Developing HIV-1 Protease inhibitors through stereospecific reactions in protein crystals

Protease inhibitors are key components in the chemotherapy of HIV infection. However, the appearance of viral mutants routinely compromises their clinical efficacy, creating a constant need for new and more potent inhibitors. Recently, a new class of epoxide-based inhibitors of HIV-1 protease was investigated and the configuration of the epoxide carbons was demonstrated to play a crucial role in determining the binding affinity. Here we report the comparison between three crystal structures at near-atomic resolution of HIV-1 protease in complex with the epoxide-based inhibitor, revealing an in-situ epoxide ring opening triggered by a pH change in the mother solution of the crystal. Increased pH in the crystal allows a stereospecific nucleophile attack of an ammonia molecule onto an epoxide carbon, with formation of a new inhibitor containing amino-alcohol functions. The described experiments open a pathway for the development of new stereospecific protease inhibitors from a reactive lead compound

Self-assembling tripeptide forming water-bound channels and hydrogels

D-Ser(tBu)-L-Phe-L-Trp is described as a self-assembling tripeptide that yields nanofibrillar hydrogels at physiological conditions (phosphate buffer at pH 7.4). The peptide is characterized by several spectroscopic methods, such as circular dichroism and fluorescence, oscillatory rheometry, and transmission electron microscopy. Single-crystal X-ray diffraction reveals supramolecular packing into water-bound channels and allows the visualization of the intermolecular interactions holding together peptide stacks

Hierarchical Self-assembly and Controlled Disassembly of a Cavitand-based Host-Guest Supramolecular Polymer

There is a considerable interest in dynamic materials featuring modular components with nano-scale dimensions and controlled responsiveness to external stimuli. Supramolecular polymers are a class of materials that fulfill nicely all these conditions. Here, we present a family of host-guest supramolecular polymers that combine the outstanding complexing properties of tetraphosphonate cavitands toward N-methylpyridinium guests with molecular switching. The designed monomer is a cavitand featuring four inward facing P=O groups at the upper rim and a single N-methylpyridinium unit at the lower rim, forming instantaneously a polymeric species in solution thanks to the high complexation constants measured for these host-guest interactions. This system has been analyzed by NMR spectroscopic and electrochemical techniques. In order to interpret the results of diffusion-sensitive experiments, we took advantage of the X-ray crystal structure obtained for the polymeric species and developed an original treatment of the PGSE data by non-linear fitting. The analysis of the experimental data identified an isodesmic polymerization model at monomer concentration below 20 mM, driven by intrachain host-guest interactions, and an additional level of tetrameric bundle aggregation above 20 mM, due to interchain dipolar and quadrupolar interactions. Two orthogonal disassembly procedures have been implemented: electrochemical reduction for the linear chains and solvent-driven dissolution for the bundles

Templating porphyrin anisotropy via magnetically aligned carbon nanotubes

The preparation and characterisation of a novel three‐dimensional organic material consisting of porphyrin arrays on carbon nanotubes embedded in an organogel is reported. Firstly, the porphyrin array was prepared through metal‐ligand coordination of a ditopic ligand (1,2‐bis(4‐pyridyl)ethane) and two bis‐Zn(II) porphyrins, linked through a pyrene core, and was studied through UV‐Vis, NMR and diffusion spectroscopies. Secondly, the porphyrin supramolecular architecture was adsorbed on pristine carbon nanotubes, greatly improving the dispersibility of the latter in organic solvents. The hybrid material was characterised by means of UV‐Vis spectroscopy, microscopic techniques and thermogravimetric analysis. Finally, by exploiting the anisotropic magnetic susceptibility of carbon nanotubes, the hybrid material was aligned under a magnetic field, the organisation of which could be maintained by in situ gelation. The resultant hybrid organogel exhibited notable optical anisotropy, suggesting an anisotropic arrangement of the porphyrin‐CNTs architectures in the macroscopic material

Microscopy and modelling investigations on the morphology of the biofilm exopolysaccharide produced by Burkholderia multivorans strain C1576

: Bacteria form very often biofilms where they embed in a self-synthesized matrix exhibiting a gel-like appearance. Matrices offer several advantages, including defence against external threats and the easiness of intercellular communication. In infections, biofilm formation enhances bacteria resistance against antimicrobials, causing serious clinical problems for patients' treatments. Biofilm matrices are composed of proteins, extracellular DNA, and polysaccharides, the latter being the major responsible for matrix architecture. The repeating unit of the biofilm polysaccharide synthesized by Burkholderia multivorans strain C1576 contains two mannoses and two sequentially linked rhamnoses, one of them 50 % methylated on C-3. Rhamnose, a 6-deoxysugar, has lower polarity than other common monosaccharides and its methylation further reduces polarity. This suggests a possible role of this polysaccharide in the biofilm matrix; in fact, computer modelling and atomic force microscopy studies evidenced intra- and inter-molecular non-polar interactions both within polysaccharides and with aliphatic molecules. In this paper, the polysaccharide three-dimensional morphology was investigated using atomic force microscopy in both solid and solution states. Independent evidence of the polymer conformation was obtained by transmission electron microscopy which confirmed the formation of globular compact structures. Finally, data from computer dynamic simulations were used to model the three-dimensional structure

Structure and Dynamics of AMPA Receptor GluA2 in Resting, Pre-Open, and Desensitized States

SummaryIonotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory signaling in the nervous system. Despite the profound importance of iGluRs to neurotransmission, little is known about the structures and dynamics of intact receptors in distinct functional states. Here, we elucidate the structures of the intact GluA2 AMPA receptor in an apo resting/closed state, in an activated/pre-open state bound with partial agonists and a positive allosteric modulator, and in a desensitized/closed state in complex with fluorowilliardiine. To probe the conformational properties of these states, we carried out double electron-electron resonance experiments on cysteine mutants and cryoelectron microscopy studies. We show how agonist binding modulates the conformation of the ligand-binding domain “layer” of the intact receptors and how, upon desensitization, the receptor undergoes large conformational rearrangements of the amino-terminal and ligand-binding domains. We define mechanistic principles by which to understand antagonism, activation, and desensitization in AMPA iGluRs

Large heterometallic coordination cages with gyrobifastigium-like geometry

Large (Mw > 10 kDa) heterometallic coordination cages with gyrobifastigium-like geometry are obtained by using metalloligands with sterically demanding FeII clathrochelate cores and four divergent pyridyl groups. Upon reaction with cis-blocked PtII and PdII complexes, MII8L4 cages are formed. The gyrobifastigium geometry of these cages is in contrast to the barrel-like structures which are typically observed for metallasupramolecular assemblies with M8L4 stoichiometry

The Intricate Structural Chemistry of MII2nLn-Type Assemblies

The reaction of cis-blocked, square-planar M-II complexes with tetratopic N-donor ligands is known to give metallasupramolecular assemblies of the formula M2nLn. These assemblies typically adopt barrel-like structures, with the ligands paneling the sides of the barrels. However, alternative structures are possible, as demonstrated by the recent discovery of a Pt8L4 cage with unusual gyrobifastigium-like geometry. To date, the factors that govern the assembly of (M2nLn)-L-II complexes are not well understood. Herein, we provide a geometric analysis of M2nLn complexes, and we discuss how size and geometry of the ligand is expected to influence the self-assembly process. The theoretical analysis is complemented by experimental studies using different cis-blocked Pt-II complexes and metalloligands with four divergent pyridyl groups. Mononuclear metalloligands gave mainly assemblies of type Pt8L4, which adopt barrel- or gyrobifastigium-like structures. Larger assemblies can also form, as evidenced by the crystallographic characterization of a Pt10L5 complex and a Pt16L8 complex. The former adopts a pentagonal barrel structure, whereas the latter displays a barrel structure with a distorted square orthobicupola geometry. The Pt16L8 complex has a molecular weight of more than 23 kDa and a diameter of 4.5 nm, making it the largest, structurally characterized M2nLn complex described to date. A dinuclear metalloligand was employed for the targeted synthesis of pentagonal Pt10L5 barrels, which are formed in nearly quantitative yields

Unexpected Gating Behaviour of an Engineered Potassium Channel Kir

In this study, we investigated the dynamics and functional characteristics of the KirBac3.1 S129R, a mutated bacterial potassium channel for which the inner pore-lining helix (TM2) was engineered so that the bundle crossing is trapped in an open conformation. The structure of this channel has been previously determined at high atomic resolution. We explored the dynamical characteristics of this open state channel using an in silico method MDeNM that combines molecular dynamics simulations and normal modes. We captured the global and local motions at the mutation level and compared these data with HDX-MS experiments. MDeNM provided also an estimation of the probability of the different opening states that are in agreement with our electrophysiological experiments. In the S129R mutant, the Arg129 mutation releases the two constriction points in the channel that existed in the wild type but interestingly creates another restriction point

Chirality Effects on Peptide Self-Assembly Unraveled from Molecules to Materials

Self-assembling short peptides are attractive minimal systems for mimicking the constituents of living systems and building (bio)materials. The combination of both D- and L-amino acids into heterochiral sequences is a versatile strategy for building durable supramolecular architectures, especially when their homochiral analogs do not self-assemble. The reasons for this divergent behavior have remained obscure until now. Here, we elucidate how and why homochiral and heterochiral peptides behave differently. We identify a key spectroscopy signature and its corresponding molecular conformation, whereby an amphiphilic structure is uniquely enabled by the peptide stereochemistry. Importantly, we unravel the self-assembly process as a continuum from the conformation of single molecules to their organization into nano- and microstructures and through to macroscopic hydrogels, which are probed for cytotoxicity in fibroblast cell culture. In this way, (bio)material properties at the macro-scale can be linked to the chemical structure of their building blocks at the angstrom scale. Nature makes pervasive use of homochirality (e.g., D-sugars and L-peptides) to assemble biomolecules, whose interactions determine life processes. D-amino acids rarely occur, and their effects are not yet completely understood. For a long time, structural complexity (e.g., polypeptides and constrained molecules) was considered a requirement for achieving defined conformations that ultimately allow biomolecule recognition and function. Here, we detail how minimalist building blocks can adopt conformations with a characteristic spectroscopic signature, whereby substitution of just one L-amino acid for its D mirror image leads to a divergent path for assembly in water. Subtle molecular variations are amplified through increasing size scale all the way to macroscopic differences that are visible to the eye. Ultimately, the design of heterochiral (bio)molecules thus provides an alternative approach to shed new light on the supramolecular interactions that define life as we know it. This work explains why and how heterochiral and homochiral tripeptides differ in their assembly in water. A characteristic spectroscopic signature is assigned to molecular conformation. We monitor the process as a continuum from the molecular scale to the macroscopic biomaterials so that the final properties are linked to chemical structure of the building blocks. This work lays the foundation for the design of supramolecular hydrogel biomaterials based on short sequences of hydrophobic D- and L-amino acids