Role of electrostatic interactions in PDZ domain ligand recognition

ABSTRACT: PDZ domains are protein-protein interaction modules that normally recognize short C-terminal peptides. The apparent requirement for a ligand with a free terminal carboxylate group has led to the proposal that electrostatic interactions with the terminus play a significant role in recognition. However, this model has been called into question by the more recent finding that PDZ domains can recognize some internal peptide motifs that occur within a specific secondary structure context. Although these motifs bind at the same interface, they lack a terminal charge. Here we have investigated the role of electrostatics in PDZ-mediated recognition in the mouse R1-syntrophin PDZ domain by examining the salt dependence of binding to both terminal and internal ligands and the effects of mutating a conserved basic residue previously proposed to play a role in electrostatic recognition. These studies indicate that direct electrostatic interactions with the peptide terminus do not play a significant energetic role in binding. Additional chemical modification studies of the peptide terminus support a model in which steric and hydrogen bonding complementarity play a primary role in recognition specificity. Peptides with a free carboxy terminus, or presented within a specific structural context, can satisfy these requirements. Cells rely on modular protein-protein recognition domains to assemble multiprotein signaling complexes. PDZ 1 (PSD-95, Dlg, ZO-1 homology) domains form a large family of such modules. PDZ domain-containing proteins play an important role in organizing signaling structures at cellcell signaling junctions such as synapses (1, 2). PDZ domains were first characterized for their ability to specifically recognize C-terminal peptide ligands, including the C-termini of receptors and channels Here we describe experiments aimed at more precisely evaluating the role of electrostatic interactions in PDZmediated peptide recognition using the Mus musculus R1-syntrophin PDZ domain as a model system. First, we have determined the sensitivity of various PDZ-mediated interactions to ionic strength, and second, we have determined the effects of mutating a conserved basic residue in the PDZ binding groove that has been proposed to play a role in electrostatic recognition. These results indicate that electrostatics do not play a significant role in recognition of the terminal carboxylate. In addition, we have examined a library of ligand variants and found that PDZ domains discriminate very tightly between ligands with minor alterations in the precise chemical structure of the C-terminus. Together, these results are consistent with a model in which steric and † This research was supported by grants from the National Institutes of Health and the David and Lucille Packard Foundation (to W.A.L.) and the Stewart Trust (to R.K.G. alcohol; Fmoc, 9-fluorenylmethoxycarbonyl; FPLC, fast performance liquid chromatography; HATU, 2-(1H-9-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HEPES, N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid; HPLC, high-pressure liquid chromatography; IPTG, isopropyl -D-thiogalactopyranoside; MeOH, methanol or methyl alcohol; Ni-NTA, nickel nitriloacetic acid; nNOS, neuronal nitric oxide synthase; PCR, polymerase chain reaction; PDZ, PSD-95, Dlg, ZO-1 homology; TEV, tobacco etch virus; TFA, trifluoroacetic acid; TIS, triisopropylsilane

Spatiotemporal Analysis of the Molecular Interaction between PICK1 and PKC

PICK1 is a protein which was initially identified as a protein kinase Cα (αPKC) binding protein using the yeast two-hybrid system. In addition to αPKC, the PICK1 complex binds to and regulates various transmembrane proteins including receptors and transporters. However, it has not been clarified when and where PICK1 binds to αPKC. We examined the spatio­temporal interaction of PICK1 and PKC using live imaging techniques and showed that the activated αPKC binds to PICK1 and transports it to the plasma membrane. Although the membrane translocation of PICK1 requires the activation of αPKC, PICK1 is retained on the membrane even after PKC moves back to the cytosol. These results suggest that the interaction between αPKC and PICK1 is transient and may not be necessary for the regulation of receptors/transporters by PICK1 or by αPKC on the membrane

Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3

Ubiquitination by HECT E3 enzymes regulates myriad processes, including tumor suppression, transcription, protein trafficking, and degradation. HECT E3s use a two-step mechanism to ligate ubiquitin to target proteins. The first step is guided by interactions between the catalytic HECT domain and the E2∼ubiquitin intermediate, which promote formation of a transient, thioester-bonded HECT∼ubiquitin intermediate. Here we report that the second step of ligation is mediated by a distinct catalytic architecture established by both the HECT E3 and its covalently linked ubiquitin. The structure of a chemically trapped proxy for an E3∼ubiquitin-substrate intermediate reveals three-way interactions between ubiquitin and the bilobal HECT domain orienting the E3∼ubiquitin thioester bond for ligation, and restricting the location of the substrate-binding domain to prioritize target lysines for ubiquitination. The data allow visualization of an E2-to-E3-to-substrate ubiquitin transfer cascade, and show how HECT-specific ubiquitin interactions driving multiple reactions are repurposed by a major E3 conformational change to promote ligation. DOI:http://dx.doi.org/10.7554/eLife.00828.001

A study on trajectory control of manipulator using power packet dispatching

Date of Conference: 18-22 Oct. 2015In the power packet dispatching system, electric power is transferred with information tags attached physically. An application of the system to robots can enable flexible and efficient power management with a dynamically varying powerline connection between power sources and loads. In this paper, a numerical simulation verifies the achievement of a trajectory control of a manipulator by power packets. As a result, it is clarified that a conventional power conversion system at the load side can be replaced by a packet generating system at the source side. This can be a step for new power management inside robots with power packets

Sequence requirement and subtype specificity in the high-affinity interaction between human frizzled and dishevelled proteins

Members of the Wnt family of lipoglycoproteins initiate signaling by binding to Frizzled (Fz) receptors, and the signal is then relayed by Disheveled (Dvl). The Dvl PDZ domain is known to interact directly with a peptide derived from the KTXXXW motif of Fz7, which is conserved in all known Fz subtypes. We found that an extended region spanning the KTXXXW motif on both its N-terminal and C-terminal sides dramatically influences the affinity of peptides derived from Fz7 for Dvl PDZ. An alanine scanning study identified the specific residues external to the KTXXXW motif that are important for high-affinity binding. In a circular dichroism analysis, mutation of some of these critical residues resulted in peptide conformational changes, suggesting that the secondary structure of the peptides contributes to Fz-Dvl PDZ binding. Of the 10 known Fz subtypes, peptides derived from only Fz1, Fz2, Fz3, Fz4, and Fz7 directly bound to Dvl PDZ domain in our study. Other Fz subtypes, including some known to be involved in Wnt/β-catenin signaling (Fz5, Fz9), did not bind to Dvl, suggesting that direct interaction with Dvl PDZ does not determine the subtype-specific functionality of Fz. Molecular modeling and circular dichroism studies indicated that the Fz peptides that bind to Dvl PDZ domain form specific conformations that are different from those of nonbinding peptides