24 research outputs found
REdiii: a pipeline for automated structure solution
High-throughput crystallographic approaches require integrated software solutions to minimize the need for manual effort. REdiii is a system that allows fully automated crystallographic structure solution by integrating existing crystallographic software into an adaptive and partly autonomous workflow engine. The program can be initiated after collecting the first frame of diffraction data and is able to perform processing, molecular-replacement phasing, chain tracing, ligand fitting and refinement without further user intervention. Preset values for each software component allow efficient progress with high-quality data and known parameters. The adaptive workflow engine can determine whether some parameters require modifications and choose alternative software strategies in case the preconfigured solution is inadequate. This integrated pipeline is targeted at providing a comprehensive and efficient approach to screening for ligand-bound co-crystal structures while minimizing repetitiveness and allowing a high-throughput scientific discovery process
Crystal structure of APOBEC3A bound to single-stranded DNA reveals structural basis for cytidine deamination and specificity
Nucleic acid editing enzymes are essential components of the immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins, and contribute to the diversification and lethality of cancers. Among these enzymes are the seven human APOBEC3 deoxycytidine deaminases, each with unique target sequence specificity and subcellular localization. While the enzymology and biological consequences have been extensively studied, the mechanism by which APOBEC3s recognize and edit DNA remains elusive. Here we present the crystal structure of a complex of a cytidine deaminase with ssDNA bound in the active site at 2.2 A. This structure not only visualizes the active site poised for catalysis of APOBEC3A, but pinpoints the residues that confer specificity towards CC/TC motifs. The APOBEC3A-ssDNA complex defines the 5\u27-3\u27 directionality and subtle conformational changes that clench the ssDNA within the binding groove, revealing the architecture and mechanism of ssDNA recognition that is likely conserved among all polynucleotide deaminases, thereby opening the door for the design of mechanistic-based therapeutics
Structural Determination of the Broadly Reactive Anti-IGHV1-69 Anti-idiotypic Antibody G6 and Its Idiotope
The heavy chain IGHV1-69 germline gene exhibits a high level of polymorphism and shows biased use in protective antibody (Ab) responses to infections and vaccines. It is also highly expressed in several B cell malignancies and autoimmune diseases. G6 is an anti-idiotypic monoclonal Ab that selectively binds to IGHV1-69 heavy chain germline gene 51p1 alleles that have been implicated in these Ab responses and disease processes. Here, we determine the co-crystal structure of humanized G6 (hG6.3) in complex with anti-influenza hemagglutinin stem-directed broadly neutralizing Ab D80. The core of the hG6.3 idiotope is a continuous string of CDR-H2 residues starting with M53 and ending with N58. G6 binding studies demonstrate the remarkable breadth of binding to 51p1 IGHV1-69 Abs with diverse CDR-H3, light chain, and antigen binding specificities. These studies detail the broad expression of the G6 cross-reactive idiotype (CRI) that further define its potential role in precision medicine
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Discovery of a human monoclonal antibody that cross-neutralizes venom phospholipase A2s from three different snake genera
Despite the considerable global impact of snakebite envenoming, available treatments remain suboptimal. Here, we report the discovery of a broadly-neutralizing human monoclonal antibody, using a phage display-based cross-panning strategy, capable of reducing the cytotoxic effects (using a mouse myoblast cell line that may demonstrate the impact of venoms on skeletal muscle) of venom phospholipase A2s from three different snake genera from different continents. This highlights the potential of utilizing monoclonal antibodies to potentially compose more effective, safer, and globally accessible polyvalent antivenoms that hopefully can be widely applicable for snakebite envenomings
Resilience trinity: safeguarding ecosystem functioning and services across three different time horizons and decision contexts
Ensuring ecosystem resilience is an intuitive approach to safeguard the functioning of ecosystems and hence the future provisioning of ecosystem services (ES). However, resilience is a multi-faceted concept that is difficult to operationalize. Focusing on resilience mechanisms, such as diversity, network architectures or adaptive capacity, has recently been suggested as means to operationalize resilience. Still, the focus on mechanisms is not specific enough. We suggest a conceptual framework, resilience trinity, to facilitate management based on resilience mechanisms in three distinctive decision contexts and time-horizons: i) reactive, when there is an imminent threat to ES resilience and a high pressure to act, ii) adjustive, when the threat is known in general but there is still time to adapt management, and iii) provident, when time horizons are very long and the nature of the threats is uncertain, leading to a low willingness to act. Resilience has different interpretations and implications at these different time horizons, which also prevail in different disciplines. Social ecology, ecology, and engineering are often implicitly focussing on provident, adjustive, or reactive resilience, respectively, but these different notions and of resilience and their corresponding social, ecological, and economic trade-offs need to be reconciled. Otherwise, we keep risking unintended consequences of reactive actions, or shying away from provident action because of uncertainties that cannot be reduced. The suggested trinity of time horizons and their decision contexts could help ensuring that longer-term management actions are not missed while urgent threats to ES are given priority
Establishing APOBEC3F as a structural target in novel antiretroviral strategies
HIV infection and the consequences it can have on human life is a tragic reality for millions of people. Since the discovery of HIV being the cause of AIDS in 1983 tremendous efforts have been made on the preventive, curative, palliative and therapeutic aspects of this disease. Antiretroviral therapy (ART) has made it possible to abolish fatal progression of AIDS in individuals but requires lifelong treatment adherence and can lead to the emergence of drug resistant strains necessitating ongoing efforts in search of novel therapeutic approaches. At the front line of major challenges remaining are the development of effective vaccines, the depletion of viral reservoirs by targeting latent infections and novel therapeutic strategies that can be translated into other disease scenarios outside the HIV/AIDS paradigm. Antiretroviral APOBEC3 proteins are intrinsic immune factors capable of potently restricting HIV-1 but are counteracted by the viral protein Vif. Targeting the Vif-APOBEC3 axis may lead to novel immunomodulating therapeutic approaches.
In this thesis the crystal structure of the catalytically active and HIV-1 Vif binding domain of the antiretroviral host factor APOBEC3F is being described. Determinants for Vif susceptibility form a contiguous negatively charged surface. Conserved sequence motifs across APOBEC3 proteins and between APOBEC3F and Vif suggest an intriguing case of molecular mimicry between viral and host protein. These motifs participate in crystallographic contacts and determine the solution state of the APOBEC3F catalytic domain. Providing insights into Vif interaction and solution behavior will establish APOBEC3F as a structural target for novel antiretroviral strategies.
High throughput cocrystallization trials are necessary during attempts at identifying binding sites of putative APOBEC3F modulating small molecules and peptides. REdiii, a fully automated pipeline encompassing every process from indexing of diffraction data to ligand placement was developed allowing fast analysis of crystallographic datasets without requiring any user input during the process. This tool leads to vast improvements in the screening
process for crystals of any ligand bound protein.HIV Infektionen und die darausfolgenden Konsequenzen sind eine tragische Realität für Millionen von Menschen. Seit der Entdeckung von HIV als Ursache von AIDS 1983, wurden bedeutende Versuche in den präventiven, kurativen, palliativen und therapeutischen Aspekten dieser Krankheit unternommen. Antiretrovirale Therapie (ART) hat es ermöglicht, der fatalen Progression von AIDS bei Erkrankten einhalt zu bieten, erfordert aber lebenslange Einhaltung der Behandlung und kann zur Emergenz von medikamentenresistenten Stämmen führen weswegen weitergehende Bemühungen hinsichtlich der Suche nach neuen therapeutischen Ansätzen notwendig sind. An der Front der gravierendsten Herausforderungen liegen die Entwicklung effektiver Impfstoffe, die Depletion viraler Reservoirs durch gezieltes Angreifen auf latente Infektionen und neue therapeutische Strategien, die auf andere Krankheitsszenarien außerhalb des HIV/AIDS Paradigmas übertragen werden können. Antiretrovirale APOBEC3 Proteine sind intrinsische Immunfaktoren in der Lage, HIV-1 wirksam zu restringieren, werden aber vom viralen Protein Vif entgegengewirkt. Gezieltes Eingreifen in die Vif-APOBEC3 Achse kann zu neuen, immunmodulierenden therapeutischen Ansätzen führen.
In dieser Schrift wird die Kristallstruktur der katalytisch aktiven und HIV-1 Vif bindenden Domäne des antiretroviralen Faktors APOBEC3F beschrieben. Determinanten der Anfälligkeit für Vif formen eine zusammenhängende negativ geladene Oberfläche. Konservierte Sequenzmotive zwischen APOBEC3 Proteinen und zwischen APOBEC3F und Vif weisen auf einen interessanten Fall von molekularem Mimikry zwischen Wirt- und Virusprotein. Diese Motive sind Teil der kristallographischen Kontakte und beeinflussen den Zustand der APOBEC3F katalytischen Domäne in Lösung. Einsichten in die Interaktion mit Vif und das Verhalten in Lösung werden APOBEC3F als ein strukturelles Ziel für neuartige antiretrovirale Strategien etablieren.
Hochdurchsatz in kristallographischen Ansätzen ist notwendig während Versuchen die Bindestellen von potenziell APOBEC3F modulierenden niedermolekularen Wirkstoffen und Peptiden zu identifizieren. REdiii, eine vollautomatisierte Pipeline, die jeden einzelnen Prozess von der Indizierung von Diffraktionsdaten zur Ligandenplazierung beinhaltet, wurde entwickelt um rapide Analyse von kristallographischen Datensätzen ohne die Notwendigkeit irgendeiner Form von nutzerseitigen Eingaben während des Prozesses zu erlauben. Dieses Werkzeug führt zu einer deutlichen Verbesserung des Screeningprozesses für Kristalle von ligandengebundenen Proteinen
The challenges with developing therapeutic monoclonal antibodies for pandemic application
A computational analysis of the structural determinants of APOBEC3\u27s catalytic activity and vulnerability to HIV-1 Vif
APOBEC3s (A3) are Zn(2+) dependent cytidine deaminases with diverse biological functions and implications for cancer and immunity. Four of the seven human A3s restrict HIV by \u27hypermutating\u27 the reverse-transcribed viral genomic DNA. HIV Virion Infectivity Factor (Vif) counters this restriction by targeting A3s to proteasomal degradation. However, there is no apparent correlation between catalytic activity, Vif binding, and sequence similarity between A3 domains. Our comparative structural analysis reveals features required for binding Vif and features influencing polynucleotide deaminase activity in A3 proteins. All Vif-binding A3s share a negatively charged surface region that includes residues previously implicated in binding the highly-positively charged Vif. Additionally, catalytically active A3s share a positively charged groove near the Zn(2+) coordinating active site, which may accommodate the negatively charged polynucleotide substrate. Our findings suggest surface electrostatics, as well as the spatial extent of substrate accommodating region, are critical determinants of substrate and Vif binding across A3 proteins with implications for anti-retroviral and anti-cancer therapeutic design
Immunotargeting of Nanocrystals by SpyCatcher Conjugation of Engineered Antibodies
Inorganic nanocrystals such as quantum dots (QDs) and upconverting nanoparticles (UCNPs) are uniquely suited for quanti-tative live-cell imaging and are typically functionalized with ligands to study specific receptors or cellular targets. Antibod-ies (Ab) are among the most useful targeting reagents owing to their high affinities and specificities, but common nanocrys-tal labeling methods may orient Ab incorrectly, be reversible or denaturing, or lead to Ab-NP complexes too large for some applications. Here, we show that SpyCatcher proteins, which bind and spontaneously form covalent isopeptide bonds with cognate SpyTag peptides, can conjugate engineered Ab to nanoparticle surfaces with control over stability, orientation, and stoichiometry. Compact SpyCatcher-functionalized QDs and UCNPs may be labeled with short-chain variable fragment Ab (scFv) engineered to bind urokinase-type plasminogen activator receptors (uPAR) that are overexpressed in many human can-cers. Confocal imaging of anti-uPAR scFv-QD conjugates shows the Ab mediates specific binding and internalization by breast cancer cells expressing uPAR. Time-lapse imaging of photostable scFv-UCNP conjugates show that Ab binding caus-es uPAR internalization with a ∼20-minute half-life on the cell surface, and uPAR is internalized to endolysosomal com-partments distinct from general membrane stains and without significant recycling to the cell surface. The controlled and stable conjugation of engineered Ab to NPs enables targeting of diverse receptors for live-cell study of their distribution, trafficking, and physiology
Immunotargeting of Nanocrystals by SpyCatcher Conjugation of Engineered Antibodies
Inorganic nanocrystals such as quantum dots (QDs) and upconverting nanoparticles (UCNPs) are uniquely suited for quantitative live-cell imaging and are typically functionalized with ligands to study specific receptors or cellular targets. Antibodies (Ab) are among the most useful targeting reagents owing to their high affinities and specificities, but common nanocrystal labeling methods may orient Ab incorrectly, be reversible or denaturing, or lead to Ab-NP complexes too large for some applications. Here, we show that SpyCatcher proteins, which bind and spontaneously form covalent isopeptide bonds with cognate SpyTag peptides, can conjugate engineered Ab to nanoparticle surfaces with control over stability, orientation, and stoichiometry. Compact SpyCatcher-functionalized QDs and UCNPs may be labeled with short-chain variable fragment Ab (scFv) engineered to bind urokinase-type plasminogen activator receptors (uPAR) that are overexpressed in many human cancers. Confocal imaging of anti-uPAR scFv-QD conjugates shows the antibody mediates specific binding and internalization by breast cancer cells expressing uPAR. Time-lapse imaging of photostable scFv-UCNP conjugates shows that Ab binding causes uPAR internalization with a ∼20 min half-life on the cell surface, and uPAR is internalized to endolysosomal compartments distinct from general membrane stains and without significant recycling to the cell surface. The controlled and stable conjugation of engineered Ab to NPs enables targeting of diverse receptors for live-cell study of their distribution, trafficking, and physiology