69 research outputs found
Ligand binding by PDZ domains
No full textThe postsynaptic density protein-95/disks large/zonula occludens-1 (PDZ) protein domain family is one of the most common proteinprotein interaction modules in mammalian cells, with paralogs present in several hundred human proteins. PDZ domains are found in most cell types, but neuronal proteins, for example, are particularly rich in these domains. The general function of PDZ domains is to bring proteins together within the appropriate cellular compartment, thereby facilitating scaffolding, signaling, and trafficking events. The many functions of PDZ domains under normal physiological as well as pathological conditions have been reviewed recently. In this review, we focus on the molecular details of how PDZ domains bind their protein ligands and their potential as drug targets in this context
Protolytic properties of polyamine wasp toxin analogues studied by 13C NMR spectroscopy
No full textAcid-base properties of the natural polyamine wasp toxin PhTX-433 (1) and seven synthetic analogues [PhTX-343 (2), PhTX-334 (3), PhTX-443 (4), PhTX-434 (5), PhTX-344 (6), PhTX-444 (7), and PhTX-333 (8)], each having four protolytic sites, were characterized by (13)C NMR spectroscopy. Nonlinear, multiparameter, simultaneous fit of all chemical shift data obtained from the NMR titration curves yielded macroscopic pK(a) values as well as intrinsic chemical shift data of all differently protonated macrospecies. Analyses of the chemical shift data demonstrated strong interactions between all four sites and provided information about complex relationships between chemical shift values and protonation state. Deprotonation of fully protonated forms starts at the central amino group of the polyamine moiety, and the extent of this trend depends on the distance to the flanking, protonated amino groups. The pK(a1) values of 1-8 are in the range 8.2-9.4. Hence, some of the toxins are incompletely protonated at the pH and ionic strength conditions used for assessment of their interactions with ionotropic glutamate and nicotinic acetylcholine receptors, and the degree of protonation is expected to have pharmacological importance in the ion-channel binding event
A sequential binding mechanism in a PDZ domain.
No full textConformational selection and induced fit are two well-known mechanisms of allosteric protein-ligand interaction. Some proteins, like ubiquitin, have recently been found to exist in multiple conformations at equilibrium, suggesting that the conformational selection may be a general mechanism of interaction, in particular for single-domain proteins. Here, we found that the PDZ2 domain of SAP97 binds its ligand via a sequential (induced fit) mechanism. We performed binding experiments using SAP97 PDZ2 and peptide ligands and observed biphasic kinetics with the stopped-flow technique, indicating that ligand binding involves at least a two-step process. By using an ultrarapid continuous-flow mixer, we then detected a hyperbolic dependence of binding rate constants on peptide concentration, corroborating the two-step binding mechanism. Furthermore, we found a similar dependence of the rate constants on both PDZ and peptide concentration, demonstrating that the PDZ2-peptide interaction involves a precomplex, which then undergoes a conformational change, and thereby follows an induced fit mechanism
Energetic pathway sampling in a protein interaction domain
No full textThe affinity and specificity of protein-ligand interactions are influenced by energeticcrosstalk within the protein domain. However, the molecular details of such intradomain allostery are still unclear. Here, we have experimentally detected and computationally predicted interactionpathways in the postsynaptic density 95/discs large/zonula occludens 1 (PDZ)-peptide ligand model system using wild-type and circularly permuted PDZ proteins. The circular permutant introduced small perturbations in the tertiary structure and a concomitant rewiring of allosteric pathways, allowing us to describe how subtle changes may reshape energetic signaling. The results were analyzed in the context of other members of the PDZ family, which were found to contain distinct interaction pathways for different peptide ligands. The data reveal a fascinating scenario whereby several energetic pathways are sampled within one single domain and distinct pathways are activated by specific protein ligands.
Ligand binding to the pdz domains of postsynaptic density protein 95
No full textCellular scaffolding and signalling is generally governed by multidomain proteins, where each
domain has a particular function. Postsynaptic density protein 95 (PSD-95) is involved in synapse formation
and is a typical example of such a multidomain protein. Protein–protein interactions of PSD95
are well studied and include the following three protein ligands: (i) N-methyl-D-aspartate-type
ionotropic glutamate receptor subunit GluN2B, (ii) neuronal nitric oxide synthase and (iii) cysteinerich
protein (CRIPT), all of which bind to one or more of the three PDZ domains in PSD-95. While
interactions for individual PDZ domains of PSD-95 have been well studied, less is known about
the influence of neighbouring domains on the function of the respective individual domain. We
therefore performed a systematic study on the ligand-binding kinetics of PSD-95 using constructs
of different size for PSD-95 and its ligands. Regarding the canonical peptide-binding pocket and relatively
short peptides (up to 15-mer), the PDZ domains in PSD-95 by and large work as individual binding
modules. However, in agreement with previous studies, residues outside of the canonical
binding pocket modulate the affinity of the ligands. In particular, the dissociation of the 101 amino
acid CRIPT from PSD-95 is slowed down at least 10-fold for full-length PSD-95 when compared with
the individual PDZ3 domain
Probing the Role of Backbone Hydrogen Bonds in Protein-Peptide Interactions by Amide-to-Ester Mutations
No full textOne of the most frequent protein-protein interaction modules in mammalian cells is the postsynaptic density 95/discs large/zonula occludens 1 (PDZ) domain, involved in scaffolding and signaling and emerging as an important drug target for several diseases. Like many other protein-protein interactions, those of the PDZ domain family involve formation of intermolecular hydrogen bonds: C-termini or internal linear motifs of proteins bind as beta-strands to form an extended antiparallel beta-sheet with the PDZ domain. Whereas extensive work has focused on the importance of the, amino acid side chains of the protein ligand, the role of the backbone hydrogen bonds in the binding reaction is not known. Using amide-to-ester substitutions to perturb the backbone hydrogen-bonding pattern, we have systematically probed putative backbone hydrogen bonds between four different PDZ domains and peptides corresponding to natural protein ligands. Amide-to-ester mutations of the three C-terminal amides of the peptide ligand severely affected the affinity with the PDZ domain, demonstrating that hydrogen bonds contribute significantly to ligand binding (apparent changes in binding energy, Delta Delta G = 1.3 to >3.8 kcal mol(-1)). This decrease in affinity was mainly due to an increase in the dissociation rate constant, but a significant decrease in the association rate constant was found for some amide-to-ester mutations Suggesting that native hydrogen bonds have begun to form in the transition state of the binding reaction. This study provides a general framework for studying the role of backbone hydrogen bonds in protein-peptide interactions and for the first time specifically addresses these for PDZ domain-peptide interactions
Side-chain interactions form late and cooperatively in the binding reaction between disordered peptides and PDZ domains
No full textPreparation of peptide thioesters through fmoc-based solid-phase peptide synthesis by using amino thioesters
No full textAn effective procedure for the synthesis of peptide alkyl thioesters by 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis was developed. The free C terminus of a fully protected peptide was coupled in solution with the free amino group of an amino thioester. This furnished the fully protected peptide thioester, which was globally deprotected to afford the desired unprotected peptide thioester. The method is compatible with labile groups such as phosphoryl and glycosyl moieties. The synthesis of peptide alkyl thioesters by 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) is developed. The free C terminus of a fully protected peptide is coupled in solution with the free amino group of an amino thioester. This furnishes the fully protected peptide thioester, which can be globally deprotected to afford the desired unprotected peptide thioester
Recent advances in the medicinal chemistry of polyamine toxins
No full textThis review describes the recent developments in the field of polyamine toxins, with focus on structure activity relationship investigations, including studies of importance of the polyamine moiety for biological activity, photolabeling studies using polyamine toxins as templates, as well as use of solid phase methods for the synthesis of polyamine toxins. The review is mainly concerned with effects of polyamine toxins on nicotinic acetylcholine receptors and ionotropic glutamate receptors
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