Co-reporter:Yuan Fu;Dr. Sreenivasa Rao Ramisetty;Nejmun Hussain ; Anne M. Baranger
ChemBioChem 2012 Volume 13( Issue 1) pp:112-119
Publication Date(Web):
DOI:10.1002/cbic.201100487
Abstract
Muscleblind-like proteins (MBNL) are RNA-binding proteins that bind to the poly(CUG) and poly(CCUG) sequences that are the causative agents of myotonic dystrophy. It has been suggested that as a result of binding to the repeating RNA sequences, MBNL1 is abnormally expressed and translocated, which leads to many of the misregulated events in myotonic dystrophy. In this work, steady-state fluorescence quenching experiments suggest that MBNL1 alters the structure of helical RNA targets upon binding, which may explain the selectivity of MBNL1 for less structured RNA sites. The removal of one pair of zinc fingers greatly impairs the binding affinity of MBNL1, which indicates that the two pairs of zinc fingers might possibly interact with RNA targets cooperatively. Alanine scanning mutagenesis results suggest that the binding energy may be distributed across the protein. Overall, the results presented here suggest that small molecules that stabilize the helical structure of poly(CUG) and poly(CCUG) RNAs will inhibit the formation of complexes with MBNL1.
Co-reporter:Yulia Benitex ;Anne M. Baranger
Journal of the American Chemical Society 2011 Volume 133(Issue 11) pp:3687-3689
Publication Date(Web):March 1, 2011
DOI:10.1021/ja102601h
The effects of modifying the electronic characteristics of nonpolar base analogues substituted at positions involved in stacking interactions between SL2 RNA and the U1A protein are described. A surprisingly large difference in the stability between complexes formed with base analogues that differ only in the position of substitution of a single fluorine atom is observed. The results of high-level ab initio calculations of the interactions between the nonpolar base analogue and the amino acid side chain correlate with the experimentally observed trends in complex stability, which suggests that changes in stacking interactions that result from varying the position and degree of fluorine substitution contribute to the effects of fluorine substitution on the stability of the U1A−SL2 RNA complex.
Co-reporter:Bethany L. Kormos;Susan N. Pieniazek;David L. Beveridge;Anne M. Baranger
Biopolymers 2011 Volume 95( Issue 9) pp:591-606
Publication Date(Web):
DOI:10.1002/bip.21616
Abstract
Molecular dynamics (MD) simulations were carried out to compare the free and bound structures of wild type U1A protein with several Phe56 mutant U1A proteins that bind the target stem loop 2 (SL2) RNA with a range of affinities. The simulations indicate the free U1A protein is more flexible than the U1A–RNA complex for both wild type and Phe56 mutant systems. A complete analysis of the hydrogen-bonding (HB) and non-bonded (VDW) interactions over the course of the MD simulations suggested that changes in the interactions in the free U1A protein caused by the Phe56Ala and Phe56Leu mutations may stabilize the helical character in loop 3, and contribute to the weak binding of these proteins to SL2 RNA. Compared with wild type, changes in HB and VDW interactions in Phe56 mutants of the free U1A protein are global, and include differences in β-sheet, loop 1 and loop 3 interactions. In the U1A–RNA complex, the Phe56Ala mutation leads to a series of differences in interactions that resonate through the complex, while the Phe56Leu and Phe56Trp mutations cause local differences around the site of mutation. The long-range networks of interactions identified in the simulations suggest that direct interactions and dynamic processes in both the free and bound forms contribute to complex stability. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 591–606, 2011.
Co-reporter:Sreenivasa Rao Ramisetty, Anne M. Baranger
Bioorganic & Medicinal Chemistry Letters 2010 Volume 20(Issue 10) pp:3134-3137
Publication Date(Web):15 May 2010
DOI:10.1016/j.bmcl.2010.03.090
The binding of a quinoline derivative (QD2) to a small RNA stem loop containing a 3′-dangling end (RNA1) has been studied. The compound was identified by first performing a similarity search of the NCI database of 250,000 compounds and then using computational docking with autodock to evaluate the binding of the resulting compounds to RNA1. Binding experiments using fluorescence and ITC methods revealed that QD2 binds cooperatively to four binding sites on RNA1 with equilibrium binding dissociation constants ranging from 8.2 (±0.3) to 12.5 (±4.2) μM. CD and UV titration experiments suggested that binding of QD2 changes the conformation of both RNA1 and the QD2 chromophore and stabilizes RNA1.
Co-reporter:Douglas M. Warui ;Anne M. Baranger
Journal of Medicinal Chemistry 2009 Volume 52(Issue 17) pp:5462-5473
Publication Date(Web):August 19, 2009
DOI:10.1021/jm900599v
We have screened the NCI diversity set library for molecules that bind specifically to stem loop 3 (SL3) RNA of the packaging element Ψ of HIV-1 using the docking programs DOCK and AutoDock, followed by MD simulations. The association of the predicted ligands with SL3 RNA was characterized using fluorescence, ITC, UV-melting, CD, and footprinting techniques. Nine ligands for SL3 RNA have been identified, four of which bind with higher affinity to SL3 RNA than to either single- or double-stranded RNA motifs. The most selective ligands, 9 (NSC252359) and 5 (NSC123111), bind SL3 RNA with dissociation constants of 11 μM and 98 μM, respectively. Compound 9 binds with 4−7-fold higher affinity to SL3 RNA than to the other tetraloops found in Ψ-RNA, SL2 and SL4 RNAs. The results suggest that both 9 and 5 bind to the stem region of SL3 RNA without large distortions of the SL3 RNA.
Co-reporter:Jonathan F. Arambula;Sreenivasa Rao Ramisetty;Anne M. Baranger;Steven C. Zimmerman
PNAS 2009 Volume 106 (Issue 38 ) pp:16068-16073
Publication Date(Web):2009-09-22
DOI:10.1073/pnas.0901824106
This work describes the rational design, synthesis, and study of a ligand that selectively complexes CUG repeats in RNA (and
CTG repeats in DNA) with high nanomolar affinity. This sequence is considered a causative agent of myotonic dystrophy type
1 (DM1) because of its ability to sequester muscleblind-like (MBNL) proteins. Ligand 1 was synthesized in two steps from commercially
available compounds, and its binding to CTG and CUG repeats in oligonucleotides studied. Isothermal titration calorimetry
studies of 1 with various sequences showed a preference toward the T-T mismatch (Kd of 390 ± 80 nM) with a 13-, 169-, and 85-fold reduction in affinity toward single C-C, A-A, and G-G mismatches, respectively.
Binding and Job analysis of 1 to multiple CTG step sequences revealed high affinity binding to every other T-T mismatch with
negative cooperativity for proximal T-T mismatches. The affinity of 1 for a (CUG)4 step provided a Kd of 430 nM with a binding stoichiometry of 1:1. The preference for the U-U in RNA was maintained with a 6-, >143-, and >143-fold
reduction in affinity toward single C-C, A-A, and G-G mismatches, respectively. Ligand 1 destabilized the complexes formed
between MBNL1N and (CUG)4 and (CUG)12 with IC50 values of 52 ± 20 μM and 46 ± 7 μM, respectively, and Ki values of 6 ± 2 μM and 7 ± 1 μM, respectively. These values were only minimally altered by the addition of competitor tRNA.
Ligand 1 does not destabilize the unrelated RNA-protein complexes the U1A-SL2 RNA complex and the Sex lethal-tra RNA complex. Thus, ligand 1 selectively destabilizes the MBNL1N-poly(CUG) complex.
Co-reporter:Divina Anunciado, Apratim Dhar, Martin Gruebele, Anne M. Baranger
Journal of Molecular Biology (20 May 2011) Volume 408(Issue 5) pp:896-908
Publication Date(Web):20 May 2011
DOI:10.1016/j.jmb.2011.02.054
The U1A–SL2 RNA complex is a model system for studying interactions between RNA and the RNA recognition motif (RRM), which is one of the most common RNA binding domains. We report here kinetic studies of dissociation of the U1A–SL2 RNA complex, using laser temperature jump and stopped-flow fluorescence methods with U1A proteins labeled with the intrinsic chromophore tryptophan. An analysis of the kinetic data suggests three phases of dissociation with time scales of ∼ 100 μs, ∼ 50 ms, and ∼ 2 s. We propose that the first step of dissociation is a fast rearrangement of the complex to form a loosely bound complex. The intermediate step is assigned to be the dissociation of the U1A–SL2 RNA complex, and the final step is assigned to a reorganization of the U1A protein structure into the conformation of the free protein. These assignments are consistent with previous proposals based on thermodynamic, NMR, and surface plasmon resonance experiments and molecular dynamics simulations. Together, these results begin to build a comprehensive model of the complex dynamic processes involved in the formation and dissociation of an RRM–RNA complex.Download high-res image (214KB)Download full-size imageHighlights► We studied the dissociation of the U1A protein–SL2 RNA complex using T-jump and stopped-flow fluorescence methods. Our kinetic data suggest a three-step mechanism of dissociation. Each step corresponds to a conformational change of the U1A protein. Our results contribute to an understanding of the complex dynamic processes involved in RRM–RNA association and dissociation.
