FraC nanopores with adjustable diameter identify the mass of opposite-charge peptides with 44 dalton resolution

A high throughput single-molecule method for identifying peptides and sequencing proteins based on nanopores could reduce costs and increase speeds of sequencing, allow the fabrication of portable home-diagnostic devices, and permit the characterization of low abundance proteins and heterogeneity in post-translational modifications. Here we engineer the size of Fragaceatoxin C (FraC) biological nanopore to allow the analysis of a wide range of peptide lengths. Ionic blockades through engineered nanopores distinguish a variety of peptides, including two peptides differing only by the substitution of alanine with glutamate. We also find that at pH 3.8 the depth of the peptide current blockades scales with the mass of the peptides irrespectively of the chemical composition of the analyte. Hence, this work shows that FraC nanopores allow direct readout of the mass of single peptide in solution, which is a crucial step towards the developing of a real-time and single-molecule protein sequencing device

Virus Evolution Reveals an Exclusive Role for LEDGF/p75 in Chromosomal Tethering of HIV

Retroviruses by definition insert their viral genome into the host cell chromosome. Although the key player of retroviral integration is viral integrase, a role for cellular cofactors has been proposed. Lentiviral integrases use the cellular protein LEDGF/p75 to tether the preintegration complex to the chromosome, although the existence of alternative host proteins substituting for the function of LEDGF/p75 in integration has been proposed. Truncation mutants of LEDGF/p75 lacking the chromosome attachment site strongly inhibit HIV replication by competition for the interaction with integrase. In an attempt to select HIV strains that can overcome the inhibition, we now have used T-cell lines that stably express a C-terminal fragment of LEDGF/p75. Despite resistance development, the affinity of integrase for LEDGF/p75 is reduced and replication kinetics in human primary T cells is impaired. Detection of the integrase mutations A128T and E170G at key positions in the LEDGF/p75–integrase interface provides in vivo evidence for previously reported crystallographic data. Moreover, the complementary inhibition by LEDGF/p75 knockdown and mutagenesis at the integrase–LEDGF/p75 interface points to the incapability of HIV to circumvent LEDGF/p75 function during proviral integration. Altogether, the data provide a striking example of the power of viral molecular evolution. The results underline the importance of the LEDGF/p75 HIV-1 interplay as target for innovative antiviral therapy. Moreover, the role of LEDGF/p75 in targeting integration will stimulate research on strategies to direct gene therapy vectors into safe landing sites

Computational design of symmetrical eight-bladed beta-propeller proteins

β-Propeller proteins form one of the largest families of protein structures, with a pseudo-symmetrical fold made up of subdomains called blades. They are not only abundant but are also involved in a wide variety of cellular processes, often by acting as a platform for the assembly of protein complexes. WD40 proteins are a subfamily of propeller proteins with no intrinsic enzymatic activity, but their stable, modular architecture and versatile surface have allowed evolution to adapt them to many vital roles. By computationally reverse-engineering the duplication, fusion and diversification events in the evolutionary history of a WD40 protein, a perfectly symmetrical homologue called Tako8 was made. If two or four blades of Tako8 are expressed as single polypeptides, they do not self-assemble to complete the eight-bladed architecture, which may be owing to the closely spaced negative charges inside the ring. A different computational approach was employed to redesign Tako8 to create Ika8, a fourfold-symmetrical protein in which neighbouring blades carry compensating charges. Ika2 and Ika4, carrying two or four blades per subunit, respectively, were found to assemble spontaneously into a complete eight-bladed ring in solution. These artificial eight-bladed rings may find applications in bionanotechnology and as models to study the folding and evolution of WD40 proteins

The transcriptional co-activator LEDGF/p75 displays a dynamic scan-and-lock mechanism for chromatin tethering

Nearly all cellular and disease related functions of the transcriptional co-activator lens epithelium-derived growth factor (LEDGF/p75) involve tethering of interaction partners to chromatin via its conserved integrase binding domain (IBD), but little is known about the mechanism of in vivo chromatin binding and tethering. In this work we studied LEDGF/p75 in real-time in living HeLa cells combining different quantitative fluorescence techniques: spot fluorescence recovery after photobleaching (sFRAP) and half-nucleus fluorescence recovery after photobleaching (hnFRAP), continuous photobleaching, fluorescence correlation spectroscopy (FCS) and an improved FCS method to study diffusion dependence of chromatin binding, tunable focus FCS. LEDGF/p75 moves about in nuclei of living cells in a chromatin hopping/scanning mode typical for transcription factors. The PWWP domain of LEDGF/p75 is necessary, but not sufficient for in vivo chromatin binding. After interaction with HIV-1 integrase via its IBD, a general protein–protein interaction motif, kinetics of LEDGF/p75 shift to 75-fold larger affinity for chromatin. The PWWP is crucial for locking the complex on chromatin. We propose a scan-and-lock model for LEDGF/p75, unifying paradoxical notions of transcriptional co-activation and lentiviral integration targeting

Development of small molecule protein-protein interaction inhibitors targeting the HIV-1 integrase - LEDGF/p75 complex.

AIDS, veroorzaakt door HIV, is een wereldwijd verspreide ziekte. Tot op de dag van vandaag is er geen geneesmiddel voor handen, maar met de comb inatie van medicijnen die de replicatie van het virus verhinderen (de zo genaamde HIV-remmers) en medicatie die de opportunistische infecties beh andelen kan de ziekte vertraagd worden. Het virus kan echter door mutati es resistent worden tegen de HIV-remmende geneesmiddelen. Daarom is de o ntwikkeling van nieuwe strategieen om het virus af te remmen noodzakelij k. Een van de nieuwe mogelijke strategieeen is de inhibitie van het integra tieproces. Tijdens deze stap van de replicatiecyclus van het virus wordt het HIV-genoom geintegreerd in het humaan genoom. Deze complexe stap wo rdt uitgevoerd door het integrase enzym van het virus via verschillende stappen, die elk een potentieel farmaceutisch doelwit vormen. LEDGF/p75 werd ontdekt als een cellulaire cofactor van het integrase enzym en na v alidatie bleek dat de interactie tussen LEDGF/p75 en integrase essentiee l is voor de integratiestap in de virale replicatiecyclus. Inhibitie van de interactie van het integrase met LEDGF/p75 is dus een nieuwe interes sante manier om HIV replicatie te remmen. Dit type van inhibitie, het ve rhinderen van een proteine-proteine-interactie, werd klassiek als onmoge lijk beschouwd maar recente successen bewijzen het tegendeel. Daarom wer d een onderzoek gestart naar inhibitoren van de HIV-1 integrase - LEDGF/ p75 interactie met behulp van computationele methodes. Hiervoor werd eer st een inleidend onderzoek gedaan gebruikmakende van de reeds gekende re mmers van proteine-proteine-interacties. In dit onderzoek werd aangetoond dat de 'regel-van-5' (ook wel gekend al s de Lipinski regels) niet van toepassing is op deze remmers. Een set va n verschillende 2-dimensionele descriptoren werd getest en de populatie van de proteine-proteine -interactie-inhibitoren werd vergeleken met dez e van andere kleine molecules. Analyse van deze resultaten leverde ons d rie descriptoren en hun grenswaarden om kleine molecules te selecteren o p een mogelijke remmende capaciteit van proteine-proteine-interacties. In een tweede luik van het computationele onderzoek toonden we aan dat k leine molecules die competitief een proteine-proteine-interactie verbrek en het proteine-proteine grensoppervlak nabootsen en dus dat wanneer de juiste aminozuren uit een proteine-proteine interface geselecteerd worde n deze gebruikt kunnen worden voor het opstellen van een farmacofoor mod el om zo een remmer voor deze proteine-proteine-interactie te vinden doo r middel van een virtuele zoektocht naar inhibitoren. Na de analyse van de bestaande proteine-proteine-interactieremmers werde n de verkregen inzichten toegepast op de HIV-1 integrase - LEDGF/p75 int eractie. Er werd een eerste virtuele zoektocht opgestart waarin de remme rs eerst werden gelterd op proteineproteine-interactieremmende capacite it. Vervolgens werden de geselecteerde molecules getest met een farmacof oor model dat gebaseerd was op verschillende kristalstructuren waarvan b elangrijkste structuur deze is van het integrase CCD gebonden met het IB D van LEDGF/p75. Verdere selectie van de molecules gebeurde met behulp v an moleculaire docking. De inhibitorische activiteit van de eerste molec ules werd getest met behulp van de ALPHA-screen assay. Optimalisatie van de eerste inhibitoren leidde to t remmers met in cellulo activiteit. Kristallograe werd gebruikt om de bindingsmodus te bevestigen. De kristalstructuren lieten ook toe om nieu we derivaten met een betere activiteit te ontwerpen. We kunnen dus beslu iten dat er tijdens dit onderzoek (uitgevoerd door de KULeuven Laborator ia voor Biomoleculaire Modellering, voor Moleculaire Virologie en Genthe rapie en voor Biokristallizatie en het Centre for Drug Design and Discov ery (CD3)) nieuwe gevalideerde molecules gevonden werden die mits optima lisatie in de toekomst farmaceutisch gebruikt kunnen worden in antiretro virale therapie.status: publishe

Protein-templated synthesis of metal-based nanomaterials

There is currently intense interest in using protein scaffolds to prepare uniform nanometre-sized clusters of metals or other inorganic material for use as sensors, imaging agents, drugs, magnetic materials or catalysts. Non-templated chemical synthesis of such clusters often results in a product of variable size and quality, but protein molecules have proved adept at nucleating and stabilizing precise nanoclusters of various kinds. Although much research has focused on natural proteins, such as the iron-storage protein ferritin, recent developments in protein design have allowed entirely novel, symmetrical proteins to be used as templates for the first time.status: publishe

FraC nanopores with adjustable diameter identify the mass of opposite-charge peptides with 44 dalton resolution

A high throughput single-molecule method for identifying peptides and sequencing proteins based on nanopores could reduce costs and increase speeds of sequencing, allow the fabrication of portable home-diagnostic devices, and permit the characterization of low abundance proteins and heterogeneity in post-translational modifications. Here we engineer the size of Fragaceatoxin C (FraC) biological nanopore to allow the analysis of a wide range of peptide lengths. Ionic blockades through engineered nanopores distinguish a variety of peptides, including two peptides differing only by the substitution of alanine with glutamate. We also find that at pH 3.8 the depth of the peptide current blockades scales with the mass of the peptides irrespectively of the chemical composition of the analyte. Hence, this work shows that FraC nanopores allow direct readout of the mass of single peptide in solution, which is a crucial step towards the developing of a real-time and single-molecule protein sequencing device.status: publishe

FraC nanopores with adjustable diameter identify the mass of opposite-charge peptides with 44 dalton resolution

A high throughput single-molecule method for identifying peptides and sequencing proteins based on nanopores could reduce costs and increase speeds of sequencing, allow the fabrication of portable home-diagnostic devices, and permit the characterization of low abundance proteins and heterogeneity in post-translational modifications. Here we engineer the size of Fragaceatoxin C (FraC) biological nanopore to allow the analysis of a wide range of peptide lengths. Ionic blockades through engineered nanopores distinguish a variety of peptides, including two peptides differing only by the substitution of alanine with glutamate. We also find that at pH 3.8 the depth of the peptide current blockades scales with the mass of the peptides irrespectively of the chemical composition of the analyte. Hence, this work shows that FraC nanopores allow direct readout of the mass of single peptide in solution, which is a crucial step towards the developing of a real-time and single-molecule protein sequencing device

Electrostatic similarities between protein and small molecule ligands facilitate the design of protein-protein interaction inhibitors.

One of the underlying principles in drug discovery is that a biologically active compound is complimentary in shape and molecular recognition features to its receptor. This principle infers that molecules binding to the same receptor may share some common features. Here, we have investigated whether the electrostatic similarity can be used for the discovery of small molecule protein-protein interaction inhibitors (SMPPIIs). We have developed a method that can be used to evaluate the similarity of electrostatic potentials between small molecules and known protein ligands. This method was implemented in a software called EleKit. Analyses of all available (at the time of research) SMPPII structures indicate that SMPPIIs bear some similarities of electrostatic potential with the ligand proteins of the same receptor. This is especially true for the more polar SMPPIIs. Retrospective analysis of several successful SMPPIIs has shown the applicability of EleKit in the design of new SMPPIIs