Aleksandr B. Sahakyan

rG4-seq reveals widespread formation of G-quadruplex structures in the human transcriptome

We introduce RNA G-quadruplex sequencing (rG4-seq), a transcriptome-wide RNA G-quadruplex (rG4) profiling method that couples rG4-mediated reverse transcriptase stalling with next-generation sequencing. Using rG4-seq on polyadenylated-enriched HeLa RNA, we generated a global in vitro map of thousands of canonical and noncanonical rG4 structures. We characterize rG4 formation relative to cytosine content and alternative RNA structure stability, uncover rG4-dependent differences in RNA folding and show evolutionarily conserved enrichment in transcripts mediating RNA processing and stability.

Cyclophilin A catalyzes proline isomerization by an electrostatic handle mechanism

Significance One of the most widespread molecular switches in biochemical pathways is based on the isomerization of the amino acid proline, a process that normally is facilitated by enzymes known as “proline isomerases.” We show that cyclophilin A, one of the most common proline isomerases, acts by a simple mechanism, which we describe as an “electrostatic handle.” In this mechanism, the enzyme creates an electrostatic environment in its catalytic site that rotates a peptide bond in the substrate by pulling the electric dipole associated with the carbonyl group preceding the peptide bond itself. Our results thus identify a specific mechanism by which electrostatics is exploited in enzyme catalysis. Proline isomerization is a ubiquitous process that plays a key role in the folding of proteins and in the regulation of their functions. Different families of enzymes, known as “peptidyl-prolyl isomerases” (PPIases), catalyze this reaction, which involves the interconversion between the cis and trans isomers of the N-terminal amide bond of the amino acid proline. However, complete descriptions of the mechanisms by which these enzymes function have remained elusive. We show here that cyclophilin A, one of the most common PPIases, provides a catalytic environment that acts on the substrate through an electrostatic handle mechanism. In this mechanism, the electrostatic field in the catalytic site turns the electric dipole associated with the carbonyl group of the amino acid preceding the proline in the substrate, thus causing the rotation of the peptide bond between the two residues. We identified this mechanism using a combination of NMR measurements, molecular dynamics simulations, and density functional theory calculations to simultaneously determine the cis-bound and trans-bound conformations of cyclophilin A and its substrate as the enzymatic reaction takes place. We anticipate that this approach will be helpful in elucidating whether the electrostatic handle mechanism that we describe here is common to other PPIases and, more generally, in characterizing other enzymatic processes.

Selective Chemical Labeling of Natural T Modifications in DNA

We present a chemical method to selectively tag and enrich thymine modifications, 5-formyluracil (5-fU) and 5-hydroxymethyluracil (5-hmU), found naturally in DNA. Inherent reactivity differences have enabled us to tag 5-fU chemoselectively over its C modification counterpart, 5-formylcytosine (5-fC). We rationalized the enhanced reactivity of 5-fU compared to 5-fC via ab initio quantum mechanical calculations. We exploited this chemical tagging reaction to provide proof of concept for the enrichment of 5-fU containing DNA from a pool that contains 5-fC or no modification. We further demonstrate that 5-hmU can be chemically oxidized to 5-fU, providing a strategy for the enrichment of 5-hmU. These methods will enable the mapping of 5-fU and 5-hmU in genomic DNA, to provide insights into their functional role and dynamics in biology.

Using side-chain aromatic proton chemical shifts for a quantitative analysis of protein structures.

Chemical shifts are receiving renewed attention in structural biology owing to the recent introduction of novel methodologies that enable their use in protein structure determination. As these approaches have so far been mostly concerned with backbone atoms, it would be highly desirable to further generalize them to also include side-chain atoms. A major motivation for this objective is that side chains play crucial roles in determining the conformational properties of protein surfaces and interior cavities, which in most cases define the specificity of biomolecular interactions. In particular, aromatic side chains are capable of forming interactions with a variety of chemical groups through hydrophobic, p–p stacking, p–anion and p–cation interactions, and often comprise the hot spots of protein–protein and protein–ligand complex formation, and protein folding. Furthermore, aromatic side chains, as sources of ring current effects, substantially influence the chemical shifts of other nuclei, including the highly exploited backbone nuclei. However, although ring-current terms are frequently included in chemical shift predictions of backbone nuclei, aromatic chemical shifts are not normally used to define the geometry of the aromatic rings themselves. Recent advances in specific labeling technologies for aromatic side chains will soon increase the number of assigned aromatic chemical shifts, thus adding new prospects to the established methodology of aromatic chemical shift measurements. The incorporation of chemical shifts of aromatic side chains in structure-determination algorithms, in addition to the backbone atoms, would make it possible to extend the use of chemical shifts in structural studies. To achieve this goal, a chemical shift prediction method for side-chain nuclei that is based solely on the configurations of proximal atoms needs to be developed. This type of predictions, which is at variance with other currently available chemical shift predictors that provide chemical shift evaluations for side-chain nuclei, are readily differentiable with respect to the atomic coordinates, and thus enable the calculation of biasing forces for the integration of the equations of motion within a molecular dynamics scheme. Prediction of aromatic side-chain chemical shifts by differentiable functions opens new opportunities to monitor a range of important processes, and will increase the scope of chemical shift usage in determining the structures of biomolecular complexes and complex biomolecular systems. To address this challenge, we present here ArShift, a chemical shift prediction method for protein side-chain aromatic H nuclei. We then demonstrate that by using only aromatic side-chain chemical shifts, structures that do not match the state from which chemical shifts are measured can be revealed. The ArShift predictions are based on known phenomenological terms that describe the effects of ring current, magnetic anisotropy, and electric field terms, which are accompanied by a set of dihedral angle terms and distance-based polynomials (see the Supporting Information). A comprehensive analysis of the aromatic chemical shift assignments available from the BMRB database is used after filtering and re-referencing steps to reduce the number of inaccurate and artifactual entries (Figures S1 and S2 in the Supporting Information). To identify the mapping between chemical shifts and structures, only structures determined by X-ray crystallography at a resolution of 2.0 or better are considered in the derivation of the geometric terms. The combination of terms used in the predictions is then optimized through a Monte Carlo approach to decrease the number of fitted coefficients, thus increasing the significance of the remaining ones (Table S1). We assessed the accuracy of the prediction method by performing individual predictions (in leave-one-out tests) for all the chemical shift entries used for deriving the coefficients. The standard deviations of the residual errors (denoted here as standard errors) for the models implemented in the ArShift package are 0.189, 0.204, 0.256, 0.191, and 0.173 ppm for PheHd, Phe-He, Phe-Hz, Tyr-Hd, and Tyr-He nuclei, respectively (Figures S3 and S4). The comparison of the ArShift standard errors and the standard deviations of the corresponding chemical shift types in the BMRB database are presented in Figure 1. Predictions for C nuclei are not reported in this work because they do not currently provide a significant improvement over those based on the average values derived from the BMRB database. The reason for this situation is most probably the neglect of the stronger isotope effects on C [*] A. B. Sahakyan, Dr. A. Cavalli, Prof. M. Vendruscolo Department of Chemistry, University of Cambridge Lensfield Road, Cambridge CB2 1EW (UK) E-mail: mv245@cam.ac.uk Dr. W. F. Vranken European Bioinformatics Institute Wellcome Trust Genome Campus Cambridge CB10 1SD (UK) [] Current Address: Structural Biology Brussels Vrije Universiteit Brussel Pleinlaan 2, 1050 Brussel (Belgium)

ALMOST: An all atom molecular simulation toolkit for protein structure determination

Almost (all atom molecular simulation toolkit) is an open source computational package for structure determination and analysis of complex molecular systems including proteins, and nucleic acids. Almost has been designed with two primary goals: to provide tools for molecular structure determination using various types of experimental measurements as conformational restraints, and to provide methods for the analysis and assessment of structural and dynamical properties of complex molecular systems. The methods incorporated in Almost include the determination of structural and dynamical features of proteins using distance restraints derived from nuclear Overhauser effect measurements, orientational restraints obtained from residual dipolar couplings and the structural restraints from chemical shifts. Here, we present the first public release of Almost, highlight the key aspects of its computational design and discuss the main features currently implemented. Almost is available for the most common Unix‐based operating systems, including Linux and Mac OS X. Almost is distributed free of charge under the GNU Public License, and is available both as a source code and as a binary executable from the project web site at http://www.open‐almost.org. Interested users can follow and contribute to the further development of Almost on http://sourceforge.net/projects/almost.

Analysis of the contributions of ring current and electric field effects to the chemical shifts of RNA bases.

Ring current and electric field effects can considerably influence NMR chemical shifts in biomolecules. Understanding such effects is particularly important for the development of accurate mappings between chemical shifts and the structures of nucleic acids. In this work, we first analyzed the Pople and the Haigh-Mallion models in terms of their ability to describe nitrogen base conjugated ring effects. We then created a database (DiBaseRNA) of three-dimensional arrangements of RNA base pairs from X-ray structures, calculated the corresponding chemical shifts via a hybrid density functional theory approach and used the results to parametrize the ring current and electric field effects in RNA bases. Next, we studied the coupling of the electric field and ring current effects for different inter-ring arrangements found in RNA bases using linear model fitting, with joint electric field and ring current, as well as only electric field and only ring current approximations. Taken together, our results provide a characterization of the interdependence of ring current and electric field geometric factors, which is shown to be especially important for the chemical shifts of non-hydrogen atoms in RNA bases.

Structural Analysis using SHALiPE to Reveal RNA G-Quadruplex Formation in Human Precursor MicroRNA

Abstract RNA G‐quadruplex (rG4) structures are of fundamental importance to biology. A novel approach is introduced to detect and structurally map rG4s at single‐nucleotide resolution in RNAs. The approach, denoted SHALiPE, couples selective 2′‐hydroxyl acylation with lithium ion‐based primer extension, and identifies characteristic structural fingerprints for rG4 mapping. We apply SHALiPE to interrogate the human precursor microRNA 149, and reveal the formation of an rG4 structure in this non‐coding RNA. Additional analyses support the SHALiPE results and uncover that this rG4 has a parallel topology, is thermally stable, and is conserved in mammals. An in vitro Dicer assay shows that this rG4 inhibits Dicer processing, supporting the potential role of rG4 structures in microRNA maturation and post‐transcriptional regulation of mRNAs.

A Conformational Ensemble Derived Using NMR Methyl Chemical Shifts Reveals a Mechanical Clamping Transition That Gates the Binding of the HU Protein to DNA

Recent improvements in the accuracy of structure-based methods for the prediction of nuclear magnetic resonance chemical shifts have inspired numerous approaches for determining the secondary and tertiary structures of proteins. Such advances also suggest the possibility of using chemical shifts to characterize the conformational fluctuations of these molecules. Here we describe a method of using methyl chemical shifts as restraints in replica-averaged molecular dynamics (MD) simulations, which enables us to determine the conformational ensemble of the HU dimer and characterize the range of motions accessible to its flexible β-arms. Our analysis suggests that the bending action of HU on DNA is mediated by a mechanical clamping mechanism, in which metastable structural intermediates sampled during the hinge motions of the β-arms in the free state are presculpted to bind DNA. These results illustrate that using side-chain chemical shift data in conjunction with MD simulations can provide quantitative information about the free energy landscapes of proteins and yield detailed insights into their functional mechanisms.

G-quadruplex structures within the 3' UTR of LINE-1 elements stimulate retrotransposition

Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in higher eukaryotes that have a significant role in shaping genomes, owing to their abundance. Here we report that guanine-rich sequences in the 3′ untranslated regions (UTRs) of hominoid-specific LINE-1 elements are coupled with retrotransposon speciation and contribute to retrotransposition through the formation of G-quadruplex (G4) structures. We demonstrate that stabilization of the G4 motif of a human-specific LINE-1 element by small-molecule ligands stimulates retrotransposition.

A geometrical parametrization of C1'-C5' RNA ribose chemical shifts calculated by density functional theory.

It has been recently shown that NMR chemical shifts can be used to determine the structures of proteins. In order to begin to extend this type of approach to nucleic acids, we present an equation that relates the structural parameters and the (13)C chemical shifts of the ribose group. The parameters in the equation were determined by maximizing the agreement between the DFT-derived chemical shifts and those predicted through the equation for a database of ribose structures. Our results indicate that this type of approach represents a promising way of establishing quantitative and computationally efficient analytical relationships between chemical shifts and structural parameters in nucleic acids.