Homology-Modelling Protein-Ligand Interactions: Allowing for Ligand-Induced Conformational Change

Current homology-modelling methods do not consider small molecules in their automated processes. Therefore, the development of a reliable tool for protein-ligand homology modelling is an important next step in generating plausible models for molecular interactions. Two automated protein-ligand homology-modelling strategies, requiring no expert knowledge from the user, are investigated here. Both employ the “induced fit” concept with flexibility in side chains and ligand. The most successful strategy superimposes the new ligand over the original ligand before homology modelling, allowing the new ligand to be taken into consideration during protein modelling (rather than after), facilitating conformational change in the local backbone if necessary. We show that this approach results in successful modelling of the ligand and key binding-site residues of angiotensin-converting enzyme 2 (ACE2) from its homologue ACE, which is not possible via conventional homology modelling or by homology modelling followed by docking. Several other difficult target complexes are also successfully modelled, reproducing native protein-ligand contacts with significantly different biological substrates and different binding-site conformations. These include the modelling of Cdk5 (cyclin-dependent kinase 5) from Cdk2, thymidine phosphorylase from a bacterial homologue, and dihydrofolate reductase from a recombinant variant with a markedly different inhibitor. In terms of average modelling quality across 82 targets, the ligand RMSD with respect to the experimental structure is 1.4 Å (and 2.0 Å for the protein binding site) for “easy” cases and 2.9 Å for the ligand (and 2.7 Å for the protein binding site) in “hard” cases. This demonstrates the importance of selecting an optimal template. Ligand-modelling accuracy is strongly dependent on target-template ligand structural similarity, rather than target-template sequence identity. However, protein-modelling accuracy is dependent on both. Our automated protein-ligand homology-modelling strategy generates a higher degree of accuracy than homology modelling followed by docking, generating an average ligand RMSD that is 1-2 Å better than docking with homology models.     

The Effects of Phosphomimetic Lid Mutation on the Thermostability of the N-terminal Domain of MDM2

The multidomain E3 ubiquitin ligase MDM2 catalyzes p53 ubiquitination by a “dual-site” docking mechanism whereby MDM2 binding to at least two distinct peptide motifs on p53 promotes ubiquitination. One protein-protein interaction occurs between the N-terminal hydrophobic pocket of MDM2 and the transactivation motif of p53, and the second interaction occurs between the acidic domain of MDM2 and a motif in the DNA-binding domain of p53. A flexible N-terminal pseudo-substrate or “lid” adjacent to the N-terminal hydrophobic pocket of MDM2 has a phosphorylation site, and there are distinct models proposed on how the phosphorylated lid could affect MDM2 function. Biochemical studies have predicted that phosphomimetic mutation will stabilize the lid on the surface of MDM2 and will “open” the hydrophobic pocket and stabilize the MDM2-p53 complex, while NMR studies proposed that phosphomimetic mutation “closes” the lid over the MDM2 pocket and inhibits MDM2-p53 complex formation. To resolve these discrepancies, we utilized a quantitative fluorescence-based dye binding assay to measure the thermal unfolding of wild-type (wt), ΔLid, and S17D N-terminal domains of MDM2 as a function of increasing ligand concentration. Our data reveal that S17D lid mutation increases, rather than decreases, the thermostability of the N-terminal domain of MDM2 in the absence or in the presence of ligand. ΔLid mutation, by contrast, increases MDM2 thermoinstability. This is consistent with biochemical data, using full-length MDM2, showing that the S17D mutation stabilizes the MDM2-p53 complex and increases the specific activity of the E3 ubiquitin ligase function of MDM2. These data indicate that phosphomimetic lid mutation results in an “opening,” rather than a “closing,” of the pocket of MDM2 and highlight the ability of small intrinsically disordered or unstructured peptide motifs to regulate the specific activity of a protein.     

Cytoplasmic oxysterol-binding proteins: sterol sensors or transporters?

Families of oxysterol-binding protein (ORP) homologues are present in eukaryotes from yeast to man. Their hallmark feature is a characteristic ligand binding domain that, for several family members, has been shown to accommodate different oxysterols and/or cholesterol. ORPs of the “long” subtype contain targeting determinants for the endoplasmic reticulum and to other organelle membranes, the most prominent of which are phosphoinositide-binding pleckstrin homology domains, while “short” ORPs comprise a ligand binding domain with little additional sequences. There is increasing evidence that both long and short ORPs can be enriched at membrane contact sites, junctions of the endoplasmic reticulum with other organelles, where they are suggested to execute regulatory or sterol transfer functions. In this review we discuss the current evidence for putative roles of ORPs as sterol sensors or transporters.     

Parameterization of an effective potential for protein–ligand binding from host–guest affinity data

Force field accuracy is still one of the “stalemates” in biomolecular modeling. Model systems with high quality experimental data are valuable instruments for the validation and improvement of effective potentials. With respect to protein–ligand binding, organic host–guest complexes have long served as models for both experimental and computational studies because of the abundance of binding affinity data available for such systems. Binding affinity data collected for cyclodextrin (CD) inclusion complexes, a popular model for molecular recognition, is potentially a more reliable resource for tuning energy parameters than hydration free energy measurements. Convergence of binding free energy calculations on CD host–guest systems can also be obtained rapidly, thus offering the opportunity to assess the robustness of these parameters. In this work, we demonstrate how implicit solvent parameters can be developed using binding affinity experimental data and the binding energy distribution analysis method (BEDAM) and validated using the Grid Inhomogeneous Solvation Theory analysis. These new solvation parameters were used to study protein–ligand binding in two drug targets against the HIV‐1 virus and improved the agreement between the calculated and the experimental binding affinities. This work illustrates how benchmark sets of high quality experimental binding affinity data and physics‐based binding free energy models can be used to evaluate and optimize force fields for protein–ligand systems. Copyright © 2015 John Wiley & Sons, Ltd.     

Le Magdalénien de la grotte « Grappin » à Arlay (Jura, France) : nouveaux regards

The “Grappin” or “Saint-Vincent's” cave, on the western slope of Jura, East of France, has been explored since 1889. From then to 1960, it has yielded substantial material dated to middle “à navettes” Magdalenian developing during the late Pleniglacial. The study of this settlement, although often mentioned, was never dealt with comprehensively until now. Due to its scientific importance for the middle Magdalenian of western Europe, the site is to be reinvestigated through a global interdisciplinary project entitled “The Tardiglacial and the start of Holocene in the Jura and its margins”. This paper will review our present knowledge of the site, radiocarbon dates and archaeological data. It also focuses on ornaments and engraved mobile art.     

Le genre Nucleolites (Echinoidea, Cassiduloidea) du Bajocien à l'Oxfordien dans le Bassin de Paris : apport des données architecturales à la systématique et à la phylogénie

The classification of the Paris basin Bajocian-Oxfordian species of the genus Nucleolites is revised. A classic point of view, until now used by authors and mainly based on the general morphology of the test, is first developed. Then, this last is modified with new architectural data, which refer to the extraxial-axial theory concerning the structure of the apical system as well as the presence of supplementary and catenal plates. These architectural data are first used facing the general morphology of the test. Such approach leads to a new point of view for the classification of the species of the genus Nucleolites. The systematics is particularly significantly simplified. The deduced and proposed phylogenetic hypothesis shows that the genus Nucleolites is composed of two parallel lineages as soon as the beginning of the genus in the Bajocian: a group with a so-called “primitive” architecture, and a group with an “advanced” one. On and after the Late Callovian, the species with a primitive organisation give birth to advanced species, which continue in younger time. In this way, the genus Nucleolites may be paraphyletic since the Late Callovian onward.     

Modeling bacterial immune systems: Strategies for expression of toxic – but useful – molecules

Protection of bacterial cells against virus infection requires expression of molecules that are able to destroy the incoming foreign DNA. However, these molecules can also be toxic for the host cell. In both restriction–modification (R–M), and the recently discovered CRISPR/Cas systems, the toxicity is (in part) avoided through rapid transition of the expression of the toxic molecules from “OFF” to “ON” state. In restriction–modification systems the rapid transition is achieved through a large binding cooperativity, and low translation rate of the control protein. On the other hand, CRISPR array expression in CRISPR/Cas systems involves a mechanism where a small decrease of unprocessed RNAs leads to a rapid increase of processed small RNAs. Surprisingly, this rapid amplification crucially depends on fast non-specific degradation of the unprocessed molecules by an unidentified nuclease, rather than on large cooperativity in protein binding. Furthermore, the major control elements that are responsible for fast transition of R–M and CRISPR/Cas systems from “OFF” to “ON” state, are also directly involved in increased stability of the steady states of these systems. We here discuss mechanisms that allow rapid transition of toxic molecules from the unproductive to the productive state in R–M and CRISPR/Cas systems. The main purpose of this discussion is to put relevant theoretical and experimental work in a perspective that points to general similarities in otherwise mechanistically very different bacterial immune systems.     

Probing the metal ion selectivity in methionine aminopeptidase via changes in the luminescence properties of the enzyme bound europium ion

We report herein, for the first time, that Europium ion (Eu³⁺) binds to the “apo” form of Escherichia coli methionine aminopeptidase (EcMetAP), and such binding results in the activation of the enzyme as well as enhancement in the luminescence intensity of the metal ion. Due to competitive displacement of the enzyme-bound Eu³⁺ by different metal ions, we could determine the binding affinities of both “activating” and “non-activating” metal ions for the enzyme via fluorescence spectroscopy. The experimental data revealed that among all metal ions, Fe²⁺ exhibited the highest binding affinity for the enzyme, supporting the notion that it serves as the physiological metal ion for the enzyme. However, the enzyme-metal binding data did not adhere to the Irving-William series. On accounting for the binding affinity vis a vis the catalytic efficiency of the enzyme for different metal ions, it appears evident that that the “coordination states” and the relative softness” of metal ions are the major determinants in facilitating the EcMetAP catalyzed reaction.     

Proposition méthodologique pour une étude des esthétiques du Mésolithique : une analyse sociologique-anthropologique de la fin du Tardiglaciaire et durant le Postglaciaire en Europe occidentale

Do the decorated productions cover esthetics, i.e. the whole of the symbolic systems of a culture of hunters-gatherers? How to leave this glance with the aesthetic requirements that we other Westerners, pose on all things… and to exceed the debate of the artistic expressions thought from the aesthetic point of view, like “works of art” objects and “stylistics” objects? It is out of question “to make a fetish” of the productions decorated “works of art” while placing them at the starting point with our reflexion, (Studying them from a functional, technological, typological, art for art, meaningful point of view…) but to associate another of dimensions of the aesthetic experiment, i.e., the context of the decorated productions.     

Baroreflex variability and “resetting”: A new perspective

A new framework is proposed for the interpretation of spontaneous cardiac baroreflex sensitivity data and the general concept of baroreflex resetting. The framework is used to explore baroreflex function along two separate lines of inquiry: one following a direct intervention in baroreflex function in individual subjects, another in a group of subjects where baroreflex function may have been compromised by coronary artery disease or aging. It is found that under baseline conditions the baroreflex is in a “free-floating” state in which the gain or “sensitivity” is highly variable, while under orthostatic stress or in the absence of or reduced vagal input the gain is more tightly controlled with an expected decline in sensitivity but a very large decline in the variability of that sensitivity. It is concluded that baroreflex “resetting” is better viewed not simply as a change in baroreflex sensitivity but rather as a change in the “focus” or “attention” of the baroreflex as expressed by an observed decline in the variability of the measured gain. The results do not support the interpretation of baroreflex “resetting” as a departure from or return to a universal “set point” as in homeostasis or open loop models.     

Figurations de l’amas stellaire des Pléiades sur deux roches gravées de la région du Mont Bego Z IX. GII. R 4 et Z IX. GIII. R 6

Two engraved rocks are situated 140 m from each other at an altitude of about 2250 m. One of them is called “female dancer”, and the other, “the Pleiads”, and both show a group of seven small pitted areas, of which one is smaller than the others and ‘ identified as the “Pleiads stellar cluster”, often depicted or mentioned during Antiquity and also throughout various historical periods.On each of these rocks, a large halberd, whose handle is oriented east-west, was traced along a natural fissure in the rock and engraved in the center of the composition. The “Pleiads stellar cluster” is represented on each of the two rocks beside the halberd : although situated to the west above the halberd blade on the “female dancer” rock, it is placed to the south and to the left of the halberd handle on “the Pleiads” rock. This difference may mark two distinct calendar data.The position of the different figures in the compositions illustrated on these two rocks, in particular inversions in the representation of some of them may indicate two distinct periods of the agricultural yearly cycle: on the rock called “female dancer”, the “heliacal raise” at summer's end, marking the beginning of the harvest and, on the rock called “the Pleiads”, the “heliacal setting”, just before winter, after the end of the harvest, when the rains soaks the ground and it is time to plow.     

Mise en place d’une équipe d’experts en physique médicale au Luxembourg : organisation, résultats qualité et radioprotection en médecine nucléaire (2002–2011)

Implementations of the statutory UE requirements concerning ionizing radiation in the medical domain (96/29/Euratom, 13th May 1996; 97/43/Euratom, 30th June 1997) have changed quality control in medical imaging departments. In Luxembourg, the Ministry of Health and the “Union of Luxembourg Hospitals” have thus created a “Medical Physics Cell” (five Medical Physics experts). It is in charge of implementing a standardized program of Quality Assurance (QA) and radiation protection relative to patients as well as staffs, on a coordinated national basis, in all five in-hospital nuclear medicine departments of the country. The program distributes QA controls between three levels, various periodicities and degrees of expertise. It sensitizes staff and medical doctors by trainings and facilitates ISO accreditation. From 2002 to 2011, in all five in-hospital departments of nuclear medicine of a small country, image quality, radiation protection and reference values of each medical equipment were defined and implemented in a standard way. In 2011, the medical physics cell, the driving force leading to the institutionalization of the discipline within the nuclear medicine departments, is considered as a partner rather than as a control body by the teams in charge of the nuclear medicine departments.     

Role of the AP2 β-Appendage Hub in Recruiting Partners for Clathrin-Coated Vesicle Assembly

Adaptor protein complex 2 α and β-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of β-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the β-appendage (the “top” and “side” sites) that bind motifs distinct from those previously identified on the α-appendage. We solved the structure of the β-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor β-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the β-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability (“matricity”). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as β-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.     

The sense of water in the blowfly Protophormia terraenovae

The gustatory system of the blowfly, Protophormia terraenovae, is a relatively simple biological model for studies on chemosensory input and behavioral output. It appears to have renewed interest as a model for studies on the role of water channels, namely aquaporins or aquaglyceroporins, in water detection. To this end, we investigated the presence of water channels, their role in “water” and “salt” cell responsiveness and the transduction mechanism involved. For the first time our electrophysiological results point to the presence of an aquaglyceroporin in the chemoreceptor membrane of the “water” cell in the blowfly taste chemosensilla whose transduction mechanism ultimately involves an intracellular calcium increase and consequently cell depolarization. This hypothesis is also supported by calcium imaging data following proper stimulation. This mechanism is triggered by “water” cell stimulation with hypotonic solutions and/or solutes such as glycerol which crosses the membrane by way of aquaglyceroporins. Behavioral output indicates that the “sense” of water in blowflies is definitely not dependent on the “water” cell only, but also on the “salt” cell sensitivity. These findings also hypothesize a new role for aquaglyceroporin in spiking cell excitability.     

La grotte de Santa Ana (Cáceres, Espagne) et l'évolution technologique au Pléistocène dans la Péninsule ibérique

The excavations carried out in the cave of Santa Ana (Cáceres, Spain), cave of the karstic network of the “Calerizo” of Cáceres, enabled us to know the existence, in stratigraphy, of the three lito-techniques modes which characterize the industrial development of lower and middle Pleistocene in the Iberian Peninsula. On standby of new research, the results obtained until now permit us two work out a diachronic assumption of technical evolution. In the Iberian Peninsula, there are only two karstic systems where we can fallow this technological development; one is the “Sierra de Atapuerca” (Burgos) and the other is the “Calerizo de Cáceres” (Cáceres).     

Configural processing of different topologically structured figures: an ERP Study

According to Chen’s theory, topological differences are perceived faster than feature differences in early visual perception. We hypothesized that topological perception is caused by the sensitivity in discriminating figures with and without “holes”. An ERP experiment was conducted utilizing a passive paradigm to investigate the differences in perceiving figures with “hole” and with “no-hole”. The results showed differences in N170 components between figures with “holes” and with “no-holes”. The inversion of the “hole” could influence the latency of N170, but the inversion of the “no-hole” could not, which indicated that global features are processed first in the “hole” perception whilst local features are given priority to the “no-hole” perception. This result was similar to studies concerning face and non-face objects, suggesting a configural processing of the “hole”.     

Understanding the influence of the protein environment on the Mn(II) centers in Superoxide Dismutases using High-Field Electron Paramagnetic Resonance

One of the most puzzling questions of manganese and iron superoxide dismutases (SODs) is what is the basis for their metal-specificity. This review summarizes our findings on the Mn(II) electronic structure of SODs and related synthetic models using high-field high-frequency electron paramagnetic resonance (HFEPR), a technique that is able to achieve a very detailed and quantitative information about the electronic structure of the Mn(II) ions. We have used HFEPR to compare eight different SODs, including iron, manganese and cambialistic proteins. This comparative approach has shown that in spite of their high structural homology each of these groups have specific spectroscopic and biochemical characteristics. This has allowed us to develop a model about how protein and metal interactions influence protein pK, inhibitor binding and the electronic structure of the manganese center. To better appreciate the thermodynamic prerequisites required for metal discriminatory SOD activity and their relationship to HFEPR spectroscopy, we review the work on synthetic model systems that functionally mimic Mn-and FeSOD. Using a single ligand framework, it was possible to obtain metal-discriminatory “activity” as well as variations in the HFEPR spectra that parallel those found in the proteins. Our results give new insights into protein-metal interactions from the perspective of the Mn(II) and new steps towards solving the puzzle of metal-specificity in SODs.