Crystal structure of paromomycin docked into the eubacterial ribosomal decoding A site

BACKGROUND: Aminoglycoside antibiotics interfere with translation in both gram-positive and gram-negative bacteria by binding to the tRNA decoding A site of the 16S ribosomal RNA. RESULTS: Crystals of complexes between oligoribonucleotides incorporating the sequence of the ribosomal A site of Escherichia coli and the aminoglycoside paromomycin have been solved at 2.5 A resolution. Each RNA fragment contains two A sites inserted between Watson-Crick pairs. The paromomycin molecules interact in an enlarged deep groove created by two bulging and one unpaired adenines. In both sites, hydroxyl and ammonium side chains of the antibiotic form 13 direct hydrogen bonds to bases and backbone atoms of the A site. In the best-defined site, 8 water molecules mediate 12 other hydrogen bonds between the RNA and the antibiotics. Ring I of paromomycin stacks over base G1491 and forms pseudo-Watson-Crick contacts with A1408. Both the hydroxyl group and one ammonium group of ring II form direct and water-mediated hydrogen bonds to the U1495oU1406 pair. The bulging conformation of the two adenines A1492 and A1493 is stabilized by hydrogen bonds between phosphate oxygens and atoms of rings I and II. The hydrophilic sites of the bulging A1492 and A1493 contact the shallow groove of G=C pairs in a symmetrical complex. CONCLUSIONS: Water molecules participate in the binding specificity by exploiting the antibiotic hydration shell and the typical RNA water hydration patterns. The observed contacts rationalize the protection, mutation, and resistance data. The crystal packing mimics the intermolecular contacts induced by aminoglycoside binding in the ribosome.     

Understanding the origins of bacterial resistance to aminoglycosides through molecular dynamics mutational study of the ribosomal A-site

Paromomycin is an aminoglycosidic antibiotic that targets the RNA of the bacterial small ribosomal subunit. It binds in the A-site, which is one of the three tRNA binding sites, and affects translational fidelity by stabilizing two adenines (A1492 and A1493) in the flipped-out state. Experiments have shown that various mutations in the A-site result in bacterial resistance to aminoglycosides. In this study, we performed multiple molecular dynamics simulations of the mutated A-site RNA fragment in explicit solvent to analyze changes in the physicochemical features of the A-site that were introduced by substitutions of specific bases. The simulations were conducted for free RNA and in complex with paromomycin. We found that the specific mutations affect the shape and dynamics of the binding cleft as well as significantly alter its electrostatic properties. The most pronounced changes were observed in the U1406C∶U1495A mutant, where important hydrogen bonds between the RNA and paromomycin were disrupted. The present study aims to clarify the underlying physicochemical mechanisms of bacterial resistance to aminoglycosides due to target mutations.     

Stacking geometry for non‐canonical G:U wobble base pair containing dinucleotide sequences in RNA: dispersion‐corrected DFT‐D study

Emergence of thousands of crystal structures of noncoding RNA molecules indicates its structural and functional diversity. RNA function is based upon a large variety of structural elements which are specifically assembled in the folded molecules. Along with the canonical Watson‐Crick base pairs, different orientations of the bases to form hydrogen‐bonded non‐canonical base pairs have also been observed in the available RNA structures. Frequencies of occurrences of different non‐canonical base pairs in RNA indicate their important role to maintain overall structure and functions of RNA. There are several reports on geometry and energetic stabilities of these non‐canonical base pairs. However, their stacking geometry and stacking stability with the neighboring base pairs are not well studied. Among the different non‐canonical base pairs, the G:U wobble base pair (G:U W:WC) is most frequently observed in the RNA double helices. Using quantum chemical method and available experimental data set we have studied the stacking geometry of G:U W:WC base pair containing dinucleotide sequences in roll‐slide parameters hyperspace for different values of twist. This study indicates that the G:U W:WC base pair can stack well with the canonical base pairs giving rise to large interaction energy. The overall preferred stacking geometry in terms of roll, twist and slide for the eleven possible dinucleotide sequences is seen to be quite dependent on their sequences. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 328–338, 2015.     

Probing the dynamics of restriction endonuclease NgoMIV‐DNA interaction by single‐molecule FRET

Many type II restriction endonucleases require two copies of their recognition sequence for optimal activity. Concomitant binding of two DNA sites by such an enzyme produces a DNA loop. Here we exploit single‐molecule Förster resonance energy transfer (smFRET) of surface‐immobilized DNA fragments to study the dynamics of DNA looping induced by tetrameric endonuclease NgoMIV. We have employed a DNA fragment with two NgoMIV recognition sites and a FRET dye pair such that upon protein‐induced DNA looping the dyes are brought to close proximity resulting in a FRET signal. The dynamics of DNA ‐ NgoMIV interactions proved to be heterogeneous, with individual smFRET trajectories exhibiting broadly different average looped state durations. Distinct types of the dynamics were attributed to different types of DNA ‐ protein complexes, mediated either by one NgoMIV tetramer simultaneously bound to two specific sites (“slow” trajectories) or by semi‐specific interactions of two DNA‐bound NgoMIV tetramers (“fast” trajectories), as well as to conformational heterogeneity of individual NgoMIV molecules.     

Linear free energy correlations for enzymatic base flipping: how do damaged base pairs facilitate specific recognition?

To efficiently maintain their genomic integrity, DNA repair glycosylases must exhibit high catalytic specificity for their cognate damaged bases using an extrahelical recognition mechanism. One possible contribution to specificity is the weak base pairing and inherent instability of damaged sites which may lead to increased extrahelicity of the damaged base and enhanced recognition of these sites. This model predicts that the binding affinity of the enzyme should increase as the thermodynamic stability of the lesion base pair decreases, because less work is required to extrude the base into its active site. We have tested this hypothesis with uracil DNA glycosylase (UDG) by constructing a series of DNA duplexes containing a single uracil (U) opposite a variety of bases (X) that formed from zero to three hydrogen bonds with U. Linear free energy (LFE) relationships were observed that correlated UDG binding affinity with the entropy and enthalpy of duplex melting, and the dynamic accessibility of the damaged site to chemical oxidation. These LFEs indicate that the increased conformational freedom of the damaged site brought about by enthalpic destabilization of the base pair promotes the formation of extrahelical states that enhance specific recognition by as much as 3000-fold. However, given the small stability differences between normal base pairs and U.A or U.G base pairs, relative base pair stability contributes little to the >10(6)-fold discrimination of UDG for uracil sites in cellular DNA. In contrast, the intrinsic instability of other more egregious DNA lesions may contribute significantly to the specificity of other DNA repair enzymes that bind to extrahelical bases.     

Molecular dynamics simulation on the allosteric analysis of the c‐di‐GMP class I riboswitch induced by ligand binding

Riboswitches are RNA molecules that regulate gene expression using conformation change, affected by binding of small molecule ligands. Although a number of ligand‐bound aptamer complex structures have been solved, it is important to know ligand‐free conformations of the aptamers in order to understand the mechanism of specific binding by ligands. In this paper, we use dynamics simulations on a series of models to characterize the ligand‐free and ligand‐bound aptamer domain of the c‐di‐GMP class I (GEMM‐I) riboswitch. The results revealed that the ligand‐free aptamer has a stable state with a folded P2 and P3 helix, an unfolded P1 helix and open binding pocket. The first Mg ions binding to the aptamer is structurally favorable for the successive c‐di‐GMP binding. The P1 helix forms when c‐di‐GMP is successive bound. Three key junctions J1/2, J2/3 and J1/3 in the GEMM‐I riboswitch contributing to the formation of P1 helix have been found. The binding of the c‐di‐GMP ligand to the GEMM‐I riboswitch induces the riboswitch's regulation through the direct allosteric communication network in GEMM‐I riboswitch from the c‐di‐GMP binding sites in the J1/2 and J1/3 junctions to the P1 helix, the indirect ones from those in the J2/3 and P2 communicating to P1 helix via the J1/2 and J1/3 media.     

Crystal structure of geneticin bound to a bacterial 16S ribosomal RNA A site oligonucleotide

Aminoglycosides are antibacterial molecules that decrease translation accuracy by binding to the decoding aminoacyl-tRNA site (A site) on 16S ribosomal RNA. We have solved the crystal structure of an RNA fragment containing the A site bound to geneticin at 2.40A resolution. Geneticin, also known as G418, is a gentamicin-related aminoglycoside: it contains three rings that are functionalized by hydroxyl, ammonium and methyl groups. The detailed comparison of the distinctive behaviour of geneticin (binding to pro- and eukaryotic A sites) with the crystallographic, biochemical and microbiological results obtained so far for aminoglycoside-A site complexes offers new insights on the system. The two sugar rings constituting the neamine part common to most of the aminoglycosides bind to the A site, as already observed in the crystal structures solved previously with paromomycin and tobramycin. The essential hydrogen bonds involving ring I (to A1408) and ring II (to the phosphate oxygen atoms of the bulged adenine bases 1492 and 1493 and to G1494) are conserved and additional contacts are observed from ring III (to phosphate oxygen atoms of G1405 and U1406). The present work illustrates a molecular basis of the range in sensitiveness exhibited by geneticin towards common point A site mutations associated to resistance phenotypes. In addition, analysis and comparisons of the structures cast light on the role played by the conserved U1406.U1495 pair in the recognition of the A site by aminoglycosides.     

Theaflavin‐3,3´‐digallate increases the antibacterial activity of β‐lactam antibiotics by inhibiting metallo‐β‐lactamase activity

Metallo‐β‐lactamases (MBLs) are some of the best known β‐lactamases produced by common Gram‐positive and Gram‐negative pathogens and are crucial factors in the rise of bacterial resistance against β‐lactam antibiotics. Although many types of β‐lactamase inhibitors have been successfully developed and used in clinical settings, no MBL inhibitors have been identified to date. Nitrocefin, checkerboard and time‐kill assays were used to examine the enzyme behaviour in vitro. Molecular docking calculation, molecular dynamics simulation, calculation of the binding free energy and ligand‐residue interaction decomposition were used for mechanistic research. The behaviour of the enzymes in vivo was investigated by a mouse infection experiment. We showed that theaflavin‐3,3´‐digallate (TFDG), a natural compound lacking antibacterial activities, can inhibit the hydrolysis of MBLs. In the checkerboard and time‐kill assays, we observed a synergistic effect of TFDG with β‐lactam antibiotics against methicillin‐resistant Staphylococcus aureus BAA1717. Molecular dynamics simulations were used to identify the mechanism of the inhibition of MBLs by TFDG, and we observed that the hydrolysis activity of the MBLs was restricted by the binding of TFDG to Gln242 and Ser369. Furthermore, the combination of TFDG with β‐lactam antibiotics showed effective protection in a mouse Staphylococcus aureus pneumonia model. These findings suggest that TFDG can effectively inhibit the hydrolysis activity of MBLs and enhance the antibacterial activity of β‐lactam antibiotics against pathogens in vitro and in vivo.     

Promiscuity in protein-RNA interactions: conformational ensembles facilitate molecular recognition in the spliceosome: conformational diversity in U2AF⁶⁵ facilitates binding to diverse RNA sequences

Here I discuss findings that suggest a universal mechanism for proteins (and RNA) to recognize and interact with various binding partners by selectively binding to different conformations that pre‐exist in the free protein's conformational ensemble. The tandem RNA recognition motif domains of splicing factor U2AF⁶⁵ fluctuate in solution between a predominately closed conformation in which the RNA binding site of one of the domains is blocked, and a lowly populated open conformation in which both RNA binding pockets are accessible. RNA binding to U2AF⁶⁵ may thus occur through the weakly populated open conformation, and the binding interaction stabilizes the open conformation. The conformational diversity observed in U2AF⁶⁵ might also facilitate binding to diverse RNA sequences as found in the polypyrimidine tracts that help define 3′ splice sites. Similar binding pathways in other systems have important consequences in biological regulation, molecular evolution, and information storage.     

Molecular dynamics simulation reveals conformational switching of water-mediated uracil-cytosine base-pairs in an RNA duplex

A 4 ns molecular dynamics simulation of an RNA duplex (r-GGACUUCGGUCC)(2 )in solution with Na+ and Cl- as counterions was performed. The X-ray structure of this duplex includes two water-mediated uracil-cytosine pairs. In contrast to the other base-pairs in the duplex the water-mediated pairs switch between different conformations. One conformation corresponds to the geometry of the water-mediated UC pairs in the duplex X-ray structure with water acting both as hydrogen-bond donor and acceptor. Another conformation is close to that of a water-mediated UC base-pair found in the X-ray structure of the 23 S rRNA sarcin/ricin domain. In this case the oxygen of the water molecule is linked to two-base donor sites. For a very short time also a direct UC base-pair and a further conformation that is similar to the one found in the RNA duplex structure but exhibits an increased H3(U)...N3(C) distance is observed. Water molecules with unusually long residence times are involved in the water-mediated conformations. These results indicate that the dynamic behaviour of the water-mediated UC base-pairs differs from that of the duplex Watson-Crick and non-canonical guanine-uracil pairs with two or three direct hydrogen bonds. The conformational variability and increased flexibility has to be taken into account when considering these base-pairs as RNA building blocks and as recognition motifs.     

Diverse mutants of HIV RRE IIB recognize wild‐type Rev ARM or Rev ARM R35G‐N40V

The binding of human immunodeficiency virus Rev protein via its arginine‐rich motif (ARM) to an internal loop in the Rev‐response element region IIB (RRE IIB) is necessary for viral replication. Many variant RNAs and ARMs that bind Rev and RRE IIB have been found. Despite the essential role of Rev asparagine 40 in recognition, the Rev ARM double‐mutant R35G‐N40V functions well in a Rev–RRE IIB reporter assay, indicating R35G‐N40V uses a distinct recognition strategy. To examine how RRE IIB may evolve specificity to wild‐type Rev ARM and R35G‐N40V, 10 RRE IIB libraries, each completely randomized in overlapping regions, were screened with wild‐type Rev ARM and R35G‐N40V using a reporter system based on bacteriophage λ N antitermination. Consistent with previous studies, a core element of RRE IIB did not vary, and substitutions occurred at conserved residues only in the presence of other substitutions. Notably, the groove‐widening, non‐canonical base‐pair G48:G71 was mutable to U48:G71 without strong loss of binding to wild‐type Rev ARM, suggesting U48:G71 performs the same role by adopting the nearly isosteric, reverse wobble base pair. Originating from RRE IIB, as few as one or two substitutions are sufficient to confer specificity to wild‐type Rev or Rev R35G‐N40. The diversity of RRE IIB mutants that maintain binding to wild‐type Rev ARM and R35G‐N40V supports neutral theories of evolution and illustrates paths by which viral RNA–protein interactions can evolve new specificities. Rev–RRE offers an excellent model with which to study the fine structure of how specificity evolves. Copyright © 2015 John Wiley & Sons, Ltd.     

Structural basis of siRNA recognition by TRBP double‐stranded RNA binding domains

The accurate cleavage of pre‐micro(mi)RNAs by Dicer and mi/siRNA guide strand selection are important steps in forming the RNA‐induced silencing complex (RISC). The role of Dicer binding partner TRBP in these processes remains poorly understood. Here, we solved the solution structure of the two N‐terminal dsRNA binding domains (dsRBDs) of TRBP in complex with a functionally asymmetric siRNA using NMR, EPR, and single‐molecule spectroscopy. We find that siRNA recognition by the dsRBDs is not sequence‐specific but rather depends on the RNA shape. The two dsRBDs can swap their binding sites, giving rise to two equally populated, pseudo‐symmetrical complexes, showing that TRBP is not a primary sensor of siRNA asymmetry. Using our structure to model a Dicer‐TRBP‐siRNA ternary complex, we show that TRBP's dsRBDs and Dicer's RNase III domains bind a canonical 19 base pair siRNA on opposite sides, supporting a mechanism whereby TRBP influences Dicer‐mediated cleavage accuracy by binding the dsRNA region of the pre‐miRNA during Dicer cleavage.     

Interaction of translation initiation factor IF1 with the E. coli ribosomal A site

Initiation Factor 1 (IF1) is required for the initiation of translation in Escherichia coli. However, the precise function of IF1 remains unknown. Current evidence suggests that IF1 is an RNA-binding protein that sits in the A site of the decoding region of 16 S rRNA. IF1 binding to 30 S subunits changes the reactivity of nucleotides in the A site to chemical probes. The N1 position of A1408 is enhanced, while the N1 positions of A1492 and A1493 are protected from reactivity with dimethyl sulfate (DMS). The N1-N2 positions of G530 are also protected from reactivity with kethoxal. Quantitative footprinting experiments show that the dissociation constant for IF1 binding to the 30 S subunit is 0.9 microM and that IF1 also alters the reactivity of a subset of Class III sites that are protected by tRNA, 50 S subunits, or aminoglycoside antibiotics. IF1 enhances the reactivity of the N1 position of A1413, A908, and A909 to DMS and the N1-N2 positions of G1487 to kethoxal. To characterize this RNA-protein interaction, several ribosomal mutants in the decoding region RNA were created, and IF1 binding to wild-type and mutant 30 S subunits was monitored by chemical modification and primer extension with allele-specific primers. The mutations C1407U, A1408G, A1492G, or A1493G disrupt IF1 binding to 30 S subunits, whereas the mutations G530A, U1406A, U1406G, G1491U, U1495A, U1495C, or U1495G had little effect on IF1 binding. Disruption of IF1 binding correlates with the deleterious phenotypic effects of certain mutations. IF1 binding to the A site of the 30 S subunit may modulate subunit association and the fidelity of tRNA selection in the P site through conformational changes in the 16 S rRNA.     

Optimization of a β‐sheet‐cap for long loop closure

Protein loops make up a large portion of the secondary structure in nature. But very little is known concerning loop closure dynamics and the effects of loop composition on fold stability. We have designed a small system with stable β‐sheet structures, including features that allow us to probe these questions. Using paired Trp residues that form aromatic clusters on folding, we are able to stabilize two β‐strands connected by varying loop lengths and composition (an example sequence: R W ITVTI – loop – KKIRV W E). Using NMR and CD, both fold stability and folding dynamics can be investigated for these systems. With the 16 residue loop peptide (sequence: R W ITVTI‐(GGGGKK)₂GGGG‐KKIRV W E) remaining folded (ΔG_U = 1.6 kJ/mol at 295K). To increase stability and extend the series to longer loops, we added an additional Trp/Trp pair in the loop flanking position. With this addition to the strands, the 16 residue loop (sequence: R W ITVRI W ‐(GGGGKK)₂GGGG‐ W KTIRV W E) supports a remarkably stable β‐sheet (ΔG_U = 6.3 kJ/mol at 295 K, T_m = ～55°C). Given the abundance of loops in binding motifs and between secondary structures, these constructs can be powerful tools for peptide chemists to study loop effects; with the Trp/Trp pair providing spectroscopic probes for assessing both stability and dynamics by NMR.     

The dynamic structural basis of differential enhancement of conformational stability by 5'- and 3'-dangling ends in RNA

Unpaired bases at the end of an RNA duplex (dangling ends) can stabilize the core duplex in a sequence-dependent manner and are important determinants of RNA folding, recognition, and functions. Using 2-aminopurine as a dangling end purine base, we have employed femtosecond time-resolved fluorescence spectroscopy, combined with UV optical melting, to quantitatively investigate the physical and structural nature of the stacking interactions between the dangling end bases and the terminal base pairs. A 3'-dangling purine base has a large subpopulation that stacks on the guanine base of the terminal GC or UG pair, either intrastrand or cross-strand depending on the orientation of the pair, thus providing stabilization of different magnitudes. On the contrary, a 5'-dangling purine base only has a marginal subpopulation that stacks on the purine of the same strand (intrastrand) but has little cross-strand stacking. Thus a 5'-dangling purine does not provide significant stabilization. These stacking structures are not static, and a dangling end base samples a range of stacked and unstacked conformations with respect to the terminal base pair. Femtosecond time-resolved anisotropy decay reveals certain hindered base conformational dynamics that occur on the picosecond to nanosecond time scales, which allow the dangling base to sample these substates. When the dangling purine is opposite to a U and is able to form a potential base pair at the end of the duplex, there is an interplay of base stacking and hydrogen-bonding interactions that depends on the orientation of the base pair relative to the adjacent GC pair. By resolving these populations that are dynamically exchanging on fast time scales, we elucidated the correlation between dynamic conformational distributions and thermodynamic stability.     

Control of box C/D snoRNP assembly by N6‐methylation of adenine

N⁶‐methyladenine is the most widespread mRNA modification. A subset of human box C/D snoRNA species have target GAC sequences that lead to formation of N⁶‐methyladenine at a key trans Hoogsteen‐sugar A·G base pair, of which half are methylated in vivo. The GAC target is conserved only in those that are methylated. Methylation prevents binding of the 15.5‐kDa protein and the induced folding of the RNA. Thus, the assembly of the box C/D snoRNP could in principle be regulated by RNA methylation at its critical first stage. Crystallography reveals that N⁶‐methylation of adenine prevents the formation of trans Hoogsteen‐sugar A·G base pairs, explaining why the box C/D RNA cannot adopt its kinked conformation. More generally, our data indicate that sheared A·G base pairs (but not Watson–Crick base pairs) are more susceptible to disruption by N⁶mA methylation and are therefore possible regulatory sites. The human signal recognition particle RNA and many related Alu retrotransposon RNA species are also methylated at N6 of an adenine that forms a sheared base pair with guanine and mediates a key tertiary interaction.