4 research outputs found
Canonical and noncanonical sites determine NPT2A binding selectivity to NHERF1 PDZ1
Na+/H+ Exchanger Regulatory Factor-1 (NHERF1) is a scaffolding protein containing 2 PDZ domains that coordinates the assembly and trafficking of transmembrane receptors and ion channels. Most target proteins harboring a C-terminus recognition motif bind more-or-less equivalently to the either PDZ domain, which contain identical core-binding motifs. However some substrates such as the type II sodium-dependent phosphate co-transporter (NPT2A), uniquely bind only one PDZ domain. We sought to define the structural determinants responsible for the specificity of interaction between NHERF1 PDZ domains and NPT2A. By performing all-atom/explicit-solvent molecular dynamics (MD) simulations in combination with biological mutagenesis, fluorescent polarization (FP) binding assays, and isothermal titration calorimetry (ITC), we found that in addition to canonical interactions of residues at 0 and -2 positions, Arg at the -1 position of NPT2A plays a critical role in association with Glu43 and His27 of PDZ1 that are absent in PDZ2. Experimentally introduced mutation in PDZ1 (Glu43Asp and His27Asn) decreased binding to NPT2A. Conversely, introduction of Asp183Glu and Asn167His mutations in PDZ2 promoted the formation of favorable interactions yielding micromolar KDs. The results describe novel determinants within both the PDZ domain and outside the canonical PDZ-recognition motif that are responsible for discrimination of NPT2A between two PDZ domains. The results challenge general paradigms for PDZ recognition and suggest new targets for drug development
Structure-Based Multiscale Approach for Identification of Interaction Partners of PDZ Domains
PDZ domains are peptide recognition
modules which mediate specific
protein–protein interactions and are known to have a complex
specificity landscape. We have developed a novel structure-based multiscale
approach which identifies crucial specificity determining residues
(SDRs) of PDZ domains from explicit solvent molecular dynamics (MD)
simulations on PDZ–peptide complexes and uses these SDRs in
combination with knowledge-based scoring functions for proteomewide
identification of their interaction partners. Multiple explicit solvent
simulations ranging from 5 to 50 ns duration have been carried out
on 28 PDZ–peptide complexes with known binding affinities.
MM/PBSA binding energy values calculated from these simulations show
a correlation coefficient of 0.755 with the experimental binding affinities.
On the basis of the SDRs of PDZ domains identified by MD simulations,
we have developed a simple scoring scheme for evaluating binding energies
for PDZ–peptide complexes using residue based statistical pair
potentials. This multiscale approach has been benchmarked on a mouse
PDZ proteome array data set by calculating the binding energies for
217 different substrate peptides in binding pockets of 64 different
mouse PDZ domains. Receiver operating characteristic (ROC) curve analysis
indicates that, the area under curve (AUC) values for binder vs nonbinder
classification by our structure based method is 0.780. Our structure
based method does not require experimental PDZ–peptide binding
data for training