of the scheme must equal that of the other half.