N-Terminus of the Third PDZ Domain of PSD-95 Orchestrates Allosteric Communication for Selective Ligand Binding

Abstract

PDZ domains constitute common models to study single-domain allostery without significant structural changes. The third PDZ domain of PSD-95 (PDZ3) is known to have selective structural features that confer unique modulatory roles to this unit. In this model system, two residues, H372 directly connected to the binding site and G330 holding an off-binding-site position, were designated to assess the effect of mutations on binding selectivity. It has been observed that the H372A and G330T-H372A mutations change ligand preferences from class I (T/S amino acid at position −2 of the ligand) to class II (hydrophobic amino acid at the same position). Alternatively, the G330T single mutation leads to the recognition of both ligand classes. We have performed a series of molecular dynamics (MD) simulations for wild-type, H372A, and G330T single mutants and a double mutant of PDZ3 in the absence and presence of both types of ligands. With the combination of free-energy difference calculations and a detailed analysis of MD trajectories, "class switching" and "class bridging" behavior of PDZ3 mutants, as well as their effects on ligand selection and binding affinities are explained. We show that the dynamics of the charged N-terminus plays a fundamental role in determining the binding preferences in PDZ3 by altering the electrostatic energy. These findings are corroborated by simulations on N-terminus-truncated versions of these systems. The dynamical allostery orchestrated by the N-terminus offers a fresh perspective to the study of communication pathways in proteins.

Dynamic Community Composition Unravels Allosteric Communication in PDZ3

“…This is a result of the close-knit character of DM complexes, having more hydrogen bonds at the binding pocket and low overall solvent accessibility, as quantified in detail in our previous study. 20 Although the averages are similar, the number of broken edges fluctuate over the course of the trajectories (Figure S3). These changes indicate that the configurations for information flow are modified throughout the MD trajectories.…”

“…In a recent study, we have investigated the relative binding energies of PDZ3 complexes using classical MD trajectory analyses and free energy perturbation calculations, and we have shown that the generated MD trajectories sample adequate conformational range to reproduce the experimentally measured binding affinities. 17,20 In particular, we have found that the interplay between protein−ligand hydrogen bonding, solvation free energy of the complex, and the dynamics of the highly charged N-terminus are the three main factors that determine the binding fate of the ligand. 20 In fact, it has been possible to compute the free energy difference values corroborating experimentally measured binding constants using various advanced simulation techniques and to break down the contribution of each of these factors by a simplified model.…”

“…17,20 In particular, we have found that the interplay between protein−ligand hydrogen bonding, solvation free energy of the complex, and the dynamics of the highly charged N-terminus are the three main factors that determine the binding fate of the ligand. 20 In fact, it has been possible to compute the free energy difference values corroborating experimentally measured binding constants using various advanced simulation techniques and to break down the contribution of each of these factors by a simplified model. However, the energy values do not suffice to draw conclusions for a mechanistic interpretation of the findings.…”

“…The MD simulations and their detailed energetic analyses have recently been reported elsewhere. 20 Briefly, protein structures are downloaded from the protein data bank (PDB) 39 (Table 1), and missing residues of ligand are inserted by using the SWISS-MODEL 40 server. Equal lengths of the structures are used whereby residue index ranges are 299−415 for the protein and 2−9 for the ligand (see Figure 1c for the sequence).…”

“…5,9−11,20−22 The dynamics of the highly charged Nterminus region (Figure 1a) has recently been shown to be important in the binding mechanism, especially due to its electrostatic contributions to the total free energy. 20 The effect of the N-terminus might be partnered with that of the Cterminus. 21 α 2 helix (Figure 1a) lines up the ligand, and its effect has been investigated by a deep mutational scan.…”