The Kinetics of PDZ Domain-Ligand Interactions and Implications for the Binding Mechanism

Abstract

PDZ domains are protein adapter modules present in a few hundred human proteins. They play important roles in scaffolding and signal transduction. PDZ domains usually bind to the C termini of their target proteins. To assess the binding mechanism of this interaction we have performed the first in-solution kinetic study for PDZ domains and peptides corresponding to target ligands. Both PDZ3 from postsynaptic density protein 95 and PDZ2 from protein tyrosine phosphatase L1 bind their respective target peptides through an apparent A ؉ B 3 A⅐B mechanism without rate-limiting conformational changes. But a mutant with a fluorescent probe (Trp) outside of the binding pocket suggests that slight changes in the structure take place upon binding in protein tyrosine phosphatase-L1 PDZ2. For PDZ3 from postsynaptic density protein 95 the pH dependence of the binding reaction is consistent with a one-step mechanism with one titratable group. The salt dependence of the interaction shows that the formation of electrostatic interactions is rate-limiting for the association reaction but not for dissociation of the complex. PDZ4 domains are found in a few hundred human proteins, either as a single domain or in arrays. These domains mediate binding to other proteins and in this way play important roles in scaffolding and signal transduction (1, 2). Structural studies have shown that the PDZ domains usually bind to the C terminus of their target proteins. A number of crystal and NMR structures of PDZ domains have been solved both with and without bound peptide (for example, Refs 3-6) ( Fig. 1). A wealth of data on different peptides binding to different PDZ domains has been obtained by screening (for example, Refs. 7 and 8) and selection (for example, Refs. 9 and 10). Such studies and those using the yeast twohybrid technique (for example, Ref. 11) provide important information on possible cellular targets for distinct PDZ domains as well as the specificity of the interaction. Moreover, theory and NMR experiments have suggested that the dynamics of PDZ domains and the residues outside of the binding pocket influence their interaction with ligands (12, 13). Despite considerable effort to clarify the structural basis for the PDZ-ligand interaction, only a handful of studies have assessed the binding energetics and specificity of PDZ-peptide interactions using proper equilibrium assays in solution (3, 11, 14 -24). Kinetics of chemical reactions not only provide "end point data" such as equilibrium constants but also yield microscopic rate constants and, more importantly, the possibility of elucidating the mechanisms of binding and probing the binding dynamics as well as the properties of the transition state of the reaction. To assess the binding mechanism, we have performed the first kinetic study of PDZ domains in solution using stopped-flow fluorimetry. The PDZ domains chosen were PDZ3 from human PSD-95, one of the most well studied PDZ domains, and the second PDZ domain from mouse protein tyrosine phosphatase-L1 (PTP-BL; also known...

Interactions outside the Boundaries of the Canonical Binding Groove of a PDZ Domain Influence Ligand Binding

“…The numbering of residues refers to full length PSD-95α without exon b and is the same as used in Doyle et al 13 The F337W mutation serves as a fluorescence probe in ligand binding experiments and does not affect the affinity of the PDZ3-peptide interaction. [16][17][18] It also does not affect the energetic coupling pattern between side-chain residues in the PDZ domain and in the peptide ligand 9 , since the coupling energy for F337W is zero with regard to a second mutation in the peptide ligand, either at the C-terminal Val 0 or at Ser -2 (not shown). The sequence corresponding to the α3-helix (residues 396-401) was deleted from the cDNA of PDZ3 to generate the α3-helix-deleted mutant PDZ3∆α3.…”

Deciphering the Kinetic Binding Mechanism of Dimeric Ligands Using a Potent Plasma-stable Dimeric Inhibitor of Postsynaptic Density Protein-95 as an Example

“…k on is the association or on-rate constant, k off is the dissociation or off-rate constant, and [A] 0 and n are the initial concentrations of the varied and constant species, respectively (20,21). The slow phase observed upon mixing PDZ1-2* with dimeric peptide, which increased up to a constant value with peptide concentration, was analyzed with a model-free polynomial equation (Equation 4), to calculate k obs at 15 M dimeric peptide by intrapolation.…”

Comparison of successive transition states for folding reveals alternative early folding pathways of two homologous proteins

“…43) as well as a short His-tag, which does not influence the folding of PDZ3 (21). Expression and purification were as described earlier (37,46). The purity was checked by SDS/PAGE and the identity, by MALDI-TOF mass spectrometry.…”