Insights into the Mechanism of Ligand Binding to Octopine Dehydrogenase from Pecten maximus by NMR and Crystallography

Octopine dehydrogenase (OcDH) from the adductor muscle of the great scallop, Pecten maximus, catalyzes the NADH dependent, reductive condensation of L-arginine and pyruvate to octopine, NAD+, and water during escape swimming and/or subsequent recovery. The structure of OcDH was recently solved and a reaction mechanism was proposed which implied an ordered binding of NADH, L-arginine and finally pyruvate. Here, the order of substrate binding as well as the underlying conformational changes were investigated by NMR confirming the model derived from the crystal structures. Furthermore, the crystal structure of the OcDH/NADH/agmatine complex was determined which suggests a key role of the side chain of L-arginine in protein cataylsis. Thus, the order of substrate binding to OcDH as well as the molecular signals involved in octopine formation can now be described in molecular detail

Solution structure of stem-loop α of the hepatitis B virus post-transcriptional regulatory element

Chronic hepatitis B virus (HBV) infections may lead to severe diseases like liver cirrhosis or hepatocellular carcinoma (HCC). The HBV post-transcriptional regulatory element (HPRE) facilitates the nuclear export of unspliced viral mRNAs, contains a splicing regulatory element and resides in the 3′-region of all viral transcripts. The HPRE consists of three sub-elements α (nucleotides 1151–1346), β1 (nucleotides 1347–1457) and β2 (nucleotides 1458–1582), which confer together full export competence. Here, we present the NMR solution structure (pdb 2JYM) of the stem-loop α (SLα, nucleotides 1292–1321) located in the sub-element α. The SLα contains a CAGGC pentaloop highly conserved in hepatoviruses, which essentially adopts a CUNG-like tetraloop conformation. Furthermore, the SLα harbours a single bulged G residue flanked by A-helical regions. The structure is highly suggestive of serving two functions in the context of export of unspliced viral RNA: binding sterile alpha motif (SAM-) domain containing proteins and/or preventing the utilization of a 3′-splice site contained within SLα

Methodik zur Simulation von Material- und Energieflüssen: Entscheidungsunterstützung für Simulationsstudien in Energieeffizienzprojekten

Die effektive Planung von Energieeffizienzmaßnahmen hat große Bedeutung für Produktionsunternehmen. Ein dazu geeignetes Planungswerkezug ist die um energetische Betrachtungen erweiterte Materialflusssimulation, mit der sich komplexe Wechselwirkungen von Material- und Energieflüssen in Fabriken untersuchen lassen. Dieser Beitrag präsentiert eine entscheidungstheoretisch fundierte Methodik zur Ausgestaltung derartiger Simulationsstudien in Umplanungsprojekten zur Energieeffizienzsteigerung

Structural snapshot of cyclic nucleotide binding domains from cyclic nucleotide-sensitive ion channels.

Cyclic nucleotide-binding domains (CNBDs) that are present in various channel proteins play crucial roles in signal amplification cascades. Although atomic resolution structures of some of those CNBDs are available, the detailed mechanism by which they confer cyclic nucleotide-binding to the ion channel pore remains poorly understood. In this review, we describe structural insights about cyclic nucleotide-binding-induced conformational changes in CNBDs and their potential coupling with channel gating

Procedure model for efficient simulation studies which consider the flows of materials and energy simultaneously

The premise of this work is that more guidance is required for the application of the simultaneous simulation of material flows and energy flows. This assumption is supported by the findings of a systematic review which showed great diversity in earlier published case studies. Yet, other existing procedure models provide little advice regarding the manifestations of specific details of a simulation study. This paper presents a novel procedure model that is meant to increase the efficiency in simulation projects by providing detailed and consistent guidance for design choices in a simulation study. Selected results of its application are shown exemplarily

Analysis of the Ion Channel Gating Mechanism in Solution by Nuclear Magnetic Resonance (NMR) Spectroscopy

Ion channels activated by cyclic nucleotides play crucial roles in signal transduction pathways. Upon binding of cyclic nucleotides to the intracellular cyclic nucleotide-binding domain (CNBD) of HCN or CNG channels (hyperpolarization-activated and cyclic nucleotide-gated channels or cyclic nucleotide-gated channels) an opening of the membrane pore occurs.To analyze the underlying gating mechanism highly resolved structures of the cyclic nucleotide-binding domains are necessary. Until now, structures of CNBDs from eukaryotic HCN channels as well as prokaryotic CNG channels are known. However, CNBD crystal structures of the HCN channels reveal no significant differences between apo and holo state1,2. In contrast, CNBD structures of the prokaryotic Mesorhizobium loti K1 channel, solved by liquid state NMR spectroscopy, show substantial rearrangements upon binding of a cyclic nucleotide3,4.Further elucidation of the gating mechanism will be done by structural analysis of an eukaryotic CNBD using liquid state NMR spectroscopy

Analysis of the Bin1 SH3 interaction with peptides derived from the hepatitis C virus protein NS5A and c-Myc reveals that NS5A can competitively displace c-Myc in vitro

Severe liver damage like cirrhosis and hepatocellular carcinoma (HCC) can be caused by manifestation of the hepatitis C virus (HCV) infection. Constitutively activated c-Myc oncogene has been shown to contribute to the establishment of HCV-mediated HCC. Interestingly, only one of many isoforms of the tumor suppressor protein Bin1 (bridging integrator 1), Bin1+12A, contains an internal, canonical SH3 binding motif that recognizes its own SH3 domain. This leads to the inability of Bin1+12A to interact with c-Myc. The expression of the Bin1+12A isoform is a main phenotype in malignant melanoma cells. We suggest that also other mechanisms that disturb the interaction of Bin1 and c-Myc might have severe consequences since the latter is tightly regulated in healthy cells. The HCV nonstructural protein 5A (NS5A) plays a key role in virus replication and assembly. NS5A plays an intercepting role in several cellular pathways, which are linked to cell growth, cell cycle control, cell survival, cellular stress response, apoptosis as well as HCC. It is known that NS5A contains a highly conserved canonical, polyproline (PxxP) SH3-binding motif, which is located between its D2 and D3 domains. This PxxP motif was described to interact with the SH3 domain of Bin1. In addition to a biophysical analysis of the canonical binding between Bin1 SH3 and the PxxP motif of NS5A [1], we identified two additional low-affinity binding sites for non-canonical SH3 binding on NS5A [2]. The hypothesis underlying the work presented here is that viral NS5A is able to sequester cellular Bin1 from c-Myc

Generic energy-enhancement module for consumption analysis of manufacturing processes in discrete event simulation

Customer demands for eco-friendly goods, energy supply bottlenecks or consumption/emission restrictions in many countries, as well as the current economic situation demand a conscious use of energy in industry. Manufacturers and suppliers need to increase efficiency of their products, processes and resources dramatically in order to reach agreed energy targets. To achieve substantial reductions it becomes necessary to establish predictive assessments of the energy demand for new production systems and whole manufacturing sites as standard operating procedures in systems engineering. This paper discusses a newly developed, generic energy-enhancement module, which renders resource consumption analyses within material flow simulation software possible

Energieflexibilität im Maschinen- und Anlagenbau: Vorgehen und Handlungshilfen für die Energiewende im Mittelstand

Maschinen- und Anlagenbauunternehmen sind gefordert, sich auf die Veränderungen im Zuge der Energiewende einzustellen, wollen sie ihre Wettbewerbsfähigkeit erhalten. Dies umfasst vor allem die Synchronisation des Energiebedarfs ihrer Produktionsprozesse mit dem marktseitigen Energieangebot. Um insbesondere mittelständische Unternehmen dabei zu unterstützen, stellt der Beitrag Vorgehen und Hilfsmittel (u. a. einen Maßnahmenkatalog) zur Identifikation, Bewertung und Auswahl geeigneter Maßnahmen vor

Hepatitis C virus NS5A is able to competitively displace c-Myc from the Bin1 SH3 domain in vitro

We studied the interaction of the SH3 domain of Bin1 with a 15-mer peptide of HCV NS5A and show its potency to competitively displace a 15-mer human c-Myc fragment, which is a physiological ligand of Bin1, using NMR spectroscopy. Fluorescence spectroscopy and ITC were employed to determine the affinity of Bin1 SH3 to NS5A(347–361), yielding a submicromolar affinity to NS5A. Our study compares the binding dynamics and affinities of the relevant regions for binding of c-Myc and NS5A to Bin1 SH3. The result gives further insights into the potential role of NS5A in Bin1-mediated apoptosis.Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd