Integrated analysis of Shank1 PDZ interactions with C-terminal and internal binding motifs.
Ontology highlight
ABSTRACT:
SUBMITTER: Ali M
PROVIDER: S-EPMC8244488 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:NHE3 directly binds Na+/H+ exchanger regulatory factor (NHERF) family scaffolding proteins that are required for many aspects of NHE3 regulation. The NHERFs bind both to an internal region (amino acids 586-660) of the NHE3 C terminus and to the NHE3 C-terminal four amino acids. The internal NHERF-binding region contains both putative Class I (-592SAV-) and Class II (-595CLDM-) PDZ-binding motifs (PBMs). Point mutagenesis showed that only the Class II motif contributes to NHERF binding. In this study, the roles in regulation of NHE3 activity of these two PBMs were investigated, revealing the following findings. 1) Interaction occurred between these binding sites because mutation of either removed nearly all NHERF binding. 2) Mutations in either significantly reduced basal NHE3 activity. Total and percent plasma membrane (PM) NHE3 protein expression was reduced in the C-terminal but not in the internal PBD mutation. 3) cGMP- and Ca2+-mediated inhibition of NHE3 was impaired in both the internal and the C-terminal PBM mutations. 4) There was a significant reduction in half-life of the PM pool of NHE3 in only the internal PBM mutation but no change in total NHE3 half-life in either. 5) There were some differences in NHE3-associating proteins in the two PBM mutations. In conclusion, NHE3 binds to NHERF proteins via both an internal Class II PBM and C-terminal Class I PBM, which interact. The former determines NHE3 stability in the PM, and the latter determines total expression and percent PM expression.
| S-EPMC5437235 | biostudies-literature
Project description:Shank proteins are among the most abundant and well-studied postsynaptic scaffold proteins. Their PDZ domain has unique characteristics as one of its loop regions flanking the ligand-binding site is uniquely long and has also been implicated in the formation of PDZ dimers. Here we report the initial characterization of the Shank1 PDZ domain by solution NMR spectroscopy. The assigned chemical shifts are largely consistent with the common features of PDZ domains in general and the available Shank PDZ crystal structures in particular. Our analysis suggests that under the conditions investigated, the domain is monomeric and the unique loop harbors a short helical segment, observed in only one of the known X-ray structures so far. Our work stresses the importance of solution-state investigations to fully decipher the functional relevance of the structural and dynamical features unique to Shank PDZ domains.
| S-EPMC9068651 | biostudies-literature
Project description:Single nucleotide polymorphisms (SNPs) are a major contributor to genetic and phenotypic variation within populations. Non-synonymous SNPs (nsSNPs) modify the sequence of proteins and can affect their folding or binding properties. Experimental analysis of all nsSNPs is currently unfeasible and therefore computational predictions of the molecular effect of nsSNPs are helpful to guide experimental investigations. While some nsSNPs can be accurately characterized, for instance if they fall into strongly conserved or well annotated regions, the molecular consequences of many others are more challenging to predict. In particular, nsSNPs affecting less structured, and often less conserved regions, are difficult to characterize. Binding sites that mediate protein-protein or other protein interactions are an important class of functional sites on proteins and can be used to help interpret nsSNPs. Binding sites targeted by the PDZ modular peptide recognition domain have recently been characterized. Here we use this data to show that it is possible to computationally identify nsSNPs in PDZ binding motifs that modify or prevent binding to the proteins containing the motifs. We confirm these predictions by experimentally validating a selected subset with ELISA. Our work also highlights the importance of better characterizing linear motifs in proteins as many of these can be affected by genetic variations.
| S-EPMC3983204 | biostudies-literature
Project description:The cytoplasmic protein Dishevelled (Dvl) and the associated membrane-bound receptor Frizzled (Fz) are essential in canonical and noncanonical Wnt signaling pathways. However, the molecular mechanisms underlying this signaling are not well understood. By using NMR spectroscopy, we determined that an internal sequence of Fz binds to the conventional peptide binding site in the PDZ domain of Dvl; this type of site typically binds to C-terminal binding motifs. The C-terminal region of the Dvl inhibitor Dapper (Dpr) and Frodo bound to the same site. In Xenopus, Dvl binding peptides of Fz and Dpr/Frodo inhibited canonical Wnt signaling and blocked Wnt-induced secondary axis formation in a dose-dependent manner, but did not block noncanonical Wnt signaling mediated by the DEP domain. Together, our results identify a missing molecular connection within the Wnt pathway. Differences in the binding affinity of the Dvl PDZ domain and its binding partners may be important in regulating signal transduction by Dvl.
| S-EPMC4381837 | biostudies-literature
Project description:PDZ domains most commonly bind the C-terminus of their protein targets. Typically the C-terminal four residues of the protein target are considered as the binding motif, particularly the C-terminal residue (P0) and third-last residue (P-2) that form the major contacts with the PDZ domain's "binding groove". We solved crystal structures of seven human PDZ domains, including five of the seven PDLIM family members. The structures of GRASP, PDLIM2, PDLIM5, and PDLIM7 show a binding mode with only the C-terminal P0 residue bound in the binding groove. Importantly, in some cases, the P-2 residue formed interactions outside of the binding groove, providing insight into the influence of residues remote from the binding groove on selectivity. In the GRASP structure, we observed both canonical and noncanonical binding in the two molecules present in the asymmetric unit making a direct comparison of these binding modes possible. In addition, structures of the PDZ domains from PDLIM1 and PDLIM4 also presented here allow comparison with canonical binding for the PDLIM PDZ domain family. Although influenced by crystal packing arrangements, the structures nevertheless show that changes in the positions of PDZ domain side-chains and the alpha B helix allow noncanonical binding interactions. These interactions may be indicative of intermediate states between unbound and fully bound PDZ domain and target protein. The noncanonical "perpendicular" binding observed potentially represents the general form of a kinetic intermediate. Comparison with canonical binding suggests that the rearrangement during binding involves both the PDZ domain and its ligand.
| S-EPMC2867013 | biostudies-literature
Project description:The specificity of protein-protein interactions in cellular signaling cascades is dependent on the sequence and intramolecular location of distinct amino acid motifs. We used the two-hybrid interaction trap to identify proteins that can associate with the PDZ motif-rich segment in the protein tyrosine phosphatase PTP-BL. A specific interaction was found with the Lin-11, Isl-1, Mec-3 (LIM) domain containing protein RIL. More detailed analysis demonstrated that the binding specificity resides in the second and fourth PDZ motif of PTP-BL and the LIM domain in RIL. Immunohistochemistry on various mouse tissues revealed a submembranous colocalization of PTP-BL and RIL in epithelial cells. Remarkably, there is also an N-terminal PDZ motif in RIL itself that can bind to the RIL-LIM domain. We demonstrate here that the RIL-LIM domain can be phosphorylated on tyrosine in vitro and in vivo and can be dephosphorylated in vitro by the PTPase domain of PTP-BL. Our data point to the presence of a double PDZ-binding interface on the RIL-LIM domain and suggest tyrosine phosphorylation as a regulatory mechanism for LIM-PDZ associations in the assembly of multiprotein complexes. These findings are in line with an important role of PDZ-mediated interactions in the shaping and organization of submembranous microenvironments of polarized cells.
| S-EPMC25295 | biostudies-literature
Project description:Inhibitors of PDZ-peptide interactions have important implications in a variety of biological processes including treatment of cancer and Parkinson's disease. Even though experimental studies have reported characterization of peptidomimetic inhibitors of PDZ-peptide interactions, the binding modes for most of them have not been characterized by structural studies. In this study we have attempted to understand the structural basis of the small molecule-PDZ interactions by in silico analysis of the binding modes and binding affinities of a set of 38 small molecules with known K(i) or K(d) values for PDZ2 and PDZ3 domains of PSD-95 protein. These two PDZ domains show differential selectivity for these compounds despite having a high degree of sequence similarity and almost identical peptide binding pockets. Optimum binding modes for these ligands for PDZ2 and PDZ3 domains were identified by using a novel combination of semi-flexible docking and explicit solvent molecular dynamics (MD) simulations. Analysis of the binding modes revealed most of the peptidomimectic ligands which had high K(i) or K(d) moved away from the peptide binding pocket, while ligands with high binding affinities remained in the peptide binding pocket. The differential specificities of the PDZ2 and PDZ3 domains primarily arise from differences in the conformation of the loop connecting βB and βC strands, because this loop interacts with the N-terminal chemical moieties of the ligands. We have also computed the MM/PBSA binding free energy values for these 38 compounds with both the PDZ domains from multiple 5 ns MD trajectories on each complex i.e. a total of 228 MD trajectories of 5 ns length each. Interestingly, computational binding free energies show good agreement with experimental binding free energies with a correlation coefficient of approximately 0.6. Thus our study demonstrates that combined use of docking and MD simulations can help in identification of potent inhibitors of PDZ-peptide complexes.
| S-EPMC3738590 | biostudies-literature
Project description:Tiam-family guanine exchange proteins are activators of the Rho GTPase Rac1 and critical for cell morphology, adhesion, migration, and polarity. These modular proteins contain a variety of signaling domains, including a single postsynaptic density-95/discs large/zonula occludens-1 (PDZ) domain. Here, we show how structural and thermodynamic approaches applied to the Tiam1 PDZ domain can be used to gain unique insights into the affinity and specificity of PDZ-ligand interactions with peptides derived from Syndecan1 and Caspr4 proteins. First, we describe a fluorescence anisotropy-based assay that can be used to determine PDZ-ligand interactions, and describe important considerations in designing binding experiments. Second, we used site-specific mutagenesis in combination with double-mutant cycle analysis to probe the binding energetics and cooperativity of residues in two ligand binding pockets (S(0) and S(-2)) that are involved in Tiam1 PDZ-ligand interactions. Peptide ligand binding results and double-mutant cycle analysis revealed that the S(0) pocket was important for Syndecan1 and Caspr4 peptide interactions and that the S(-2) pocket provided selectivity for the Syndecan1 ligand. Finally, we devised a "peptide evolution" strategy whereby a Model consensus peptide was "evolved" into either the Syndecan1 or Caspr4 peptide by site-directed mutagenesis. These results corroborated the PDZ mutational analysis of the S(0) pocket and identified the P(-4) position in the ligand as critical for Syndecan1 affinity and selectivity. Together, these studies show that a combined structural and thermodynamic approach is powerful for obtaining insights into the origin of Tiam1 PDZ-ligand domain affinity and specificity.
| S-EPMC4137555 | biostudies-literature
Project description:Mutations in cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, cause cystic fibrosis. To investigate interactions of CFTR in living cells, we measured the diffusion of quantum dot-labeled CFTR molecules by single particle tracking. In multiple cell lines, including airway epithelia, CFTR diffused little in the plasma membrane, generally not moving beyond 100-200 nm. However, CFTR became mobile over micrometer distances after 1) truncations of the carboxy terminus, which contains a C-terminal PDZ (PSD95/Dlg/ZO-1) binding motif; 2) blocking PDZ binding by C-terminal green fluorescent protein fusion; 3) disrupting CFTR association with actin by expression of a mutant EBP50/NHERF1 lacking its ezrin binding domain; or 4) skeletal disruption by latrunculin. CFTR also became mobile when the cytoskeletal adaptor protein binding capacity was saturated by overexpressing CFTR or its C terminus. Our data demonstrate remarkable and previously unrecognized immobilization of CFTR in the plasma membrane and provide direct evidence that C-terminal coupling to the actin skeleton via EBP50/ezrin is responsible for its immobility.
| S-EPMC1679663 | biostudies-literature
Project description:Synaptic adhesion-like molecules (SALMs) are a family of cell adhesion molecules involved in neurite outgrowth and synapse formation. Of the five family members, only SALM1, -2, and -3 contain a cytoplasmic C-terminal PDZ-binding motif. We have found that SALM1 is unique among the SALMs because deletion of its PDZ-binding motif (SALM1ΔPDZ) blocks its surface expression in heterologous cells. When expressed in hippocampal neurons, SALM1ΔPDZ had decreased surface expression in dendrites and the cell soma but not in axons, suggesting that the PDZ-binding domain may influence cellular trafficking of SALMs to specific neuronal locations. Endoglycosidase H digestion assays indicated that SALM1ΔPDZ is retained in the endoplasmic reticulum (ER) in heterologous cells. However, when the entire C-terminal tail of SALM1 was deleted, SALM1 was detected on the cell surface. Using serial deletions, we identified a region of SALM1 that contains a putative dileucine ER retention motif, which is not present in the other SALMs. Mutation of this DXXXLL motif allowed SALM1 to leave the ER and enhanced its surface expression in heterologous cells and neurons. An increase in the number of protrusions at the dendrites and cell body was observed when this SALM1 mutant was expressed in hippocampal neurons. With electron microscopy, these protrusions appeared to be irregular, enlarged spines and filopodia. Thus, enrichment of SALM1 on the cell surface affects dendritic arborization, and intracellular motifs regulate its dendritic versus axonal localization.
| S-EPMC3281672 | biostudies-literature