Determination of Configuration and Conformation of a Reserpine Derivative with Seven Stereogenic Centers Using Molecular Dynamics with RDC‐Derived Tensorial Constraints

NMR-based determination of the configuration of complex molecules containing many stereocenters is often not possible using traditional NOE data and coupling patterns. Making use of residual dipolar couplings (RDCs), we were able to determine the relative configuration of a natural product containing seven stereocenters, including a chiral amine lacking direct RDC data. To identify the correct relative configuration out of 32 possible ones, experimental RDCs were used in three different approaches for data interpretation: by fitting experimental data based singular value decomposition (SVD) using a single alignment tensor and either (i) a single conformer or (ii) multiple conformers, or alternatively (iii) using molecular dynamics simulations with tensorial orientational constraints (MDOC). Even though in all three approaches one and the same configuration could be selected and clear discrimination between possible configurations was achieved, the experimental data was not fully satisfied by the methods based on single tensor approaches. While these two approaches are faster, only MDOC is able to fully reproduce experimental results, as the obtained conformational ensemble adequately covers the conformational space necessary to describe the molecule with inherent flexibility

Knottin cyclization: impact on structure and dynamics

<p>Abstract</p> <p>Background</p> <p>Present in various species, the knottins (also referred to as inhibitor cystine knots) constitute a group of extremely stable miniproteins with a plethora of biological activities. Owing to their small size and their high stability, knottins are considered as excellent leads or scaffolds in drug design. Two knottin families contain macrocyclic compounds, namely the cyclotides and the squash inhibitors. The cyclotide family nearly exclusively contains head-to-tail cyclized members. On the other hand, the squash family predominantly contains linear members. Head-to-tail cyclization is intuitively expected to improve bioactivities by increasing stability and lowering flexibility as well as sensitivity to proteolytic attack.</p> <p>Results</p> <p>In this paper, we report data on solution structure, thermal stability, and flexibility as inferred from NMR experiments and molecular dynamics simulations of a linear squash inhibitor EETI-II, a circular squash inhibitor MCoTI-II, and a linear analog lin-MCoTI. Strikingly, the head-to-tail linker in cyclic MCoTI-II is by far the most flexible region of all three compounds. Moreover, we show that cyclic and linear squash inhibitors do not display large differences in structure or flexibility in standard conditions, raising the question as to why few squash inhibitors have evolved into cyclic compounds. The simulations revealed however that the cyclization increases resistance to high temperatures by limiting structure unfolding.</p> <p>Conclusion</p> <p>In this work, we show that, in contrast to what could have been intuitively expected, cyclization of squash inhibitors does not provide clear stability or flexibility modification. Overall, our results suggest that, for squash inhibitors in standard conditions, the circularization impact might come from incorporation of an additional loop sequence, that can contribute to the miniprotein specificity and affinity, rather than from an increase in conformational rigidity or protein stability. Unfolding simulations showed however that cyclization is a stabilizing factor in strongly denaturing conditions. This information should be useful if one wants to use the squash inhibitor scaffold in drug design.</p

Parmbsc1: a refined force field for DNA simulations

We present parmbsc1, a force field for DNA atomistic simulation, which has been parameterized from high-level quantum mechanical data and tested for nearly 100 systems (representing a total simulation time of ~140 μs) covering most of DNA structural space. Parmbsc1 provides high-quality results in diverse systems. Parameters and trajectories are available at http://mmb.irbbarcelona.org/ParmBSC1/

TAR-RNA Recognition by a Novel Cyclic Aminoglycoside Analogue

Die Bildung des Tat-Protein/TAR RNA-Komplexes ist ein entscheidender Schritt in der Regulation der Expression des HI-Virus (Human Immunodeficiency Virus, HIV). Für eine vollständige Transkription des viralen Gens ist die Interaktion des Tat/TARKomplexes mit dem positiven Transkriptionsfaktor-Komplex P-EFTb (Positive Transcription Elongation Factor) über dessen Cyclin T1-Komponente (CycT1) notwendig. Durch Mutagenesestudien wurde die Hexanukleotid-Schleife der TAR RNA als Kontaktstelle für die Wechselwirkung mit CycT1 identifiziert. Zur Entwicklung neuer Arzneimittel gegen das HIV stellt die Störung des Zusammenspiels zwischen dem Tat/CycT1-Komplex und der TAR RNA ein lohnendes Ziel dar. Positiv geladene Verbindungen wie Aminoglycoside oder Peptidmimetika binden an die TAR RNA und brechen so den Tat/TAR-Komplex auf. In dieser Arbeit wird die Bestimmung der dreidimensionalen Struktur des Komplexes zwischen der HIV-2 TAR RNA und einem Neooligoaminodeoxysaccharid mit Hilfe der NMR-Spektroskopie beschrieben. Im Gegensatz zu anderen Aminoglycosiden wechselwirkt diese neuartige Verbindung gleichzeitig mit den für die Bindung des Tat-Proteins verantwortlichen Resten des Bulges wie auch mit dem Adenosin 35 der Hexanukleotid-Schleife der TAR RNA. Diese Schleifenregion erfährt bei der Bildung des Komplexes mit dem Aminoglycosid eine große konformationelle Änderung. Dieser neue Bindungsmodus eröffnet zusammen mit der einfachen synthetischen Zugänglichkeit von Neooligoaminodeoxysaccharid-Derivaten die Möglichkeit, eine neue Klasse von TAR RNA bindenden Molekülen zu entwerfen. Diese könnten gleichzeitig die Bildung des binären Tat/TAR- wie auch des ternären Tat/TAR/CycT1-Komplexes durch Störung der Schleifen- und Bulge-Region der RNA verhindern.The formation of the Tat-protein/TAR RNA complex is a crucial step in the regulation of Human Immunodeficiency Virus (HIV)-gene expression. To obtain fulllength viral transcripts the Tat/TAR complex has to recruit the positive transcription elongation factor complex (P-EFTb), which interacts with TAR through its CyclinT1 (CycT1) component. Mutational studies identified the TAR hexanucleotide loop as a crucial region for contacting CyclinT1. Interfering with the interaction between the Tat/CycT1 complex and the TAR RNA is an attractive strategy for the design of anti- HIV drugs. Positively charged molecules, like aminoglycosides or peptidomimetics, bind the TAR RNA, disrupting the Tat/TAR complex. Here, we investigate the complex between the HIV-2 TAR RNA and a neooligoaminodeoxysaccharide by NMR spectroscopy. In contrast to other aminoglycosides, this novel aminoglycoside analogue contacts simultaneously the bulge residues required for Tat binding and the A35 residue of the hexanucleotide loop. Upon complex formation, the loop region undergoes profound conformational changes. The novel binding mode, together with the easy accessibility of derivatives for the neooligoaminodeoxysaccharide, could open the way to the design of a new class of TAR RNA binders, which simultaneously inhibit the formation of both the Tat/TAR binary complex and the Tat/TAR/CyclinT1 ternary complex by obstructing both the bulge and loop regions of the RNA