Detection and Quantitative Analysis of Two Independent Binding Modes of a Small Ligand Responsible for DC-SIGN Clustering

DC-SIGN (dendritic cell-specific ICAM-3 grabbing non-integrin) is a C-type lectin receptor (CLRs) present, mainly in dendritic cells (DCs), as one of the major pattern recognition receptors (PRRs). This receptor has a relevant role in viral infection processes. Recent approaches aiming to block DC-SIGN have been presented as attractive anti-HIV strategies. DC-SIGN binds mannose or fucose-containing carbohydrates from viral proteins such as the HIV envelope glycoprotein gp120. We have previously demonstrated that multivalent dendrons bearing multiple copies of glycomimetic ligands were able to inhibit DC-SIGN-dependent HIV infection in cervical explant models. Optimization of glycomimetic ligands requires detailed characterization and analysis of their binding modes because they notably influence binding affinities. In a previous study we characterized the binding mode of DC-SIGN with ligand 1, which shows a single binding mode as demonstrated by NMR and X-ray crystallography. In this work we report the binding studies of DC-SIGN with pseudotrisaccharide 2, which has a larger affinity. Their binding was analysed by TR-NOESY and STD NMR experiments, combined with the CORCEMA-ST protocol and molecular modelling. These studies demonstrate that in solution the complex cannot be explained by a single binding mode. We describe the ensemble of ligand bound modes that best fit the experimental data and explain the higher inhibition values found for ligand

The Chromodomain of LIKE HETEROCHROMATIN PROTEIN 1 Is Essential for H3K27me3 Binding and Function during Arabidopsis Development

Polycomb group (PcG) proteins are essential to maintain gene expression patterns during development. Transcriptional repression by PcG proteins involves trimethylation of H3K27 (H3K27me3) by Polycomb Repressive Complex 2 (PRC2) in animals and plants. PRC1 binds to H3K27me3 and is required for transcriptional repression in animals, but in plants PRC1-like activities have remained elusive. One candidate protein that could be involved in PRC1-like functions in plants is LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), because LHP1 associates with genes marked by H3K27me3 in vivo and has a chromodomain that binds H3K27me3 in vitro. Here, we show that disruption of the chromodomain of Arabidopsis thaliana LHP1 abolishes H3K27me3 recognition, releases gene silencing and causes similar phenotypic alterations as transcriptional lhp1 null mutants. Therefore, binding to H3K27me3 is essential for LHP1 protein function

Structure-based methods for virtual screening, protein design, and protein function studies

The average time a drug requires to successfully appear into the market is around 15-20 years. Structure-based techniques play a determinant role in many moments of this long process and computer-based approaches are becoming always more popular, for they are cheap and fast. Computer-based approaches to structure-based drug discovery are also very common in Academia, both in the field of development and application. Moreover, most of the software currently used in this field is being developed in Academia. In this doctoral thesis two main topics are covered: virtual screening by the means of docking and the application of molecular dynamics simulations to macromolecular systems of interest. Two projects employed virtual screening: the search for inhibitors of cathepsin B and the search for allosteric effectors of cathepsin K. The two projects are ideally linked because in both the active site of the enzyme was not targeted. In the cathepsin B project, the so called occluding loop was chosen to show that inhibition of this enzyme could be obtained without targeting the active site. In the cathepsin K project, a putative allosteric pocket was targeted. The pocket was found by a method that considers the residue conservation in evolutionarily linked protein families and then confirmed by two independent structure-based methods. Two other projects involved molecular dynamics simulations of biological macromolecules: designed armadillo repeat proteins and the CRY1 protein of Arabidopsis. In both projects, the evaluation of local and conformational flexibility played a major role. The first one employed static homology modelling and molecular dynamics simulations not only to generate a putative structure of an armadillo repeat protein, but also for improving its dynamical behavior, which could not have been inferred from a static structure only. The second project helped to explain the effect of a single-point mutation of the CRY1 protein of Arabidopsis thaliana. Molecular dynamics simulations of the two systems were run and the difference of activity between them was explained in terms of different flexibility of the protein surface close to the mutation. Die durchschnittliche Zeit, die ein Medikament braucht, um erfolgreich auf den Markt zu kommen, beträgt 15-20 Jahre. Struktur-basierte Methoden spielen eine entscheidende Rolle an vielen Stellen dieses langen Prozesses und computer-basierte Ansätze werden immer beliebter, da sie kostengünstig und schnell sind. Computer-basierte Ansätze für die struktur-basierte Identifikation neuer Medikamente werden sehr häufig in der Wissenschaft verwendet, sowohl auf dem Gebiet der Entwicklung als auch für Anwendungen. Darüber hinaus wird ein Grossteil der aktuellen Software dieses Gebiets in der Wissenschaft entwickelt. In dieser Doktorarbeit werden zwei Hauptthemen behandelt: virtuelles Screening durch Docken und die Anwendung von Moleküldynamik-Simulationen auf makromolekulare Systeme. Bei zwei Projekten wurde virtuelles Screening verwendet: die Suche nach Inhibitoren von cathepsin B und die Suche nach allosterischen Effektoren von cathepsin K. Die zwei Projekte sind ideal miteinander verbunden, da beide nicht auf das aktive Zentrum der Enzyme abzielten. Im cathepsin B Projekt wurde die sogenannte “occluding loop” gewählt um zu zeigen, dass das Enzym inhibiert werden kann, ohne das aktive Zentrum des Enzyms anzugreifen. Im cathepsin K Projekt wurde eine mutmassliche allosterische Bindungsstelle ausgewählt. Die Bindungstasche wurde mit Hilfe einer Methode gefunden, die den Erhalt der Aminosäuresequenz bei evolutionär verbundenen Proteinfamilien berücksichtigt, und anschliessend durch zwei unabhängige struktur-basierte Verfahren bestätigt. Zwei weitere Projekte beinhalten Moleküldynamik-Simulationen von biologischen Makromolekülen: entworfene Armadillo Repeat-Proteine und das CRY1 Protein der Arabidopsis. In beiden Projekten spielte die Evaluation der lokalen und konformationellen Flexibilität eine zentrale Rolle. Beim ersten wurden statisches Homologie-Modelling und Moleküldynamik-Simulationen verwendet, um nicht nur eine mutmassliche Struktur eines Armadillo Wiederholungsproteins zu generieren, sondern auch sein dynamisches Verhalten zu verbessern, das allein aus einer statischen Struktur nicht hätte abgeleitet werden können. Das zweite Projekt diente der Erklärung der Auswirkung einer Einzelmutation des CRY1 Proteins von Arabidopsis thaliana. Moleküldynamik-Simulationen beider Systeme (Wildtype und Mutierter) wurden durchgeführt und der Unterschied ihren Aktivitäten mit einer unterschiedlichen Flexibilität der Proteinober#äche in örtlicher Nähe zur Mutation erklärt

A Gain-of-Function Mutation of Arabidopsis CRYPTOCHROME1 Promotes Flowering1[W][OA]

Plants use different classes of photoreceptors to collect information about their light environment. Cryptochromes are blue light photoreceptors that control deetiolation, entrain the circadian clock, and are involved in flowering time control. Here, we describe the cry1-L407F allele of Arabidopsis (Arabidopsis thaliana), which encodes a hypersensitive cryptochrome1 (cry1) protein. Plants carrying the cry1-L407F point mutation have elevated expression of CONSTANS and FLOWERING LOCUS T under short-day conditions, leading to very early flowering. These results demonstrate that not only the well-studied cry2, with an unequivocal role in flowering promotion, but also cry1 can function as an activator of the floral transition. The cry1-L407F mutants are also hypersensitive toward blue, red, and far-red light in hypocotyl growth inhibition. In addition, cry1-L407F seeds are hypersensitive to germination-inducing red light pulses, but the far-red reversibility of this response is not compromised. This demonstrates that the cry1-L407F photoreceptor can increase the sensitivity of phytochrome signaling cascades. Molecular dynamics simulation of wild-type and mutant cry1 proteins indicated that the L407F mutation considerably reduces the structural flexibility of two solvent-exposed regions of the protein, suggesting that the hypersensitivity might result from a reduced entropic penalty of binding events during downstream signal transduction. Other nonmutually exclusive potential reasons for the cry1-L407F gain of function are the location of phenylalanine-407 close to three conserved tryptophans, which could change cry1’s photochemical properties, and stabilization of ATP binding, which could extend the lifetime of the signaling state of cry1

Aspergillus Galactomannan Enzyme-Linked Immunosorbent Assay Cross-Reactivity Caused by Invasive Geotrichum capitatum

We report three cases of invasive Geotrichum capitatum infection in patients with acute leukemia for which an enzyme-linked immunosorbent assay (ELISA) for Aspergillus galactomannan was positive, with no evidence of aspergillosis. Supernatants obtained from suspensions of 17 G. capitatum strains gave positive reactions with the Aspergillus galactomannan ELISA. These clinical and laboratory data seem to suggest that G. capitatum produces a soluble antigen that is cross-reactive with Aspergillus galactomannan

A double-headed cathepsin B inhibitor devoid of warhead

Most synthetic inhibitors of peptidases have been targeted to the active site for inhibiting catalysis through reversible competition with the substrate or by covalent modification of catalytic groups. Cathepsin B is unique among the cysteine peptidase for the presence of a flexible segment, known as the occluding loop, which can block the primed subsites of the substrate binding cleft. With the occluding loop in the open conformation cathepsin B acts as an endopeptidase, and it acts as an exopeptidase when the loop is closed. We have targeted the occluding loop of human cathepsin B at its surface, outside the catalytic center, using a high-throughput docking procedure. The aim was to identify inhibitors that would interact with the occluding loop thereby modulating enzyme activity without the help of chemical warheads against catalytic residues. From a large library of compounds, the in silico approach identified [2-[2-(2,4-dioxo-1,3-thiazolidin-3-yl)ethylamino]-2-oxoethyl] 2-(furan-2-carbonylamino) acetate, which fulfills the working hypothesis. This molecule possesses two distinct binding moieties and behaves as a reversible, double-headed competitive inhibitor of cathepsin B by excluding synthetic and protein substrates from the active center. The kinetic mechanism of inhibition suggests that the occluding loop is stabilized in its closed conformation, mainly by hydrogen bonds with the inhibitor, thus decreasing endoproteolytic activity of the enzyme. Furthermore, the dioxothiazolidine head of the compound sterically hinders binding of the C-terminal residue of substrates resulting in inhibition of the exopeptidase activity of cathepsin B in a physiopathologically relevant pH range