Abstract:Previous studies of the N-terminal PDZ tandem from PSD-95 produced divergent models and failed to identify interdomain contacts stabilizing the structure. We used ensemble and single-molecule FRET along with replica-exchange molecular dynamics to fully characterize the energy landscape. Simulations and experiments identified two conformations: an open-like conformation with a small contact interface stabilized by salt bridges, and a closed-like conformation with a larger contact interface stabilized by surface… Show more
“…This is further supported by the strong similarity of chemical shifts when comparing Xph15 and Xph20 binding to PDZ domain 1 alone and to the tandem. There should also be minimal impact on the dynamic interconversion of two major domain arrangements of the PDZ1 and PDZ2 domains that has been recently described 38 .Fig. 4Docking models.…”
Designing highly specific modulators of protein-protein interactions (PPIs) is especially challenging in the context of multiple paralogs and conserved interaction surfaces. In this case, direct generation of selective and competitive inhibitors is hindered by high similarity within the evolutionary-related protein interfaces. We report here a strategy that uses a semi-rational approach to separate the modulator design into two functional parts. We first achieve specificity toward a region outside of the interface by using phage display selection coupled with molecular and cellular validation. Highly selective competition is then generated by appending the more degenerate interaction peptide to contact the target interface. We apply this approach to specifically bind a single PDZ domain within the postsynaptic protein PSD-95 over highly similar PDZ domains in PSD-93, SAP-97 and SAP-102. Our work provides a paralog-selective and domain specific inhibitor of PSD-95, and describes a method to efficiently target other conserved PPI modules.
“…This is further supported by the strong similarity of chemical shifts when comparing Xph15 and Xph20 binding to PDZ domain 1 alone and to the tandem. There should also be minimal impact on the dynamic interconversion of two major domain arrangements of the PDZ1 and PDZ2 domains that has been recently described 38 .Fig. 4Docking models.…”
Designing highly specific modulators of protein-protein interactions (PPIs) is especially challenging in the context of multiple paralogs and conserved interaction surfaces. In this case, direct generation of selective and competitive inhibitors is hindered by high similarity within the evolutionary-related protein interfaces. We report here a strategy that uses a semi-rational approach to separate the modulator design into two functional parts. We first achieve specificity toward a region outside of the interface by using phage display selection coupled with molecular and cellular validation. Highly selective competition is then generated by appending the more degenerate interaction peptide to contact the target interface. We apply this approach to specifically bind a single PDZ domain within the postsynaptic protein PSD-95 over highly similar PDZ domains in PSD-93, SAP-97 and SAP-102. Our work provides a paralog-selective and domain specific inhibitor of PSD-95, and describes a method to efficiently target other conserved PPI modules.
“…S10). These experimental structures include, in particular, two chains of the same crystal structure from PDB (ID: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=3GSL) , a structure determined by FRET presented by Bowen et al , and two further structures, an open‐like and a closed‐like conformation, resulting from the refinement of the same FRET structure by DMD simulations and presented by Sanabria and coworkers (5 structures in total).…”
Section: Resultsmentioning
confidence: 99%
“…Also, this study came to the conclusion that the same supramodular structure is maintained in the full‐length protein as in the isolated tandem . The FRET structure was subsequently refined with discrete molecular dynamic simulation and was found to be the average of many possible interdomain orientations, with two structures—an open‐like and a closed‐like conformation—emerging as the energetically most favorable ones . This study also identified the ultraweak interdomain interactions in the two most dominant conformations that stabilize the supramodular structure of the PDZ12 tandem.…”
mentioning
confidence: 80%
“…Correspondence of the generated ensembles to the experimentally determined FRET data was determined by calculating the correlation between interdomain Cɑ atom–atom distances and the three sets of mean interdye distances reported by Sanabria and coworkers . In their work, they fit the experimentally measured ensemble time correlated single photon counting (eTCSPC) decays to three individual conformers with a three‐Gaussian distributed model, thus obtaining three sets of data containing 10 mean interdomain interdye distances for each of the 10 PDZ12 tandem variant they designed.…”
The postsynaptic density protein‐95 (PSD‐95) regulates synaptic plasticity through interactions mediated by its peptide‐binding PDZ domains. The two N‐terminal PDZ domains of PSD‐95 form an autonomous structural unit, and their interdomain orientation and dynamics depend on ligand binding. To understand the mechanistic details of the effect of ligand binding, we generated conformational ensembles using available experimentally determined nuclear Overhauser effect interatomic distances and S2 order parameters. In our approach, the fast dynamics of the two domains is treated independently. We find that intradomain structural changes induced by ligand binding modulate the probability of the occurrence of specific domain–domain orientations. Our results suggest that the β2‐β3 loop in the PDZ domains is a key regulatory region, which influences both intradomain motions and supramodular rearrangement.
“…Atomistic MD (molecular dynamics) simulations have been extensively used to investigate biomolecular complexes 18 . Several Recent studies have used MD simulations for understanding dynamics of ligand binding 19 , dynamics of multi PDZ domains 20 and allosteric interactions [21][22] associated with C-terminal peptide recognition by PDZ domains. However, internal peptide recognition has not been studied in detail.…”
PDZ domains are important peptide recognition modules which usually recognize short C-terminal stretches of their interaction partners, but certain PDZ domains can also recognize internal peptides in the interacting proteins. Due to the scarcity of data on internal peptide recognition and lack of understanding of the mechanistic details of internal peptide recognition, identification of PDZ domains capable of recognizing internal peptides has been a difficult task.Since Par-6 PDZ domain can recognize both C-terminal and internal peptides, we have carried out multiple explicit solvent MD simulations of 1 μs duration on free and peptide bound Par-6 PDZ to decipher mechanistic details of internal peptide recognition. These simulations have been analyzed to identify residues which play a crucial role in internal peptide recognition by PDZ domains. Based on the conservation profile of the identified residues, we have predicted 47 human PDZ domains to be capable of recognizing internal peptides in human. We have also investigated how binding of CDC42 to the CRIB domain adjacent to the Par6 PDZ allosterically modulate the peptide recognition by Par6 PDZ. Our MD simulations on CRIB-Par6_PDZ didomain in isolation as well as in complex with CDC42, indicate that in absence of CDC42 the adjacent CRIB domain induces open loop conformation of PDZ facilitating internal peptide recognition. On the other hand, upon binding of CDC42 to the CRIB domain, Par6 PDZ adopts closed loop conformation required for recognition of C-terminus peptides. These results provide atomistic details of how binding of interaction partners onto adjacent domains can allosterically regulate substrate binding to PDZ domains. In summary, MD simulations provide novel insights into the modulation of substrate recognition preference of PDZ by specific peptides, adjacent domains and binding of interaction partners at allosteric sites.
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