An Optimised Small‐Molecule Stabiliser of the 14‐3‐3–PMA2 Protein–Protein Interaction

Modulation of protein–protein interactions (PPIs) is a highly demanding, but also a very promising approach in chemical biology and targeted drug discovery. In contrast to inhibiting PPIs with small, chemically tractable molecules, stabilisation of these interactions can only be achieved with complex natural products, like rapamycin, FK506, taxol, forskolin, brefeldin and fusicoccin. Fusicoccin stabilises the activatory complex of the plant H⁺‐ATPase PMA2 and 14‐3‐3 proteins. Recently, we have shown that the stabilising effect of fusicoccin could be mimicked by a trisubstituted pyrrolinone (pyrrolidone1, 1 ). Here, we report the synthesis, functional activity and crystal structure of derivatives of 1 that stabilise the 14‐3‐3–PMA2 complex. With a limited compound collection three modifications that are important for activity enhancement could be determined: 1) conversion of the pyrrolinone scaffold into a pyrazole, 2) introduction of a tetrazole moiety to the phenyl ring that contacts PMA2, and 3) addition of a bromine to the phenyl ring that exclusively contacts the 14‐3‐3 protein. The crystal structure of a pyrazole derivative of 1 in complex with 14‐3‐3 and PMA2 revealed that the more rigid core of this molecule positions the stabiliser deeper into the rim of the interface, enlarging especially the contact surface to PMA2. Combination of the aforementioned features gave rise to a molecule ( 37 ) that displays a threefold increase in stabilising the 14‐3‐3–PMA2 complex over 1 . Compound 37 and the other active derivatives show no effect on two other important 14‐3‐3 protein–protein interactions, that is, with CRaf and p53. This is the first study that describes the successful optimisation of a PPI stabiliser identified by screening.     

Metal‐Mediated Self‐Assembly of a β‐Sandwich Protein

The β‐sandwich cupredoxin Plastocyanin (Pc) was found to self‐assemble in the presence of Zn²⁺, a known mediator of protein–protein interfaces. Diffraction‐quality crystals of Pc grew from solutions containing zinc acetate as the sole precipitant. Di‐ and trinuclear zinc sites contribute to the crystal contacts in this structure. A different crystal form, also involving numerous zinc bridging ions, was obtained in the presence of poly(ethylene glycol) 8 000. Comparison of the two crystal forms reveals the effect of macromolecular crowding on self‐assembly. Solution‐state structural characterisation of the Zn²⁺‐mediated Pc oligomers was performed by using a combination of chemical shift perturbation mapping and small‐angle X‐ray scattering. The data indicate the formation of dimers in solution. The implications for metal‐mediated assembly and crystallisation are discussed.     

Potassium p‐nitro­phenyl sulfate

The crystal structure of the title compound, K⁺·C₆H₄NO₆S⁻, is built up from p‐nitro­phenyl sulfate anions and potassium cations. Adjacent anions form dimers, which are linked together in a three‐dimensional network via short C—H⋯O contacts. The coordination sphere of the K⁺ ions may be described as a distorted square antiprism. The crystal structure is further stabilized by π–π stacking interactions between the aryl rings.     

High‐Resolution Structural Characterization of a Helical α/β‐Peptide Foldamer Bound to the Anti‐Apoptotic Protein Bcl‐xL

Get into the groove : The first high‐resolution structure of a foldamer bound to a protein target is described (see picture; foldamer in sticks). The foldamer consists of α‐ and β‐amino acid residues and is bound to the anti‐apoptotic protein Bcl‐x_L. The overall binding mode and key interactions observed in the foldamer/Bcl‐x_L complex mimic those seen in complexes of Bcl‐x_L with natural α‐peptide ligands. Additional contacts in the foldamer/Bcl‐x_L complex involving β‐amino acid residues appear to contribute to binding affinity.

     

The peptide NCbz‐Val‐Tyr‐OMe and aromatic π–π interactions

The peptide N‐benzyloxycarbonyl‐L‐valyl‐L‐tyrosine methyl ester or NCbz‐Val‐Tyr‐OMe (where NCbz is N‐benzyloxycarbonyl and OMe indicates the methyl ester), C₂₃H₂₈N₂O₆, has an extended backbone conformation. The aromatic rings of the Tyr residue and the NCbz group are involved in various attractive intra‐ and intermolecular aromatic π–π interactions which stabilize the conformation and packing in the crystal structure, in addition to N—H...O and O—H...O hydrogen bonds. The aromatic π–π interactions include parallel‐displaced, perpendicular T‐shaped, perpendicular L‐shaped and inclined orientations.     

Reconstitution of the Cytb5–CytP450 Complex in Nanodiscs for Structural Studies using NMR Spectroscopy

Cytochrome P450s (P450s) are a superfamily of enzymes responsible for the catalysis of a wide range of substrates. Dynamic interactions between full‐length membrane‐bound P450 and its redox partner cytochrome b₅ (cytb₅) have been found to be important for the enzymatic activity of P450. However, the stability of the circa 70 kDa membrane‐bound complex in model membranes renders high‐resolution structural NMR studies particularly difficult. To overcome these challenges, reconstitution of the P450–cytb₅ complex in peptide‐based nanodiscs, containing no detergents, has been demonstrated, which are characterized by size exclusion chromatography and NMR spectroscopy. In addition, NMR experiments are used to identify the binding interface of the P450–cytb₅ complex in the nanodisc. This is the first successful demonstration of a protein–protein complex in a nanodisc using NMR structural studies and should be useful to obtain valuable structural information on membrane‐bound protein complexes.     

Invariom‐model refinement and Hirshfeld surface analysis of well‐ordered solvent‐free dibenzo‐21‐crown‐7

Crown ethers and their supramolecular derivatives are well‐known chelators and scavengers for a variety of cations, most notably heavier alkali and alkaline‐earth ions. Although they are widely used in synthetic chemistry, available crystal structures of uncoordinated and solvent‐free crown ethers regularly suffer from disorder. In this study, we present the X‐ray crystal structure analysis of well‐ordered solvent‐free crystals of dibenzo‐21‐crown‐7 (systematic name: dibenzo[b ,k ]‐1,4,7,10,13,16,19‐heptaoxacycloheneicosa‐2,11‐diene, C₂₂H₂₈O₇). Because of the quality of the crystal and diffraction data, we have chosen invarioms, in addition to standard independent spherical atoms, for modelling and briefly discuss the different refinement results. The electrostatic potential, which is directly deducible from the invariom model, and the Hirshfeld surface are analysed and complemented with interaction‐energy computations to characterize intermolecular contacts. The boat‐like molecules stack along the a axis and are arranged as dimers of chains, which assemble as rows to form a three‐dimensional structure. Dispersive C—H…H—C and C—H…π interactions dominate, but nonclassical hydrogen bonds are present and reflect the overall rather weak electrostatic influence. A fingerprint plot of the Hirshfeld surface summarizes and visualizes the intermolecular interactions. The insight gained into the crystal structure of dibenzo‐21‐crown‐7 not only demonstrates the power of invariom refinement, Hirshfeld surface analysis and interaction‐energy computation, but also hints at favourable conditions for crystallizing solvent‐free crown ethers.     

Synthesis,crystal structure and study of the crystal packing in the complex bis(4‐aminopyridine‐κN1)dichloridocobalt(II)

Despite the large number of reported crystalline structures of coordination complexes bearing pyridines as ligands, the relevance of π–π interactions among these hereroaromatic systems in the stabilization of their supramolecular structures and properties is not very well documented in the recent literature. The title compound, [CoCl₂(C₅H₆N₂)₂], was obtained as bright‐blue crystals suitable for single‐crystal X‐ray diffraction analysis from the reaction of 4‐aminopyridine with cobalt(II) chloride in ethanol. The new complex was fully characterized by a variety of spectroscopic techniques and single‐crystal X‐ray diffraction. The crystal structure showed a tetrahedral complex stabilized mainly by bidimensional motifs constructed by π–π interactions with large horizontal displacements between the 4‐aminopyridine units, and N—H…Cl hydrogen bonds. Other short contacts, such as C—H…Cl interactions, complete the three‐dimensional arrangement. The supramolecular investigation was extended by statistical studies using the Cambridge Structural Database and a Hirshfeld surface analysis.     

Poly(L‐lysine)‐Induced Aggregation of Single‐Strand Oligo‐DNA‐Modified Gold Nanoparticles

Single‐strand oligo‐DNA‐modified Au nanoparticles (AuNPs) undergo aggregation in the presence of poly(L ‐lysine) (PLL), which is attributed to the interactions between the oligo‐DNA and PLL. These interactions between the oligo‐DNA and PLL were identified to be electrostatic when the lysine residues of PLL were positively charged and to be hydrogen bonding when the residues were deprotonated. The aggregation was promoted with an increase in the pH value at a pH level lower than the pK_a value of PLL (pK_a≈10.0) due to the gradual deprotonation of the lysine residues and thus suppressed electrostatic interactions between the positively charged lysine residues of PLL and the negatively charged backbone phosphate groups of the oligo‐DNA. At pH levels higher than the pK_a value of PLL, the aggregation was identified to be dominated by the hydrogen bonds between the bases of the oligo‐DNA and the deprotonated lysine residues of PLL. This study prompts the possibility that the spectral, and thus color, change of AuNPs upon aggregation can be used as a probe to follow the interactions between oligo‐DNA and polypeptides.     

Synthesis,spectroscopic (FT–IR and UV–Vis), crystallographic and theoretical studies,and a molecular docking simulation of an imatinib‐like template

The aim of the present study was to report the crystal structure and spectroscopic, electronic, supramolecular and electrostatic properties of a new polymorph of 4‐(pyridin‐2‐yl)pyrimidin‐2‐amine (C₉H₈N₄). The compound was synthesized under microwave irradiation. The single‐crystal X‐ray structure analysis revealed an angle of 13.36 (8)° between the planes of the rings, as well as molecules linked by Nsp²—H…N hydrogen bonds forming dimers along the crystal. The material was analyzed by FT–IR vibrational spectroscopy, while a computational approach was used to elucidate the vibrational frequency couplings. The existence of Nsp²—H…N hydrogen bonds in the crystal was confirmed spectroscopically by the IR peaks from the N—H stretching vibration shifting to lower wavenumbers in the solid state relative to those in the gas phase. The supramolecular studies confirmed the formation of centrosymmetric R₂²(8) rings, which correspond to the formation of dimers that stack parallel to the b direction. Other weak C—H…π interactions, essential for crystal growth, were found. The UV–Vis spectroscopic analysis showed a donor–acceptor process, where the amino group acts as a donor and the pyridine and pyrimidine rings act as acceptors. The reactive sites of the molecule were identified and their quantitative values were defined using the electrostatic potential model proposed in the multifunctional wave function analyzer multiwfn. The calculated interaction energies between pairs of molecules were used to visualize the electrostatic terms as the leading factors against the dispersion factors in the crystal‐growth process. The docking results showed that the amino group of the pyrimidine moiety was simultaneously anchored by hydrogen‐bonding interactions with the Asp427 and His407 protein residues. This compound could be key for the realization of a series of syntheses of molecules that could be used as possible inhibitors of chronic myelogenous leukemia.     

Crystal structures of four δ‐keto esters and a Cambridge Structural Database analysis of cyano–halogen interactions

The revived interest in halogen bonding as a tool in pharmaceutical cocrystals and drug design has indicated that cyano–halogen interactions could play an important role. The crystal structures of four closely related δ‐keto esters, which differ only in the substitution at a single C atom (by H, OMe, Cl and Br), are compared, namely ethyl 2‐cyano‐5‐oxo‐5‐phenyl‐3‐(piperidin‐1‐yl)pent‐2‐enoate, C₁₉H₂₂N₂O₃, (1), ethyl 2‐cyano‐5‐(4‐methoxyphenyl)‐5‐oxo‐3‐(piperidin‐1‐yl)pent‐2‐enoate, C₂₀H₂₄N₂O₄, (2), ethyl 5‐(4‐chlorophenyl)‐2‐cyano‐5‐oxo‐3‐(piperidin‐1‐yl)pent‐2‐enoate, C₁₉H₂₁ClN₂O₃, (3), and the previously published ethyl 5‐(4‐bromophenyl)‐2‐cyano‐5‐oxo‐3‐(piperidin‐1‐yl)pent‐2‐enoate, C₁₉H₂₁BrN₂O₃, (4) [Maurya, Vasudev & Gupta (2013). RSC Adv. 3 , 12955–12962]. The molecular conformations are very similar, while there are differences in the molecular assemblies. Intermolecular C—H...O hydrogen bonds are found to be the primary interactions in the crystal packing and are present in all four structures. The halogenated derivatives have additional aromatic–aromatic interactions and cyano–halogen interactions, further stabilizing the molecular packing. A database analysis of cyano–halogen interactions using the Cambridge Structural Database [CSD; Groom & Allen (2014). Angew. Chem. Int. Ed. 53 , 662–671] revealed that about 13% of the organic molecular crystals containing both cyano and halogen groups have cyano–halogen interactions in their packing. Three geometric parameters for the C—X...N[triple‐bond]C interaction (X = F, Cl, Br or I), viz. the N...X distance and the C—X...N and C—N...X angles, were analysed. The results indicate that all the short cyano–halogen contacts in the CSD can be classified as halogen bonds, which are directional noncovalent interactions.     

One‐pot Preparation,Spectroscopic and Structural Characterization of Mercury(II) Complexes of Bulky Diimines with Halides and Pseudohalides

The preparation and characterization of two novel HgCl₂ and Hg(SCN)₂ complexes with bis[N‐(2‐tert‐butylphenyl)imine]acenaphthene is here described. One‐pot reaction techniques were used, leading to high yields of 75 % and 81 %, respectively. The complexes were characterized by microanalysis, i.r. and ¹HNMR spectroscopy, and by single crystal X‐ray diffraction. The structures of the complexes present similar characteristics, the most outstanding being the formation of dimers via intermolecular interaction. Whereas the HgCl₂ complex shows a unidimensional network due to strong π–π interactions, its Hg(SCN)₂ counterpart displays a supramolecular arrangement resulting from non classical hydrogen bond formation.     

Ubiquitin Associates with the N‐Terminal Domain of Nerve Growth Factor: The Role of Copper(II) Ions

Many biochemical pathways involving nerve growth factor (NGF), a neurotrophin with copper(II) binding abilities, are regulated by the ubiquitin (Ub) proteasome system. However, whether NGF binds Ub and the role played by copper(II) ions in modulating their interactions have not yet been investigated. Herein NMR spectroscopy, circular dichroism, ESI‐MS, and titration calorimetry are employed to characterize the interactions of NGF with Ub. NGF_1–14, which is a short model peptide encompassing the first 14 N‐terminal residues of NGF, binds the copper‐binding regions of Ub (K_D=8.6 10^?5 m ). Moreover, the peptide undergoes a random coil–polyproline type II helix structural conversion upon binding to Ub. Notably, copper(II) ions inhibit NGF_1–14/Ub interactions. Further experiments performed with the full‐length NGF confirmed the existence of a copper(II)‐dependent association between Ub and NGF and indicated that the N‐terminal domain of NGF was a valuable paradigm that recapitulated many traits of the full‐length protein.     

Exploring the (Very Flat) Potential Energy Landscape of R−Br⋅⋅⋅π Interactions with Accurate CCSD(T) and SAPT Techniques

Halogen bonds involving an aromatic moiety as an acceptor, otherwise known as R?X???π interactions, have increasingly been recognized as being important in materials and in protein–ligand complexes. These types of interactions have been the subject of many recent investigations, but little is known about the ways in which the strengths of R?X???π interactions vary as a function of the relative geometries of the interacting pairs. Here we use the accurate CCSD(T) and SAPT2+3δMP2 methods to investigate the potential energy landscapes for systems of HBr, HCCBr, and NCBr complexed with benzene. It is found that only the separation between the complexed molecules have a strong effect on interaction strength while other geometric parameters, such as tilting and shifting R?Br???π donor relative to the benzene plane, affect these interactions only mildly. Importantly, it is found that the C_6v (T‐shaped) configuration is not the global minimum for any of the dimers investigated.     

DOX: A new computational protocol for accurate prediction of the protein–ligand binding structures

Molecular docking techniques have now been widely used to predict the protein–ligand binding modes, especially when the structures of crystal complexes are not available. Most docking algorithms are able to effectively generate and rank a large number of probable binding poses. However, it is hard for them to accurately evaluate these poses and identify the most accurate binding structure. In this study, we first examined the performance of some docking programs, based on a testing set made of 15 crystal complexes with drug statins for the human 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (HMGR). We found that most of the top ranking HMGR–statin binding poses, predicted by the docking programs, were energetically unstable as revealed by the high theoretical‐level calculations, which were usually accompanied by the large deviations from the geometric parameters of the corresponding crystal binding structures. Subsequently, we proposed a new computational protocol, DOX, based on the joint use of molecular Docking, ONIOM, and eXtended ONIOM (XO) methods to predict the accurate binding structures for the protein–ligand complexes of interest. Our testing results demonstrate that the DOX protocol can efficiently predict accurate geometries for all 15 HMGR‐statin crystal complexes without exception. This study suggests a promising computational route, as an effective alternative to the experimental one, toward predicting the accurate binding structures, which is the prerequisite for all the deep understandings of the properties, functions, and mechanisms of the protein–ligand complexes. © 2015 Wiley Periodicals, Inc.     

Conformationally Programmable Chiral Foldamers with Compact and Extended Domains Controlled by Monomer Structure

Foldamers are an important class of abiotic macromolecules, with potential therapeutic applications in the disruption of protein–protein interactions. The majority adopt a single conformational motif such as a helix. A class of foldamer is now introduced where the choice of heterocycle within each monomer, coupled with a strong conformation‐determining dipole repulsion effect, allows both helical and extended conformations to be selected. Combining these monomers into hetero‐oligomers enables highly controlled exploration of conformational space and projection of side‐chains along multiple vectors. The foldamers were rapidly constructed via an iterative deprotection‐cross‐coupling sequence, and their solid‐ and solution‐phase conformations were analysed by X‐ray crystallography and NMR and CD spectroscopy. These molecules may find applications in protein surface recognition where the interface does not involve canonical peptide secondary structures.     

Do Intrinsically Disordered Proteins Possess High Specificity in Protein–Protein Interactions?

Specific protein–protein interactions are critical to cellular function. Structural flexibility and disorder‐to‐order transitions upon binding enable intrinsically disordered proteins (IDPs) to overcome steric restrictions and form complementary binding interfaces, and thus, IDPs are widely considered to have high specificity and low affinity for molecular recognition. However, flexibility may also enable IDPs to form complementary binding interfaces with misbinding partners, resulting in a great number of nonspecific interactions. Consequently, it is questionable whether IDPs really possess high specificity. In this work, we investigated this question from a thermodynamic viewpoint. We collected mutant thermodynamic data for 35 ordered protein complexes and 43 disordered protein complexes. We found that the enthalpy–entropy compensation for disordered protein complexes was more complete than that for ordered protein complexes. We further simulated the binding processes of ordered and disordered protein complexes under mutations. Simulation data confirmed the observation of experimental data analyses and further revealed that disordered protein complexes possessed smaller changes in binding free energy than ordered protein complexes under the same mutation perturbations. Therefore, interactions of IDPs are more malleable than those of ordered proteins due to their structural flexibility in the complex. Our results provide new clues for exploring the relationship between protein flexibility, adaptability, and specificity.     

Detection and characterization of nonspecific,sparsely populated binding modes in the early stages of complexation

A method is proposed to study protein–ligand binding in a system governed by specific and nonspecific interactions. Strong associations lead to narrow distributions in the proteins configuration space; weak and ultraweak associations lead instead to broader distributions, a manifestation of nonspecific, sparsely populated binding modes with multiple interfaces. The method is based on the notion that a discrete set of preferential first‐encounter modes are metastable states from which stable (prerelaxation) complexes at equilibrium evolve. The method can be used to explore alternative pathways of complexation with statistical significance and can be integrated into a general algorithm to study protein interaction networks. The method is applied to a peptide–protein complex. The peptide adopts several low‐population conformers and binds in a variety of modes with a broad range of affinities. The system is thus well suited to analyze general features of binding, including conformational selection, multiplicity of binding modes, and nonspecific interactions, and to illustrate how the method can be applied to study these problems systematically. The equilibrium distributions can be used to generate biasing functions for simulations of multiprotein systems from which bulk thermodynamic quantities can be calculated. © 2015 Wiley Periodicals, Inc.     

Br...O contacts and π–π stacking dominate the packing in methyl 4‐bromo‐3,5‐dimethoxybenzoate

The title compound, C₁₀H₁₁BrO₄, a useful precursor to pharmaceutically active isocoumarin and isochroman derivatives, crystallizes with two unique molecules in the asymmetric unit. A π–π stacking interaction links the planar molecules in the asymmetric unit. Additional π–π contacts stack pairs of molecules along the c axis. A feature of the crystal packing is the presence of a number of short Br...O contacts. A particularly unusual arrangement involves the formation of dimers, with pairs of Br...O contacts imposing a close Br...Br interaction and generating five‐membered rings within an eight‐membered ring formed by two Br...O contacts. Only two comparable arrangements have been reported previously. The Br...O contacts combine with weak C—H...O hydrogen bonds to form corrugated sheets of molecules approximately parallel to (001). These sheets are stacked along the c axis by π–π interactions to generate a three‐dimensional network.