Computational assessment of the effect of allosteric mutations on the dynamics of PDZ domains

PDZ domain-containing proteins are involved in intercellular interactions such as trafficking, signaling, cell to cell communication and organization of signaling complexes. PDZ domains are themselves small proteins which typically consist of 90 to 100 amino acids. However, the extra α helix structure at the carboxyl terminus introduces a selective structural feature to the third PDZ domain of PSD-95 which has a stabilizing effect and participates in allosteric communication. PDZ domains are the most commonly studied models to understand single domain allostery without resulting in significant structural changes. One change triggers another change at distal site, and the source of the ‘changes’ are localized perturbations such as a binding event, posttranslational modification, a mutation or light absorption. Mutations can alter the stabilization of the protein and result ON or OFF state for ligand binding. They can also cause a change in the active site and affect the ligand preference. Here we investigate the reasons leading to the allosteric regulation of mutations and their effect on the ligand preferences. By using third PDZ domain of postsynaptic density 95 (PSD-95) as a model system H372 directly connected to the binding site and G330 with a somewhat removed position were selected to assess the effect of allosteric mutations on the dynamics. In the literature, it was observed that the H372A and G330T/H372A mutations change ligand preferences from class I (T/S amino acid preference at position 2 of the ligand) to class II (hydrophobic amino acid preference at position 2 of the ligand). On the other hand, the G330T mutation leads to the recognition of both class I and class II types of ligands. Therefore, H372A is a ‘switching mutation’ while G330T mutation is ‘class bridging’. We have performed 200 ns molecular dynamics simulations for wild-type, H372A, G330T single mutants and a double mutant of third PDZ domain in the absence and presence of both types of ligands. The comparative study helps to identify the changes in the dynamics that are effective in the onset and prevention of allosteric communication. With the combination of free energy difference calculations and a detailed analysis of MD trajectories, the behavior of the PDZ domain under the mutations, which are ‘class bridging’(G330T) and ‘class changing’(H372A), and their effects on the ligand preferences and binding affinities are explained. We show that the ensemble view of allostery provides a better description of site-to-site coupling rather than a pathway view that assumes a direct connection between the effector and binding site

N-terminus of the third PDZ domain of PSD-95 orchestrates allosteric communication for selective ligand binding

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