Exploring the molecular basis of selectivity in A1 adenosine receptors agonists: a case study

Adenosine is a naturally occurring purine nucleoside that has a wide variety of well-documented regulatory functions and physiological roles. Selective activation of the adenosine A₁ receptor has drawn attention in drug discovery for the therapeutic effects on neural and cardiovascular disorders. We have developed a model of the human A₁ adenosine receptor using bovine rhodopsin as a template. A flexible docking approach has been subsequently carried out for evaluating the molecular interactions of twenty-one selective A₁ agonists with the receptor model. The results of these studies are consistent with mutational and biochemical data. In particular, they highlight a wide hydrogen-bonding network between the nucleoside portion of the ligands and the A₁ receptor as well as key amino acids for hydrophobic interactions with the different N⁶-groups of the agonists. The models presented here provide a detailed molecular map for the selective stimulation of the adenosine A₁ receptor subtype and a steady basis for the rational design of new A₁ selective ligands.     

Targeting specific PDZ domains of PSD-95; structural basis for enhanced affinity and enzymatic stability of a cyclic peptide

A cyclic peptide, Tyr-Lys-c[-Lys-Thr-Glu(betaAla)-]-Val, incorporating a beta-Ala lactam side chain linker and designed to target the PDZ domains of the postsynaptic density protein 95 (PSD-95), has been synthesized and structurally characterized by NMR while free and bound to the PDZ1 domain of PSD-95. While bound, the lactam linker of the peptide makes a number of unique contacts outside the canonical PDZ binding motif, providing a novel target for PDZ-domain specificity as well as producing a 10-fold enhancement in binding affinity. Additionally, the cyclization greatly enhances the enzymatic stability, increasing the duration that the peptide inhibits the association between PSD-95 and glutamate receptors, effectively inhibiting the clustering of kainate receptors for over 14 hr after application. Highly specific regulation of kainate receptor action may provide a novel route for treatment of drug addiction and epilepsy.     

Side-chain interactions form late and cooperatively in the binding reaction between disordered peptides and PDZ domains

Intrinsically disordered proteins are very common and mediate numerous protein-protein and protein-DNA interactions. While it is clear that these interactions are instrumental for the life of the mammalian cell, there is a paucity of data regarding their molecular binding mechanisms. Here we have used short peptides as a model system for intrinsically disordered proteins. Linear free energy relationships based on rate and equilibrium constants for the binding of these peptides to ordered target proteins, PDZ domains, demonstrate that native side-chain interactions form mainly after the rate-limiting barrier for binding and in a cooperative fashion. This finding suggests that these disordered peptides first form a weak encounter complex with non-native interactions. The data do not support the recent notion that the affinities of intrinsically disordered proteins toward their targets are generally governed by their association rate constants. Instead, we observed the opposite for peptide-PDZ interactions, namely, that changes in K(d) correlate with changes in k(off).     

A flexible docking procedure for the exploration of peptide binding selectivity to known structures and homology models of PDZ domains

PDZ domains are important scaffolding modules that typically bind to the C-termini of their interaction partners. Several structures of such complexes have been solved, revealing a conserved binding site in the PDZ domain and an extended conformation of the bound peptide. A compendium of information regarding PDZ complexes demonstrates that dissimilar C-terminal peptides bind to the same PDZ domain, and different PDZ domains can bind the same peptides. A detailed understanding of the PDZ-peptide recognition is needed to elucidate this complexity. To this end, we have designed a family of docking protocols for PDZ domains (termed PDZ-DocScheme) that is based on simulated annealing molecular dynamics and rotamer optimization, and is applicable to the docking of long peptides (20-40 rotatable bonds) to both known PDZ structures and to the more complicated problem of homology models of these domains. The resulting protocol reproduces the structures of PDZ complexes with peptides 4-8 amino acids long within 1-2 A from the experimental structure when the docking is performed to the original structure. If the structure of the target PDZ domain is an apo structure or a homology model, the docking protocol yields structures within 3 A in 9 out of 12 test cases. The automated docking procedure PDZ-DocScheme can serve in the generation of a structural context for validation of PDZ domain specificity from mutagenesis and ligand binding data.     

Tyrosine sulfation: a modulator of extracellular protein-protein interactions

Tyrosine sulfation is a post-translational modification of many secreted and membrane-bound proteins. Its biological roles have been unclear. Recent work has implicated tyrosine sulfate as a determinant of protein-protein interactions involved in leukocyte adhesion, hemostasis and chemokine signaling.     

Phosphorylation-driven protein-protein interactions: a protein kinase sensing system

A highly flexible protein kinase sensing system is described that furnishes severalfold changes in fluorescence in response to phosphorylation. A library of Src kinase peptide substrates was prepared that contained different environmentally sensitive fluorophores positioned at various sites on the active site directed sequence. Robust changes in fluorescent intensity were observed in the presence of a phosphotyrosine binding domain protein (Lck SH2 domain), which furnishes a hydrophobic environment for the fluorophore. This protein kinase sensing system has the advantages that the fluorescent indicator can be unobtrusively positioned on the peptide substrate, and that different environmentally sensitive fluorophores with distinct photophysical properties can be employed.     

A systematic family-wide investigation reveals that ~30% of mammalian PDZ domains engage in PDZ-PDZ interactions

PDZ domains are independently folded modules that typically mediate protein-protein interactions by binding to the C termini of their target proteins. However, in a few instances, PDZ domains have been reported to dimerize with other PDZ domains. To investigate this noncanonical-binding mode further, we used protein microarrays comprising virtually every mouse PDZ domain to systematically query all possible PDZ-PDZ pairs. We then used fluorescence polarization to retest and quantify interactions and coaffinity purification to test biophysically validated interactions in the context of their full-length proteins. Overall, we discovered 37 PDZ-PDZ interactions involving 46 PDZ domains (~30% of all PDZ domains tested), revealing that dimerization is a more frequently used binding mode than was previously appreciated. This suggests that many PDZ domains evolved to form multiprotein complexes by simultaneously interacting with more than one ligand.     

Proline-rich proteins--deriving a basis for residue-based selectivity in polyphenolic binding

(1)H NMR titration experiments have been used to establish that minimal proline-based models show enhanced binding selectivity towards phenol in CDCl(3), relative to other similarly protected amino acid residues. Cooperative binding effects appear to play a role, with sarcosine models affording binding constants to phenol intermediate to those obtained from proline models and other amino acid models. The mechanism for binding, based on DFT calculations and the application of Hunter's molecular recognition toolbox model, cannot be solely attributed to hydrogen bond strength, and appears to be mediated through C-H-pi bonds and the rotational freedom of the amide substrate.     

Photoactivable peptides for identifying enzyme-substrate and protein-protein interactions

Photoactivated cross-linking of peptides to proteins is a useful strategy for identifying enzyme-substrate and protein-protein interactions in cell lysates as demonstrated by studies on the human hypoxia inducible factor system.     

Detecting protein-protein interactions by isotope-edited infrared spectroscopy: a numerical approach

We present a theoretical and numerical analysis of the vibrational coupling between isotope-edited amino acids in protein dimers. Depending on the presence and magnitude of coupling between 13Calpha=O peptide bond oscillators, characteristic level splittings of vibrational eigenstates are predicted. For the example of the Gramicidin A ion channel polypeptide, we observe typical IR fingerprints for the head-to-head and the antiparallel double-helical conformation of the dimer. We suggest that these findings can be used to clearly identify the structure of polypeptide aggregates using a particularly simple isotope substitution pattern.     

Utility of formaldehyde cross-linking and mass spectrometry in the study of protein-protein interactions

For decades, formaldehyde has been routinely used to cross-link proteins in cells, tissue, and in some instances, even entire organisms. Due to its small size, formaldehyde can readily permeate cell walls and membranes, resulting in efficient cross-linking, i.e. the formation of covalent bonds between proteins, DNA, and other reactive molecules. Indeed, formaldehyde cross-linking is an instrumental component of many mainstream analytical/cell biology techniques including chromatin immunoprecipitation (ChIP) of protein-DNA complexes found in nuclei; immunohistological analysis of protein expression and localization within cells, tissues, and organs; and mass spectrometry (MS)-compatible silver-staining methodologies used to visualize low abundance proteins in polyacrylamide gels. However, despite its exquisite suitability for use in the analysis of protein environments within cells, formaldehyde has yet to be commonly employed in the directed analysis of protein-protein interactions and cellular networks. The general purpose of this article is to discuss recent advancements in the use of formaldehyde cross-linking in combination with MS-based methodologies. Key advantages and limitations to the use of formaldehyde over other cross-linkers and technologies currently used to study protein-protein interactions are highlighted, and formaldehyde-based experimental approaches that are proving very promising in their ability to accurately and efficiently identify novel protein-protein and multiprotein interaction complexes are presented.     

Predicting protein-protein interactions using graph invariants and a neural network

The PDZ domain of proteins mediates a protein-protein interaction by recognizing the hydrophobic C-terminal tail of the target protein. One of the challenges put forth by the DREAM (Discussions on Reverse Engineering Assessment and Methods) 2009 Challenge consists of predicting a position weight matrix (PWM) that describes the specificity profile of five PDZ domains to their target peptides. We consider the primary structures of each of the five PDZ domains as a numerical sequence derived from graph-theoretic models of each of the individual amino acids in the protein sequence. Using available PDZ domain databases to obtain known targets, the graph-theoretic based numerical sequences are then used to train a neural network to recognize their targets. Given the challenge sequences, the target probabilities are computed and a corresponding position weight matrix is derived. In this work we present our method. The results of our method placed second in the DREAM 2009 challenge.     

Application of a green fluorescent fusion protein to study protein-protein interactions by electrophoretic methods

A screening procedure for protein-protein interactions in cellular extracts using a green fluorescent protein (GFP) and affinity capillary electrophoresis (ACE) was established. GFP was fused as a fluorescent indicator to the C-terminus of a cyclophilin (rDmCyp20) from Drosophila melanogaster. Cyclophilins (Cyps) belong to the ubiquitously distributed enzyme family of peptidyl-prolyl cis/trans isomerases (PPlases) and are well known as cellular targets of the immunosuppressive drug cyclosporin A (CsA). The PPlase activity of the GFP fused rDmCyp20 as well as the high affinity to CsA remain intact. Using native gel electrophoresis and ACE mobility-shift assays, it was demonstrated that the known moderate affinity of Cyp20 to the capsid protein p24 of HIV-1 was detectable in the case of rDmCyp20 fused to the fluorescent tag. For the p24 / rDmCyp20-GFP binding an ACE method was established which allowed to determine a dissociation constant of Kd = 20+/-1.5 x 10(-6) M. This result was verified by size-exclusion chromatography and is in good agreement with published data for the nonfused protein. Moreover the fusion protein was utilized to screen rDmCyp20-protein interactions by capillary electrophoresis in biological matrices. A putative ligand of rDmCyp20 in crude extracts of embryonic D. melanogaster was discovered by mobility-shift assays using native gel electrophoresis with fluorescence imaging and ACE with laser-induced fluorescence detection. The approach seems applicable to a wide range of proteins and offers new opportunities to screen for moderate protein-protein interactions in biological samples.     

Chemical basis for alkali cation selectivity in potassium-channel proteins

The determination of the crystal structure of a K⁺-selective channel protein from Streptomyces lividans reveals how the rapid movement of K⁺ across membranes is catalyzed by a large family of pore-forming proteins. Many features of the structure mirror hypotheses, predictions and models of K⁺ channels developed over the past four decades of functional analysis.     

Thermodynamic analysis of a hydrophobic binding site: probing the PDZ domain with nonproteinogenic peptide ligands

[structure: see text] Isothermal titration calorimetry (ITC) is used to study the thermodynamic consequences of systematically modifying the hydrophobic character of a single residue in a series of protein-binding ligands. By substituting standard and nonproteinogenic aliphatic amino acids for the C-terminal valine of the hexapeptide KKETEV, binding to the third PDZ domain (PDZ3) of the PSD-95 protein is characterized by distinct changes in the Gibbs free energy (DeltaG), enthalpy (DeltaH), and entropy (TDeltaS) parameters. One notable observation is that peptide binding affinity can be improved with a nonstandard residue.     

The laser-induced blue state of bacteriorhodopsin: mechanistic and color regulatory roles of protein-protein interactions, protein-lipid interactions, and metal ions

In this paper we characterize the mechanistic roles of the crystalline purple membrane (PM) lattice, the earliest bacteriorhodopsin (BR) photocycle intermediates, and divalent cations in the conversion of PM to laser-induced blue membrane (LIBM; lambda(max)= 605 nm) upon irradiation with intense 532 nm pulses by contrasting the photoconversion of PM with that of monomeric BR solubilized in reduced Triton X-100 detergent. Monomeric BR forms a previously unreported colorless monomer photoproduct which lacks a chromophore band in the visible region but manifests a new band centered near 360 nm similar to the 360 nm band in LIBM. The 360 nm band in both LIBM and colorless monomer originates from a Schiff base-reduced retinyl chromophore which remains covalently linked to bacterioopsin. Both the PM-->LIBM and monomer-->colorless monomer photoconversions are mediated by similar biphotonic mechanisms, indicating that the photochemistry is localized within single BR monomers and is not influenced by BR-BR interactions. The excessively large two-photon absorptivities (> or =10(6) cm(4) s molecule(-1) photon(-1)) of these photoconversions, the temporal and spectral characteristics of pulses which generate LIBM in high yield, and an action spectrum for the PM-->LIBM photoconversion all indicate that the PM-->LIBM and Mon-->CMon photoconversions are both mediated by a sequential biphotonic mechanism in which is the intermediate which absorbs the second photon. The purple-->blue color change results from subsequent conformational perturbations of the PM lattice which induce the removal of Ca(2+) and Mg(2+) ions from the PM surface.     

Drug-like inhibitors of protein-protein interactions: a structural examination of effective protein mimicry

Protein-protein interactions represent targets for drug discovery that are highly relevant in a biological sense, but have proven difficult in a practical sense. Nevertheless, there have been recent successes in discovering drug-like small molecule inhibitors of protein-protein systems. To build on this progress, it is worth analyzing successful cases to understand at a molecular level the strategies by which these compounds effectively interfere with protein-protein pairing. A commonly observed situation is one wherein the small molecule acts as a direct mimic of one of the protein partners. This review focuses exclusively on cases where this strategy is employed, and examines the structural characteristics of the binding sites and the conformational attributes of the small molecule ligands. Common traits shared among these successful examples are identified, and formulated into potentially useful guidance for drug discovery efforts within this target class.     

p-Xylene-selective metal-organic frameworks: a case of topology-directed selectivity

Para-disubstituted alkylaromatics such as p-xylene are preferentially adsorbed from an isomer mixture on three isostructural metal-organic frameworks: MIL-125(Ti) ([Ti(8)O(8)(OH)(4)(BDC)(6)]), MIL-125(Ti)-NH(2) ([Ti(8)O(8)(OH)(4)(BDC-NH(2))(6)]), and CAU-1(Al)-NH(2) ([Al(8)(OH)(4)(OCH(3))(8)(BDC-NH(2))(6)]) (BDC = 1,4-benzenedicarboxylate). Their unique structure contains octahedral cages, which can separate molecules on the basis of differences in packing and interaction with the pore walls, as well as smaller tetrahedral cages, which are capable of separating molecules by molecular sieving. These experimental data are in line with predictions by molecular simulations. Additional adsorption and microcalorimetric experiments provide insight in the complementary role of the two cage types in providing the para selectivity.     

Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism

Identification of protein binding partners is one of the key challenges of proteomics. We recently introduced a screen for detecting protein-protein interactions based on reassembly of dissected fragments of green fluorescent protein fused to interacting peptides. Here, we present a set of comaintained Escherichia coli plasmids for the facile subcloning of fusions to the green fluorescent protein fragments. Using a library of antiparallel leucine zippers, we have shown that the screen can detect very weak interactions (K(D) approximately 1 mM). In vitro kinetics show that the reassembly reaction is essentially irreversible, suggesting that the screen may be useful for detecting transient interactions. Finally, we used the screen to discriminate cognate from noncognate protein-ligand interactions for tetratricopeptide repeat domains. These experiments demonstrate the general utility of the screen for larger proteins and elucidate mechanistic details to guide the further use of this screen in proteomic analysis. Additionally, this work gives insight into the positional inequivalence of stabilizing interactions in antiparallel coiled coils.     

Hydrogen bonds and ionic interactions in Guanidine/Guanidinium complexes: a computational case study

It is frequently said that hydrogen bonds (HBs) are enhanced by ionic interactions and in this article we intend to determine the degree at which this reinforcement happens. Considering our interest in the Guanidine(neutral)/Guanidinium(cation) system and its particular nature, all the possible 1:1 complexes with the Chloride(anion)/Hydrochloric acid(neutral) system have been studied at different levels of computation (B3LYP with 6-31+G* and TZVP basis sets; MP2 with 6-31+G*, 6-311++G** and aug-cc-pVDZ basis sets; CBS-QB3 and G3MP2). The nature of these interactions established in all the systems and, when possible, at all the levels of computation used in this study, has been analyzed using Atoms in Molecules and Natural Bond Orbital methodologies. By examining the interaction energy, the electron density at the bond critical bonds, the atomic energy, the charge transfer, the orbital energy, and the deformation energy we can conclude that HBs are stronger when the ionic interaction is stronger. Thus, both interactions do not work in an independent manner but one reinforces the other to different degrees depending on the nature of the charges present. Several correlations with the interaction energy have been found and a partition of the contributions of both the HB and ionic forces to the total interactions is proposed.