Present and future of NMR for RNA-protein complexes: A perspective of integrated structural biology.
J Magn Reson. 2014 Apr;241:126-36. doi: 10.1016/j.jmr.2013.10.007.
Nucleic acids are gaining enormous importance as key molecules in almost all biological processes. Most nucleic acids do not act in isolation but are generally associated with proteins to form high-molecular-weight nucleoprotein complexes. In this perspective article I focus on the structural studies of supra-molecular ribonucleoprotein (RNP) assemblies in solution by a combination of state-of-the-art TROSY-based NMR experiments and other structural biology techniques. I discuss ways how to combine sparse NMR data with low-resolution structural information from small-angle scattering, fluorescence and electron paramagnetic resonance spectroscopy to obtain the structure of large RNP particles by an integrated structural biology approach. In the last section I give a perspective for the study of RNP complexes by solid-state NMR.
The structure of the box C/D enzyme reveals regulation of RNA methylation.
Lapinaite, A., Simon, B., Skjaerven, L., Rakwalska-Bange, M., Gabel, F. & Carlomagno, T.
Nature. 2013 Oct 24;502(7472):519-23. doi: 10.1038/nature12581. Epub 2013 Oct 13.
Post-transcriptional modifications are essential to the cell life cycle, as they affect both pre-ribosomal RNA processing and ribosome assembly. The box C/D ribonucleoprotein enzyme that methylates ribosomal RNA at the 2'-O-ribose uses a multitude of guide RNAs as templates for the recognition of rRNA target sites. Two methylation guide sequences are combined on each guide RNA, the significance of which has remained unclear. Here we use a powerful combination of NMR spectroscopy and small-angle neutron scattering to solve the structure of the 390 kDa archaeal RNP enzyme bound to substrate RNA. We show that the two methylation guide sequences are located in different environments in the complex and that the methylation of physiological substrates targeted by the same guide RNA occurs sequentially. This structure provides a means for differential control of methylation levels at the two sites and at the same time offers an unexpected regulatory mechanism for rRNA folding.
A Suite of Solid-State NMR Experiments for RNA Intranucleotide Resonance Assignment in a 21 kDa Protein-RNA Complex.
Marchanka, A., Simon, B. & Carlomagno, T.
Angew Chem Int Ed Engl. 2013 Jul 26. doi: 10.1002/anie.201304779.
Intranucleotide resonance of the 26mer box C/D RNA in complex with the L7Ae protein were assigned by solid-state NMR (ssNMR; see picture) spectroscopy. This investigation opens the way for studying RNA in large protein-RNA complexes by ssNMR spectroscopy.
Accounting for conformational variability in protein-ligand docking with NMR-guided rescoring.
Skjaerven, L., Codutti, L., Angelini, A., Grimaldi, M., Latek, D., Monecke, P., Dreyer, M.K. & Carlomagno, T.
J Am Chem Soc. 2013 Apr 17;135(15):5819-27. doi: 10.1021/ja4007468. Epub 2013 Apr8.
A key component to success in structure-based drug design is reliable information on protein-ligand interactions. Recent development in NMR techniques has accelerated this process by overcoming some of the limitations of X-ray crystallography and computational protein-ligand docking. In this work we present a new scoring protocol based on NMR-derived interligand INPHARMA NOEs to guide the selection of computationally generated docking modes. We demonstrate the performance in a range of scenarios, encompassing traditionally difficult cases such as docking to homology models and ligand dependent domain rearrangements. Ambiguities associated with sparse experimental information are lifted by searching a consensus solution based on simultaneously fitting multiple ligand pairs. This study provides a previously unexplored integration between molecular modeling and experimental data, in which interligand NOEs represent the key element in the rescoring algorithm. The presented protocol should be widely applicable for protein-ligand docking also in a different context from drug design and highlights the important role of NMR-based approaches to describe intermolecular ligand-receptor interactions.
Structural principles of RNA catalysis in a 2'-5' lariat-forming ribozyme.
Carlomagno, T., Amata, I., Codutti, L., Falb, M., Fohrer, J., Masiewicz, P. & Simon, B.
J Am Chem Soc. 2013 Mar 20;135(11):4403-11. doi: 10.1021/ja311868t. Epub 2013 Mar8.
RNA-catalyzed lariat formation is present in both eukaryotes and prokaryotes. To date we lack structural insights into the catalytic mechanism of lariat-forming ribozymes. Here, we study an artificial 2'-5' AG1 lariat-forming ribozyme that shares the sequence specificity of lariat formation with the pre-mRNA splicing reaction. Using NMR, we solve the structure of the inactive state of the ribozyme in the absence of magnesium. The reaction center 5'-guanosine appears to be part of a helix with an exceptionally widened major groove, while the lariat-forming A48 is looped out at the apex of a pseudoknot. The model of the active state built by mutational analysis, molecular modeling, and small-angle X-ray scattering suggests that A48 is recognized by a conserved adenosine, juxtaposed to the 5'-guanosine in one base-pair step distance, while the G1-N7 coordinates a magnesium ion essential for the activation of the nucleophile. Our findings offer implications for lariat formation in RNA enzymes including the mechanism of the recognition of the branch-site adenosine.
Protein-RNA Interfaces Probed by (1) H-Detected MAS Solid-State NMR Spectroscopy.
Asami, S., Rakwalska-Bange, M., Carlomagno, T. & Reif, B.
Angew Chem Int Ed Engl. 2013 Feb 18;52(8):2345-9. doi: 10.1002/anie.201208024.Epub 2013 Jan 18.
Both protonated and deuterated samples were employed in the study of the L7Ae box C/D RNA complex by (1) H-detected solid-state NMR spectroscopy. This approach yielded high-resolution spectra and was used to determine the intermolecular interface and extract structural parameters with high accuracy.
Towards Improved Quality of GPCR Models by Usage of Multiple Templates and Profile-Profile Comparison.
Latek, D., Pasznik, P., Carlomagno, T. & Filipek, S.
PLoS One. 2013;8(2):e56742. doi: 10.1371/journal.pone.0056742. Epub 2013 Feb 28.
G-protein coupled receptors (GPCRs) are targets of nearly one third of the drugs at the current pharmaceutical market. Despite their importance in many cellular processes the crystal structures are available for less than 20 unique GPCRs of the Rhodopsin-like class. Fortunately, even though involved in different signaling cascades, this large group of membrane proteins has preserved a uniform structure comprising seven transmembrane helices that allows quite reliable comparative modeling. Nevertheless, low sequence similarity between the GPCR family members is still a serious obstacle not only in template selection but also in providing theoretical models of acceptable quality. An additional level of difficulty is the prediction of kinks and bulges in transmembrane helices. Usage of multiple templates and generation of alignments based on sequence profiles may increase the rate of success in difficult cases of comparative modeling in which the sequence similarity between GPCRs is exceptionally low. Here, we present GPCRM, a novel method for fast and accurate generation of GPCR models using averaging of multiple template structures and profile-profile comparison. In particular, GPCRM is the first GPCR structure predictor incorporating two distinct loop modeling techniques: Modeller and Rosetta together with the filtering of models based on the Z-coordinate. We tested our approach on all unique GPCR structures determined to date and report its performance in comparison with other computational methods targeting the Rhodopsin-like class. We also provide a database of precomputed GPCR models of the human receptors from that class. AVAILABILITY: GPCRM SERVER AND DATABASE: http://gpcrm.biomodellab.eu.
Synthesis and biological evaluation of a water-soluble derivative of the potent V-ATPase inhibitor archazolid.
Persch, E., Basile, T., Bockelmann, S., Huss, M., Wieczorek, H., Carlomagno, T. & Menche, D.
Bioorg Med Chem Lett. 2012 Dec 15;22(24):7735-8. doi: 10.1016/j.bmcl.2012.09.081.Epub 2012 Oct 13.
The water-solubility of the highly potent V-ATPase inhibitors archazolid A and the glucosylated derivative archazolid C was studied in the presence of a wide range of cosolvents, revealing very low solubilites. The first water-soluble analogue was then designed, synthesized, and evaluated for V-ATPase inhibitory activity in vitro.
Phf19 links methylated Lys36 of histone H3 to regulation of Polycomb activity.
Ballare, C., Lange, M., Lapinaite, A., Martin, G.M., Morey, L., Pascual, G., Liefke, R., Simon, B., Shi, Y., Gozani, O., Carlomagno, T., Benitah, S.A. & Di Croce, L.
Nat Struct Mol Biol. 2012 Dec;19(12):1257-65. doi: 10.1038/nsmb.2434. Epub 2012Oct 28.
Polycomb-group proteins are transcriptional repressors with essential roles in embryonic development. Polycomb repressive complex 2 (PRC2) contains the methyltransferase activity for Lys27. However, the role of other histone modifications in regulating PRC2 activity is just beginning to be understood. Here we show that direct recognition of methylated histone H3 Lys36 (H3K36me), a mark associated with activation, by the PRC2 subunit Phf19 is required for the full enzymatic activity of the PRC2 complex. Using NMR spectroscopy, we provide structural evidence for this interaction. Furthermore, we show that Phf19 binds to a subset of PRC2 targets in mouse embryonic stem cells and that this is required for their repression and for H3K27me3 deposition. These findings show that the interaction of Phf19 with H3K36me2 and H3K36me3 is essential for PRC2 complex activity and for proper regulation of gene repression in embryonic stem cells.
The description of protein internal motions aids selection of ligand binding poses by the INPHARMA method.
Stauch, B., Orts, J. & Carlomagno, T.
J Biomol NMR. 2012 Nov;54(3):245-56. doi: 10.1007/s10858-012-9662-1. Epub 2012Sep 22.
Protein internal motions influence observables of NMR experiments. The effect of internal motions occurring at the sub-nanosecond timescale can be described by NMR order parameters. Here, we report that the use of order parameters derived from Molecular Dynamics (MD) simulations of two holo-structures of Protein Kinase A increase the discrimination power of INPHARMA, an NMR based methodology that selects docked ligand orientations by maximizing the correlation of back-calculated to experimental data. By including internal motion in the back-calculation of the INPHARMA transfer, we obtain a more realistic description of the system, which better represents the experimental data. Furthermore, we propose a set of generic order parameters, derived from MD simulations of globular proteins, which can be used in the back-calculation of INPHARMA NOEs for any protein-ligand complex, thus by-passing the need of obtaining system-specific order parameters for new protein-ligand complexes.
Understanding the inhibitory effect of highly potent and selective archazolides binding to the vacuolar ATPase.
Dreisigacker, S., Latek, D., Bockelmann, S., Huss, M., Wieczorek, H., Filipek, S., Gohlke, H., Menche, D. & Carlomagno, T.
J Chem Inf Model. 2012 Aug 27;52(8):2265-72. doi: 10.1021/ci300242d. Epub 2012Jul 25.
Vacuolar ATPases are a potential therapeutic target because of their involvement in a variety of severe diseases such as osteoporosis or cancer. Archazolide A (1) and related analogs have been previously identified as selective inhibitors of V-ATPases with potency down to the subnanomolar range. Herein we report on the determination of the ligand binding mode by a combination of molecular docking, molecular dynamics simulations, and biochemical experiments, resulting in a sound model for the inhibitory mechanism of this class of putative anticancer agents. The binding site of archazolides was confirmed to be located in the equatorial region of the membrane-embedded V(O)-rotor, as recently proposed on the basis of site-directed mutagenesis. Quantification of the bioactivity of a series of archazolide derivatives, together with the docking-derived binding mode of archazolides to the V-ATPase, revealed favorable ligand profiles, which can guide the development of a simplified archazolide analog with potential therapeutic relevance.
NMR in natural products: understanding conformation, configuration and receptor interactions.
Nat Prod Rep. 2012 May 1;29(5):536-54. doi: 10.1039/c2np00098a. Epub 2012 Mar 29.
Covering: up to 2011. Natural products are of tremendous importance in both traditional and modern medicine. For medicinal chemistry natural products represent a challenge, as their chemical synthesis and modification are complex processes, which require many, often stereo-selective, synthetic steps. A prerequisite for the design of analogs of natural products, with more accessible synthetic routes, is the availability of their bioactive conformation. Nuclear Magnetic Resonance (NMR) spectroscopy and X-ray crystallography are the two techniques of choice to investigate the structure of natural products. In this review, I describe the most recent advances in NMR to study the conformation of natural products either free in solution or bound to their cellular receptors. In chapter 2, I focus on the use of residual dipolar couplings (RDC). On the basis of a few examples, I discuss the benefit of complementing classical NMR parameters, such as NOEs and scalar couplings, with dipolar couplings to simultaneously determine both the conformation and the relative configuration of natural products in solution. Chapter 3 is dedicated to the study of the structure of natural products in complex with their cellular receptors and is further divided in two sections. In the first section, I describe two solution-state NMR methodologies to investigate the binding mode of low-affinity ligands to macromolecular receptors. The first approach, INPHARMA (Interligand Noes for PHArmacophore Mapping), is based on the observation of interligand NOEs between two small molecules binding competitively to a common receptor. INPHARMA reveals the relative binding mode of the two ligands, thus allowing ligand superimposition. The second approach is based on paramagnetic relaxation enhancement (PRE) of ligand resonances in the presence of a receptor containing a paramagnetic center. In the second section, I focus on solid-state NMR spectroscopy as a tool to access the bioactive conformation of natural products in complex with macromolecular receptors.
Hyperpolarized binding pocket nuclear overhauser effect for determination of competitive ligand binding.
Lee, Y., Zeng, H., Mazur, A., Wegstroth, M., Carlomagno, T., Reese, M., Lee, D., Becker, S., Griesinger, C. & Hilty, C.
Angew Chem Int Ed Engl. 2012 May 21;51(21):5179-82. doi: 10.1002/anie.201201003.Epub 2012 Apr 12.
Protein-mediated polarization transfer: Ligands L1 and L2 that competitively bind to a protein are subject to indirect spin-polarization transfer through the binding site of the protein. If protons H(L1) of one ligand are hyperpolarized by dynamic nuclear polarization (DNP, see picture), signal intensities in the NMR spectrum of the second ligand become enhanced. The relative build-up of signal of the second ligand yields information on its binding epitope.
An NMR-based scoring function improves the accuracy of binding pose predictions by docking by two orders of magnitude.
Orts, J., Bartoschek, S., Griesinger, C., Monecke, P. & Carlomagno, T.
J Biomol NMR. 2012 Jan;52(1):23-30. doi: 10.1007/s10858-011-9590-5. Epub 2011 Dec14.
Low-affinity ligands can be efficiently optimized into high-affinity drug leads by structure based drug design when atomic-resolution structural information on the protein/ligand complexes is available. In this work we show that the use of a few, easily obtainable, experimental restraints improves the accuracy of the docking experiments by two orders of magnitude. The experimental data are measured in nuclear magnetic resonance spectra and consist of protein-mediated NOEs between two competitively binding ligands. The methodology can be widely applied as the data are readily obtained for low-affinity ligands in the presence of non-labelled receptor at low concentration. The experimental inter-ligand NOEs are efficiently used to filter and rank complex model structures that have been pre-selected by docking protocols. This approach dramatically reduces the degeneracy and inaccuracy of the chosen model in docking experiments, is robust with respect to inaccuracy of the structural model used to represent the free receptor and is suitable for high-throughput docking campaigns.
Computational techniques are valuable tools for the discovery of protein-protein interaction inhibitors: the 14-3-3sigma case.
Corradi, V., Mancini, M., Santucci, M.A., Carlomagno, T., Sanfelice, D., Mori, M., Vignaroli, G., Falchi, F., Manetti, F., Radi, M. & Botta, M.
Bioorg Med Chem Lett. 2011 Nov 15;21(22):6867-71. Epub 2011 Sep 10.
Targeting the binding site of 14-3-3 proteins lets the release of partner proteins involved in cell cycle progression, apoptosis, cytoskeletal rearrangement and transcriptional regulation and may therefore be regarded as an alternative strategy to integrate conventional therapeutic approaches against cancer. In the present work, we report the identification of two new small molecule inhibitors of 14-3-3sigma/c-Abl protein-protein interaction (BV01 and BV101) discovered by means of computational methods. The most interesting compound (BV01) showed a lethal dose (LD(50)) in the low micromolar range against Ba/F3 murine cell lines expressing the Imatinib (IM)-sensitive wild type Bcr-Abl construct and the IM-resistant Bcr-Abl mutation T315I. BV01 interaction with 14-3-3sigma was demonstrated by NMR studies and elucidated by docking. It blocked the binding domain of 14-3-3sigma, hence promoting the release of the partner protein c-Abl (the one not involved in Bcr rearrangement), and its translocation to both the nuclear compartment and mitochondrial membranes to induce a pro-apoptotic response. Our results advance BV01 as a confirmed hit compound capable of eliciting apoptotic death of Bcr-Abl-expressing cells by interfering with 14-3-3sigma/c-Abl protein-protein interaction.
Recognition of 2'-O-methylated 3'-end of piRNA by the PAZ domain of a Piwi protein.
Simon, B., Kirkpatrick, J.P., Eckhardt, S., Reuter, M., Rocha, E.A., Andrade-Navarro, M.A., Sehr, P., Pillai, R.S. & Carlomagno, T.
Structure. 2011 Feb 9;19(2):172-80. Epub 2011 Jan 20.
Piwi proteins are germline-specific Argonautes that associate with small RNAs called Piwi-interacting RNAs (piRNAs), and together with these RNAs are implicated in transposon silencing. The PAZ domain of Argonaute proteins recognizes the 3'-end of the RNA, which in the case of piRNAs is invariably modified with a 2'-O-methyl group. Here, we present the solution structure of the PAZ domain from the mouse Piwi protein, MIWI, in complex with an 8-mer piRNA mimic. The methyl group is positioned in a hydrophobic cavity made of conserved amino acids from strand beta7 and helix alpha3, where it is contacted by the side chain of methionine-382. Our structure is similar to that of Ago-PAZ, but subtle differences illustrate how the PAZ domain has evolved to accommodate distinct 3' ends from a variety of RNA substrates.
Structure of the K-turn U4 RNA: a combined NMR and SANS study.
Falb, M., Amata, I., Gabel, F., Simon, B. & Carlomagno, T.
Nucleic Acids Res. 2010 Oct;38(18):6274-85. doi: 10.1093/nar/gkq380. Epub 2010May 13.
K-turn motifs are universal RNA structural elements providing a binding platform for proteins in several cellular contexts. Their characteristic is a sharp kink in the phosphate backbone that puts the two helical stems of the protein-bound RNA at an angle of 60 degrees . However, to date no high-resolution structure of a naked K-turn motif is available. Here, we present the first structural investigation at atomic resolution of an unbound K-turn RNA (the spliceosomal U4-Kt RNA) by a combination of NMR and small-angle neutron scattering data. With this study, we wish to address the question whether the K-turn structural motif assumes the sharply kinked conformation in the absence of protein binders and divalent cations. Previous studies have addressed this question by fluorescence resonance energy transfer, biochemical assays and molecular dynamics simulations, suggesting that the K-turn RNAs exist in equilibrium between a kinked conformation, which is competent for protein binding, and a more extended conformation, with the population distribution depending on the concentration of divalent cations. Our data shows that the U4-Kt RNA predominantly assumes the more extended conformation in the absence of proteins and divalent cations. The internal loop region is well structured but adopts a different conformation from the one observed in complex with proteins. Our data suggests that the K-turn consensus sequence does not per se code for the kinked conformation; instead the sharp backbone kink requires to be stabilized by protein binders.
Interaction of epothilone B (patupilone) with microtubules as detected by two-dimensional solid-state NMR spectroscopy.
Kumar, A., Heise, H., Blommers, M.J., Krastel, P., Schmitt, E., Petersen, F., Jeganathan, S., Mandelkow, E.M., Carlomagno, T., Griesinger, C. & Baldus, M.
Angew Chem Int Ed Engl. 2010 Oct 4;49(41):7504-7. Europe PMC
Peptide ligands selected with CD4-induced epitopes on native dualtropic HIV-1 envelope proteins mimic extracellular coreceptor domains and bind to HIV-1 gp120 independently of coreceptor usage.
Dervillez, X., Klaukien, V., Durr, R., Koch, J., Kreutz, A., Haarmann, T., Stoll, M., Lee, D., Carlomagno, T., Schnierle, B., Mobius, K., Konigs, C., Griesinger, C. & Dietrich, U.
J Virol. 2010 Oct;84(19):10131-8. Epub 2010 Jul 21.
During HIV-1 entry, binding of the viral envelope glycoprotein gp120 to the cellular CD4 receptor triggers conformational changes resulting in exposure of new epitopes, the highly conserved CD4-induced (CD4i) epitopes that are essential for subsequent binding to chemokine receptor CCR5 or CXCR4. Due to their functional conservation, CD4i epitopes represent attractive viral targets for HIV-1 entry inhibition. The aim of the present study was to select peptide ligands for CD4i epitopes on native dualtropic (R5X4) HIV-1 envelope (Env) glycoproteins by phage display. Using CD4-activated retroviral particles carrying Env from the R5X4 HIV-1 89.6 strain as the target, we performed screenings of random peptide phage libraries under stringent selection conditions. Selected peptides showed partial identity with amino acids in the extracellular domains of CCR5/CXCR4, including motifs rich in tyrosines and aspartates at the N terminus known to be important for gp120 binding. A synthetic peptide derivative (XD3) corresponding to the most frequently selected phages was optimized for Env binding on peptide arrays. Interestingly, the optimized peptide could bind specifically to gp120 derived from HIV-1 strains with different coreceptor usage, competed with binding of CD4i-specific monoclonal antibody (MAb) 17b, and interfered with entry of both a CCR5 (R5)-tropic and a CXCR4 (X4)-tropic Env pseudotyped virus. This peptide ligand therefore points at unique properties of CD4i epitopes shared by gp120 with different coreceptor usage and could thus serve to provide new insight into the conserved structural details essential for coreceptor binding for further drug development.
The tubulin-bound structure of the antimitotic drug tubulysin.
Kubicek, K., Grimm, S.K., Orts, J., Sasse, F. & Carlomagno, T.
Angew Chem Int Ed Engl. 2010 Jun 28;49(28):4809-12. doi: 10.1002/anie.200906828. Europe PMC
The binding mode of side chain- and C3-modified epothilones to tubulin.
Erdelyi, M., Navarro-Vazquez, A., Pfeiffer, B., Kuzniewski, C.N., Felser, A., Widmer, T., Gertsch, J., Pera, B., Diaz, J.F., Altmann, K.H. & Carlomagno, T.
ChemMedChem. 2010 Jun 7;5(6):911-20.
The tubulin-binding mode of C3- and C15-modified analogues of epothilone A (Epo A) was determined by NMR spectroscopy and computational methods and compared with the existing structural models of tubulin-bound natural Epo A. Only minor differences were observed in the conformation of the macrocycle between Epo A and the C3-modified analogues investigated. In particular, 3-deoxy- (compound 2) and 3-deoxy-2,3-didehydro-Epo A (3) were found to adopt similar conformations in the tubulin-binding cleft as Epo A, thus indicating that the 3-OH group is not essential for epothilones to assume their bioactive conformation. None of the available models of the tubulin-epothilone complex is able to fully recapitulate the differences in tubulin-polymerizing activity and microtubule-binding affinity between C20-modified epothilones 6 (C20-propyl), 7 (C20-butyl), and 8 (C20-hydroxypropyl). Based on the results of transferred NOE experiments in the presence of tubulin, the isomeric C15 quinoline-based Epo B analogues 4 and 5 show very similar orientations of the side chain, irrespective of the position of the nitrogen atom in the quinoline ring. The quinoline side chain stacks on the imidazole moiety of beta-His227 with equal efficiency in both cases, thus suggesting that the aromatic side chain moiety in epothilones contributes to tubulin binding through strong van der Waals interactions with the protein rather than hydrogen bonding involving the heteroaromatic nitrogen atom. These conclusions are in line with existing tubulin polymerization and microtubule-binding data for 4, 5, and Epo B.
Synthesis and biological characterization of argyrin F.
Bulow, L., Nickeleit, I., Girbig, A.K., Brodmann, T., Rentsch, A., Eggert, U., Sasse, F., Steinmetz, H., Frank, R., Carlomagno, T., Malek, N.P. & Kalesse, M.
ChemMedChem. 2010 Jun 7;5(6):832-6. Europe PMC
Elucidation of the structure and intermolecular interactions of a reversible cyclic-peptide inhibitor of the proteasome by NMR spectroscopy and molecular modeling.
Stauch, B., Simon, B., Basile, T., Schneider, G., Malek, N.P., Kalesse, M. & Carlomagno, T.
Angew Chem Int Ed Engl. 2010 May 25;49(23):3934-8. Europe PMC
Intermolecular protein-RNA interactions revealed by 2D 31P-15N magic angle spinning solid-state NMR spectroscopy.
Jehle, S., Falb, M., Kirkpatrick, J.P., Oschkinat, H., van Rossum, B.J., Althoff, G. & Carlomagno, T.
J Am Chem Soc. 2010 Mar 24;132(11):3842-6.
The structural investigation of large RNP complexes by X-ray crystallography can be a difficult task due to the flexibility of the RNA and of the protein-RNA interfaces, which may hinder crystallization. In these cases, NMR spectroscopy is an attractive alternative to crystallography, although the large size of typical RNP complexes may limit the applicability of solution NMR. Solid-state NMR spectroscopy, however, is not subject to any intrinsic limitations with respect to the size of the object under investigation, with restrictions imposed solely by the sensitivity of the instrumentation. In addition, it does not require large, well-ordered crystals and can therefore be applied to flexible, partially disordered complexes. Here we show for the first time that solid-state NMR spectroscopy can be used to probe intermolecular interactions at the protein-RNA interface in RNP complexes. Distances between the (15)N nuclei of the protein backbone and the (31)P nuclei of the RNA backbone can be measured in TEDOR experiments and used as restraints in structure calculations. The distance measurement is accurate, as proven for the test case of the L7Ae-box C/D RNA complex, for which a crystal structure is available. The results presented here reveal the as yet unexplored potential of solid-state NMR spectroscopy in the investigation of large RNP complexes.
Modular architecture of Munc13/calmodulin complexes: dual regulation by Ca2+ and possible function in short-term synaptic plasticity.
Rodriguez-Castaneda, F., Maestre-Martinez, M., Coudevylle, N., Dimova, K., Junge, H., Lipstein, N., Lee, D., Becker, S., Brose, N., Jahn, O., Carlomagno, T. & Griesinger, C.
EMBO J. 2010 Feb 3;29(3):680-91. doi: 10.1038/emboj.2009.373. Epub 2009 Dec 10.
Ca(2+) signalling in neurons through calmodulin (CaM) has a prominent function in regulating synaptic vesicle trafficking, transport, and fusion. Importantly, Ca(2+)-CaM binds a conserved region in the priming proteins Munc13-1 and ubMunc13-2 and thus regulates synaptic neurotransmitter release in neurons in response to residual Ca(2+) signals. We solved the structure of Ca(2+)(4)-CaM in complex with the CaM-binding domain of Munc13-1, which features a novel 1-5-8-26 CaM-binding motif with two separated mobile structural modules, each involving a CaM domain. Photoaffinity labelling data reveal the same modular architecture in the complex with the ubMunc13-2 isoform. The N-module can be dissociated with EGTA to form the half-loaded Munc13/Ca(2+)(2)-CaM complex. The Ca(2+) regulation of these Munc13 isoforms can therefore be explained by the modular nature of the Munc13/Ca(2+)-CaM interactions, where the C-module provides a high-affinity interaction activated at nanomolar [Ca(2+)](i), whereas the N-module acts as a sensor at micromolar [Ca(2+)](i). This Ca(2+)/CaM-binding mode of Munc13 likely constitutes a key molecular correlate of the characteristic Ca(2+)-dependent modulation of short-term synaptic plasticity.
Drug Design for G-Protein-Coupled Receptors by a Ligand-Based NMR Method.
Bartoschek, S., Klabunde, T., Defossa, E., Dietrich, V., Stengelin, S., Griesinger, C., Carlomagno, T., Focken, I. & Wendt, K.U.
Angew Chem Int Ed Engl. 2010 49, 1426-1429 Europe PMC
(1)H, (13)C and (15)N resonance assignments of the Calmodulin-Munc13-1 peptide complex.
Rodriguez-Castaneda, F., Coudevylle, N., Becker, S., Brose, N., Carlomagno, T. & Griesinger, C.
Biomolecular NMR Assignments 2010 4, 45-48
Ca(2+)-Calmodulin binding to the variable N-terminal region of the diacylglycerol/phorbol ester-binding UNC13/Munc13 family of proteins modulates the short-term synaptic plasticity characteristics in neurons. Here, we report the sequential backbone and side chain resonance assignment of the Ca(2+)-Calmodulin/Munc13-1(458-492) peptide complex at pH 6.8 and 35 degrees C (BMRB No. 15470).
The INPHARMA technique for pharmacophore mapping: A theoretical guide to the method.
Orts, J., Griesinger, C. & Carlomagno, T.
J Magn Reson. 2009 Sep;200(1):64-73. Epub 2009 Jun 12.
During the process of drug discovery, INPHARMA can be used to derive the structure of receptor/lead compound complexes binding to each other with a K(d) in the microM to mM range. To be successful, the methodology needs adjustment of various parameters that depend on the physical constants of the binding event and on the receptor size. Here we present a thorough theoretical analysis of the INPHARMA interligand NOE effect in dependence of experimental parameters and physical constants. This analysis helps the experimentalist to choose the correct experimental parameters and consequentially to achieve optimal performance of the methodology.
An efficient strategy for the determination of the three-dimensional architecture of ribonucleoprotein complexes by the combination of a few easily accessible NMR and biochemical data: intermolecular recognition in a U4 spliceosomal complex.
Li, P., Kirkpatrick, J. & Carlomagno, T.
J Mol Biol. 2009 May 1;388(2):283-98. Epub 2009 Mar 10.
Ribonucleoprotein (RNP) complexes are involved in several cellular processes, including RNA processing, transcription and translation. RNP structures are often dynamic in nature, undergoing significant remodeling during the course of their function. Visualization of the three-dimensional arrangement of single components in the complex and characterization of the intermolecular interactions are essential for understanding the mechanisms of operation. Crystallization either is not always achievable for these highly dynamic RNP particles or requires trimming the complex to a stable, well-structured core that lacks the flexible, regulatory domains. Alternative techniques that can provide structural information for complexes in solution under native conditions, where they retain their natural dynamic properties, are needed. In this study, we explored the possibility of using a combination of NMR, biochemical data and molecular modeling to generate an accurate high-resolution model of RNP complexes. We applied this strategy to the ternary hPrp31 (human Prp31)-15.5K-U4 5'-SL (stem-loop) spliceosomal complex, which, due to its large size and instability and because of the difficulty in obtaining isotopically labeled hPrp31, is not amenable to complete structure determination by NMR. We designed a protocol where the protein-protein interaction surface is defined for 15.5K by NMR data, while the relative orientations of the U4 RNA and the hPrp31 protein are described by mutational and cross-linking data. Using these data in a restrained ensemble docking protocol, we obtained a model for the ternary complex that reveals a novel rationale for the hierarchical assembly of the complex. Comparison of the docking model with the crystal structure recently obtained for a trimmed version of the complex reveals the high accuracy of the docking model, even down to an atomic level. This work shows that the architecture of large RNP complexes is within reach by NMR investigation in solution even for those cases where a traditional structural determination cannot be performed.
Probing mutation-induced structural perturbations by refinement against residual dipolar couplings: application to the U4 spliceosomal RNP complex.
Kirkpatrick, J.P., Li, P. & Carlomagno, T.
Chembiochem. 2009 Apr 17;10(6):1007-14.
Confident interpretation of biochemical experiments performed with mutated proteins relies on verification of the integrity of the mutant structures. We present a simple and rapid refinement protocol for comparing the structures of mutated and wild-type proteins. Our approach involves measurement of residual dipolar couplings, and only requires assignment of the backbone resonances of the mutant species. We demonstrate application of the protocol to a mutant of the 15.5K protein, a core component of the U4 spliceosomal ribonucleoprotein (RNP) complex. Confirmation of the unperturbed structure of the mutated protein prompted re-examination of a previous mutagenesis study and indicated that the interpretation of mutant binding affinities in terms of direct interfacial contacts should be applied with caution.
NMR assignments of HIV-2 TAR RNA.
Carlomagno, T., Amata, I., Williamson, J.R. & Hennig, M.
Biomol NMR Assign. 2008 Dec;2(2):167-9. Epub 2008 Oct 12.
We report nearly complete assignment for all (1)H, (13)C, (31)P, and (15)N resonances in the 30-nucleotide stem-loop HIV-2 TAR RNA located at the 5' end of all viral mRNAs.
Field dependent dynamic nuclear polarization with radicals in aqueous solution.
Höfer P, Parigi G, Luchinat C, Carl P, Guthausen G, Reese M, Carlomagno, T., Griesinger C, Bennati M.
J Am Chem Soc. 2008/03 130(11) 3254-3255 Europe PMC
Conformational Preferences of Natural and C3-Modified Epothilones in Aqueous Solution.
Erdelyi, M., Pfeiffer, B., Hauenstein, K., Fohrer, J., Gertsch, J., Altmann, K.H. & Carlomagno, T.
J Med Chem. 2008 Feb 14;.
The conformational properties of the microtubule-stabilizing agent epothilone A ( 1a) and its 3-deoxy and 3-deoxy-2,3-didehydro derivatives 2 and 3 have been investigated in aqueous solution by a combination of NMR spectroscopic methods, Monte Carlo conformational searches, and NAMFIS calculations. The tubulin-bound conformation of epothilone A ( 1a), as previously proposed on the basis of solution NMR data, was found to represent a significant fraction of the ensemble of conformations present for the free ligands in aqueous solution.
Construction of a liquid-state NMR DNP shuttle spectrometer: First experimental results and evaluation of optimal performance characteristics.
Reese, M., Lennartz, D., Marquardsen, T., Hoefer, P., Tavernier, A., Carl, P., Schippmann, T., Bennati, M., Carlomagno, T., Engelke, F. & Griesinger, C.
Appl Magn Reson 2008 34(3-4) 301-311
Crystallography-independent determination of ligand binding modes.
Orts, J., Tuma, J., Reese, M., Grimm, S.K., Monecke, P., Bartoschek, S., Schiffer, A., Wendt, K.U., Griesinger, C. & Carlomagno, T.
Angew Chem Int Ed Engl. 2008;47(40):7736-40. Europe PMC
Specific methyl group protonation for the measurement of pharmacophore-specific interligand NOE interactions.
Orts, J., Grimm, S.K., Griesinger, C., Wendt, K.U., Bartoschek, S. & Carlomagno, T.
Chemistry. 2008;14(25):7517-20. Europe PMC
Argyrin a reveals a critical role for the tumor suppressor protein p27(kip1) in mediating antitumor activities in response to proteasome inhibition.
Nickeleit I, Zender S, Sasse F, Geffers R, Brandes G, Sörensen I, Steinmetz H, Kubicka S, Carlomagno, T., Menche D, Gütgemann I, Buer J, Gossler A, Manns MP, Kalesse M, Frank R, Malek NP.
Cancer Cell 2008 14(1) 23-35 Europe PMC
Studies of dynamic nuclear polarization with nitroxides in aqueous solution.
Hoefer, P., Carl, P., Guthausen, G., Prisner, T., Reese, M., Carlomagno, T., Griesinger, C. & Bennati, M.
Appl Magn Reson 2008 34(3-4) 393-398
Simultaneous determination of the conformation and relative configuration of archazolide a by using nuclear overhauser effects, J couplings, and residual dipolar couplings.
Fares, C., Hassfeld, J., Menche, D. & Carlomagno, T.
Angew Chem Int Ed Engl. 2008;47(20):3722-6. Europe PMC
13C-detection in RNA bases: revealing structure-chemical shift relationships.
Fares, C., Amata, I. & Carlomagno, T.
J Am Chem Soc. 2007 Dec 26;129(51):15814-23. Epub 2007 Dec 4.
The chemical shifts of the unprotonated carbons in the proton-deficient nucleobases of RNA are rarely reported, despite the valuable information that they contain about base-pairing and base-stacking. We have developed 13C-detected 2D-experiments to identify the unprotonated 13C in the RNA bases and have assigned all the base nuclei of uniformly 13C,15N-labeled HIV-2 TAR-RNA. The 13C chemical shift distributions revealed perturbations correlated with the base-pairing and base-stacking properties of all four base-types. From this work, we conclude that the information contained in the chemical shift perturbations within the base rings can provide valuable restraint information for solving RNA structures, especially in conformational averaged regions, where NOE-based information is not available.
Binding of the human Prp31 Nop domain to a composite RNA-protein platform in U4 snRNP.
Liu, S., Li, P., Dybkov, O., Nottrott, S., Hartmuth, K., Luhrmann, R., Carlomagno, T. & Wahl, M.C.
Science. 2007 Apr 6;316(5821):115-20.
Although highly homologous, the spliceosomal hPrp31 and the nucleolar Nop56 and Nop58 (Nop56/58) proteins recognize different ribonucleoprotein (RNP) particles. hPrp31 interacts with complexes containing the 15.5K protein and U4 or U4atac small nuclear RNA (snRNA), whereas Nop56/58 associate with 15.5K-box C/D small nucleolar RNA complexes. We present structural and biochemical analyses of hPrp31-15.5K-U4 snRNA complexes that show how the conserved Nop domain in hPrp31 maintains high RNP binding selectivity despite relaxed RNA sequence requirements. The Nop domain is a genuine RNP binding module, exhibiting RNA and protein binding surfaces. Yeast two-hybrid analyses suggest a link between retinitis pigmentosa and an aberrant hPrp31-hPrp6 interaction that blocks U4/U6-U5 tri-snRNP formation.
High-resolution solid-state NMR structure of an anticancer agent.
Lange, A., Schupp, T., Petersen, F., Carlomagno, T. & Baldus, M.
ChemMedChem. 2007 Apr;2(4):522-7.
We demonstrate that solid-state NMR methods can be used to rapidly determine the high-resolution 3D structure of Epothilone B in the polycrystalline state. The solid-state NMR structures exhibit an average heavy atom RMSD to the mean structure of 0.14 A. The 3D-structural analysis leads to stereospecific assignments and provides insight into the influence of intermolecular interactions upon ssNMR chemical-shift parameters. Our results pave the way to the study of ligand-microtubule interactions in a noncrystalline and insoluble environment at atomic level.
Structural basis of the activity of the microtubule-stabilizing agent epothilone a studied by NMR spectroscopy in solution.
Reese, M., Sanchez-Pedregal, V.M., Kubicek, K., Meiler, J., Blommers, M.J., Griesinger, C. & Carlomagno, T.
Angew Chem Int Ed Engl. 2007;46(11):1864-8. Europe PMC
The tubulin-bound conformation of discodermolide derived by NMR studies in solution supports a common pharmacophore model for epothilone and discodermolide.
Sanchez-Pedregal, V.M., Kubicek, K., Meiler, J., Lyothier, I., Paterson, I. & Carlomagno, T.
Angew Chem Int Ed Engl. 2006 Nov 13;45(44):7388-94. Europe PMC
Calculation of the dependence of homo- and heteronuclear 3J and 2J scalar couplings for the determination of the 2'-hydroxy conformation in RNA.
Fohrer, J., Reinscheid, U., Hennig, M. & Carlomagno, T.
Angew Chem Int Ed Engl. 2006 Oct 27;45(42):7033-6. Europe PMC
Stereochemical determination of Archazolid A and B, highly potent vacuolar-type ATPase inhibitors from the Myxobacterium Archangium gephyra.
Hassfeld, J., Fares, C., Steinmetz, H., Carlomagno, T. & Menche, D.
Org Lett. 2006 Oct 12;8(21):4751-4.
[structure: see text] The relative and absolute stereochemistry of the structurally unique 24-membered myxobacterial macrolides archazolid A and B, highly potent vacuolar-type ATPase (V-ATPase) inhibitors in vitro and in vivo, was determined on the basis of a combination of extensive high-field NMR studies, including J-based configuration analysis, molecular modeling, and chemical methods.
SHARP-TACSY: triple-band tailored correlated spectroscopy for base-to-sugar transfer in nucleic acid residues with intermediate time scale motions.
Fares, C. & Carlomagno, T.
J Am Chem Soc. 2006 Aug 2;128(30):9856-62.
Established experiments to identify the sugar-to-base connectivity in isotopically labeled RNA require long transfer periods and are inefficient for residues undergoing intermediate time scale motions (microsecond to millisecond). Here, an alternative transfer experiment is introduced, whereby the C1'-N1/9-C6/8 spin system is selectively brought to the so-called Hartmann-Hahn condition using selectiveheteronuclear planar triple-band tailored correlated spectroscopy (SHARP-TACSY). Results are shown for the fully labeled 30-mer oligonucleotide TAR RNA with particular attention placed on residues from and close to the bulge and the loop. For these residues, the faster relaxation can be attributed to exchange contributions stemming from transient stacking and unstacking of the bases and/or from the isomerization of the ribose sugar pucker. The new experiment shows improved signal-to-noise for residues exhibiting large microsecond-millisecond time scale motions with respect to established experiments, thus providing a valid alternative for resonance assignment in mobile RNA regions.
Influence of the 2'-hydroxyl group conformation on the stability of A-form helices in RNA.
Fohrer, J., Hennig, M. & Carlomagno, T.
J Mol Biol. 2006 Feb 17;356(2):280-7. Epub 2005 Dec 1.
The 2'-hydroxyl group plays fundamental roles in both the structure and the function of RNA, and is the major determinant of the conformational and thermodynamic differences between RNA and DNA. Here, we report a conformational analysis of 2'-OH groups of the HIV-2 TAR RNA by means of NMR scalar coupling measurements in solution. Our analysis supports the existence of a network of water molecules spanning the minor groove of an RNA A-form helix, as has been suggested on the basis of a high-resolution X-ray study of an RNA duplex. The 2'-OH protons of the lower stem nucleotides of the TAR RNA project either towards the O3' or towards the base, where the 2'-OH group can favorably participate in H-bonding interactions with a water molecule situated in the nucleotide base plane. We observe that the k(ex) rate of the 2'-OH proton with the bulk solvent anti-correlates with the base-pair stability, confirming the involvement of the 2'-OH group in a collective network of H-bonds, which requires the presence of canonical helical secondary structure. The methodology and conformational analysis presented here are broadly applicable and facilitate future studies aimed to correlate the conformation of the 2'-OH group with both the structure and the function of RNA and RNA-ligand complexes.
A thorough dynamic interpretation of residual dipolar couplings in ubiquitin.
Lakomek, N.A., Carlomagno, T., Becker, S., Griesinger, C. & Meiler, J.
J Biomol NMR. 2006 Feb;34(2):101-15.
The presence of slow motions with large amplitudes, as detected by measurements based on residual dipolar couplings [Peti, W., Meiler, J., Brueschweiler, R. and Griesinger, C. (2002) J. Am. Chem. Soc., 124, 5822-5833], has stirred up much discussion in recent years. Based on ubiquitin NH residual dipolar couplings (rdcs) measured in 31 different alignment conditions, a model-free analysis of structure and dynamics [Meiler, J., Peti, W., Prompers, J., Griesinger, C. and Brueschweiler, R. (2001) J. Am. Chem. Soc., 123, 6098-6107] is presented. Starting from this broad experimental basis, rdc-based order parameters with so far unattained accuracy were determined. These rdc-based order parameters underpin the presence of new modes of motion slower than the inverse overall tumbling correlation time. Amplitudes and anisotropies of the motion were derived. The effect of structural noise on the results was proven to be negligible.
TAR-RNA recognition by a novel cyclic aminoglycoside analogue.
Raghunathan, D., Sanchez-Pedregal, V.M., Junker, J., Schwiegk, C., Kalesse, M., Kirschning, A. & Carlomagno, T.
Nucleic Acids Res. 2006 Jul 19;34(12):3599-608. Print 2006.
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 full-length viral transcripts the Tat/TAR complex has to recruit the positive transcription elongation factor complex (P-EFTb), which interacts with TAR through its cyclin T1 (CycT1) component. Mutational studies identified the TAR hexanucleotide loop as a crucial region for contacting CycT1. 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/CycT1 ternary complex by obstructing both the bulge and loop regions of the RNA.
Bio-Nuclear Magnetic Resonance.
Carlomagno, T., Baldus, M. & Griesinger, C.
In "Comprehensive Medicinal Chemistry II", Triggle, D. & Taylor, J.B. (2006), Elsevier, Oxford
Transferred Cross-Correlated Relaxation: Application to Drug/Target Complexes.
Carlomagno, T. & Griesinger, C.
In "Handbook of Modern Magnetic Resonance: Pharmaceutical Sciences" (2006)
The NMR spectra of biomolecules without and especially with isotopic labeling provide a wealth of information about interatomic distances and angular geometries that can be used as conformational restraints in structure determination. In this review we will focus on the application of cross-correlated relaxation rates to obtain structural information on a small-molecule/protein complex that has pharmaceutical implications. Cross-correlated relaxation complements information derived from NOEs. The chosen complex: epothilone/tubulin could not be subjected to X-ray crystallography. Electron microscopy can only be performed on tubulin sheets induced with Zn ions, which show an assembly of the tubulin dimers that is not found under physiological conditions. A structure of epothilone when bound to tubulin has been determined by electron crystallography. However, the resolution is too low to deduce the conformation of epothilone in a unique way. Thus, NMR constitutes a unique tool for the investigation of this drug/protein complex to obtain a highresolution structure of the drug molecule in the bound state.
Side-chain orientation and hydrogen-bonding imprint supra-Tau(c) motion on the protein backbone of ubiquitin.
Lakomek, N.A., Fares, C., Becker, S., Carlomagno, T., Meiler, J. & Griesinger, C.
Angew Chem Int Ed Engl. 2005 Dec 2;44(47):7776-8. Europe PMC
The INPHARMA method: protein-mediated interligand NOEs for pharmacophore mapping.
Sanchez-Pedregal, V.M., Reese, M., Meiler, J., Blommers, M.J., Griesinger, C. & Carlomagno, T.
Angew Chem Int Ed Engl. 2005 Jul 4;44(27):4172-5. Europe PMC
Identification of a novel pharmacophore for peptide toxins interacting with K+ channels.
Verdier, L., Al-Sabi, A., Rivier, J.E., Olivera, B.M., Terlau, H. & Carlomagno, T.
J Biol Chem. 2005 Jun 3;280(22):21246-55. Epub 2005 Mar 30.
KappaM-conotoxin RIIIK blocks TSha1 K+ channels from trout with high affinity by interacting with the ion channel pore. As opposed to many other peptides targeting K+ channels, kappaM-RIIIK does not possess a functional dyad. In this study we combine thermodynamic mutant cycle analysis and docking calculations to derive the binding mode of kappaM-conotoxin RIIIK to the TSha1 channel. The final model reveals a novel pharmacophore, where no positively charged side chain occludes the channel pore. Instead the positive-charged residues of the toxin form a basic ring; kappaM-RIIIK is anchored to the K+ channel via electrostatic interactions of this basic ring with the loop and pore helix residues of the channel. The channel amino acid Glu-354 is likely to be a fundamental determinant of the selectivity of kappaM-RIIIK for the TSha1 channel. The Cgamma-OH of Hyp-15 is in contact with the carbonyls of the selectivity filter, disturbing the charge distribution pattern necessary for the coordination of K+ ions. This novel, experimentally based pharmacophore model proves the existence of diverse binding modes of peptidic toxins to K+ channels and underlines the role of intermolecular electrostatic interactions involving channel loop side chains in determining the selectivity of toxins for specific K+ channel types.
Assignment and NOE analysis of 2'-hydroxyl protons in RNA: implications for stabilization of RNA A-form duplexes.
Hennig, M., Fohrer, J. & Carlomagno, T.
J Am Chem Soc. 2005 Feb 23;127(7):2028-9.
The ribose 2'-OH hydroxyl group distinguishes RNA from DNA. The 2'-OH hydroxyl protons are responsible for differences in conformation, hydration, and thermodynamic stability of RNA and DNA oligonucleotides. Additionally, the 2'-OH group plays a central role in RNA catalysis. This important group lies in the shallow groove of RNA, where it is involved in a network of hydrogen bonds with water molecules stabilizing RNA A-form duplexes. Structural and dynamical information on 2'-OH hydroxyl protons is essential to understand their respective roles. Here we report the 2'-OH hydroxyl proton assignments for a 30mer RNA, the HIV-2 transactivation region, in water using solution NMR techniques. We provide structural information on 2'-OH hydroxyl groups in the form of orientational preferences contradicting the paradigm that the 2'-OH hydroxyl typically points away from the ribose H1' proton.
Ligand-target interactions: what can we learn from NMR?
Annu Rev Biophys Biomol Struct. 2005;34:245-66.
The conformation of the ligand in complex with a macromolecular target can be studied by nuclear magnetic resonance (NMR) in solution for both tightly and weakly forming complexes. In the weak binding regime (k(off) > 10(4) Hz), the structure of the bound ligand is accessible also for very large complexes (>100 kDa), which are not amenable to NMR studies in the tight binding regime. Here I review the state-of-the-art NMR methodology used for screening ligands and for the structural investigation of bound ligand conformations, in both tight and weak binding regimes. The advantages and disadvantages of each approach are critically described. The NMR methodology used to investigate transiently forming complexes has expanded considerably in the past few years, opening new possibilities for a detailed description of ligand-target interactions. Novel methods for the determination of the bound ligand conformation, in particular transferred cross-correlated relaxation, are thoroughly reviewed, and their advantages with respect to established methodology are discussed, using the epothilone-tubulin complex as a primary example.
KappaM-conotoxin RIIIK, structural and functional novelty in a K+ channel antagonist.
Al-Sabi, A., Lennartz, D., Ferber, M., Gulyas, J., Rivier, J.E., Olivera, B.M., Carlomagno, T. & Terlau, H.
Biochemistry. 2004 Jul 13;43(27):8625-35.
Venomous organisms have evolved a variety of structurally diverse peptide neurotoxins that target ion channels. Despite the lack of any obvious structural homology, unrelated toxins that interact with voltage-activated K(+) channels share a dyad motif composed of a lysine and a hydrophobic amino acid residue, usually a phenylalanine or a tyrosine. kappaM-Conotoxin RIIIK (kappaM-RIIIK), recently characterized from the cone snail Conus radiatus, blocks Shaker and TSha1 K(+) channels. The functional and structural study presented here reveals that kappaM-conotoxin RIIIK blocks voltage-activated K(+) channels with a novel pharmacophore that does not comprise a dyad motif. Despite the quite different amino acid sequence and no overlap in the pharmacological activity, we found that the NMR solution structure of kappaM-RIIIK in the C-terminal half is highly similar to that of mu-conotoxin GIIIA, a specific blocker of the skeletal muscle Na(+) channel Na(v)1.4. Alanine substitutions of all non-cysteine residues indicated that four amino acids of kappaM-RIIIK (Leu1, Arg10, Lys18, and Arg19) are key determinants for interaction with K(+) channels. Following the hypothesis that Leu1, the major hydrophobic amino acid determinant for binding, serves as the hydrophobic partner of a dyad motif, we investigated the effect of several mutations of Leu1 on the biological function of kappaM-RIIIK. Surprisingly, both the structural and mutational analysis suggested that, uniquely among well-characterized K(+) channel-targeted toxins, kappaM-RIIIK blocks voltage-gated K(+) channels with a pharmacophore that is not organized around a lysine-hydrophobic amino acid dyad motif.
Structure of the NCoA-1/SRC-1 PAS-B domain bound to the LXXLL motif of the STAT6 transactivation domain.
Razeto, A., Ramakrishnan, V., Litterst, C.M., Giller, K., Griesinger, C., Carlomagno, T., Lakomek, N., Heimburg, T., Lodrini, M., Pfitzner, E. & Becker, S.
J Mol Biol. 2004 Feb 13;336(2):319-29.
Signal transducer and activator of transcription 6 (STAT6) regulates transcriptional activation in response to interleukin-4 (IL-4) by direct interaction with coactivators. The CREB-binding protein (p300/CBP) and the nuclear coactivator 1 (NCoA-1), a member of the p160/steroid receptor coactivator family, bind independently to specific regions of the STAT6 transactivation domain and act as coactivators. The interaction between STAT6 and NCoA-1 is mediated by an LXXLL motif in the transactivation domain of STAT6. To define the mechanism of coactivator recognition, we determined the crystal structure of the NCoA-1 PAS-B domain in complex with the STAT6 LXXLL motif. The amphipathic, alpha-helical STAT6 LXXLL motif binds mostly through specific hydrophobic interactions to NCoA-1. A single amino acid of the NCoA-1 PAS-B domain establishes hydrophilic interactions with the STAT6 peptide. STAT6 interacts only with the PAS-B domain of NCoA-1 but not with the homologous regions of NCoA-2 and NCoA-3. The residues involved in binding the STAT6 peptide are strongly conserved between the different NCoA family members. Therefore surface complementarity between the hydrophobic faces of the STAT6 fragment and of the NCoA-1 PAS-B domain almost exclusively defines the binding specificity between the two proteins.
Measuring the chi 1 torsion angle in protein by CH-CH cross-correlated relaxation: a new resolution-optimised experiment.
Carlomagno, T., Bermel, W. & Griesinger, C.
J Biomol NMR. 2003 Oct;27(2):151-7.
Here we introduce an experiment with high sensitivity and resolution for the measurement of CH-CH dipolar-dipolar cross-correlated relaxation rates (CCRR) in protein side-chains. The new methodology aims to the determination of structural and dynamical parameters around the torsion angle chi(1) by measuring C(alpha)H(alpha)-C(beta)H(beta) cross-correlated relaxation rates. The method is validated on the protein ubiquitin: the chi(1) angles determined from the CCRR data are compared with the chi(1) angles of a previously determined NMR structure. The agreement between the two data sets is excellent for most residues. The few discrepancies that were found between the CCR-derived chi(1) angles and the angles of the previously determined NMR structure could be explained by taking internal motion into account. The new methodology represents a very powerful tool to determine both structure and dynamics of protein side-chains in only one experiment.
Structural studies on a twin-arginine signal sequence.
Kipping, M., Lilie, H., Lindenstrauss, U., Andreesen, J.R., Griesinger, C., Carlomagno, T. & Bruser, T.
FEBS Lett. 2003 Aug 28;550(1-3):18-22.
Translocation of folded proteins across biological membranes can be mediated by the so-called 'twin-arginine translocation' (Tat) system. To be translocated, Tat substrates require N-terminal signal sequences which usually contain the eponymous twin-arginine motif. Here we report the first structural analysis of a twin-arginine signal sequence, the signal sequence of the high potential iron-sulfur protein from Allochromatium vinosum. Nuclear magnetic resonance (NMR) analyses of amide proton resonances did not indicate a signal sequence structure. Accordingly, data from H/D exchange matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry showed that the amide protons of the signal sequence exchange rapidly, indicating the absence of secondary structure in the signal sequence up to L29. We conclude that the conserved twin-arginine motif does not form a structure by itself or as a result of intramolecular interactions.
Derivation of dihedral angles from CH-CH dipolar-dipolar cross-correlated relaxation rates: a C-C torsion involving a quaternary carbon atom in epothilone A bound to tubulin.
Carlomagno, T., Sanchez, V.M., Blommers, M.J. & Griesinger, C.
Angew Chem Int Ed Engl. 2003 Jun 6;42(22):2515-7. Europe PMC
The high-resolution solution structure of epothilone A bound to tubulin: an understanding of the structure-activity relationships for a powerful class of antitumor agents.
Carlomagno, T., Blommers, M.J., Meiler, J., Jahnke, W., Schupp, T., Petersen, F., Schinzer, D., Altmann, K.H. & Griesinger, C.
Angew Chem Int Ed Engl. 2003 Jun 6;42(22):2511-5. Europe PMC
A novel PH-cT-COSY methodology for measuring JPH coupling constants in unlabeled nucleic acids. application to HIV-2 TAR RNA.
Carlomagno, T., Hennig, M. & Williamson, J.R.
J Biomol NMR. 2002 Jan;22(1):65-81.
A quantitative analysis of JPH scalar couplings in nucleic acids is difficult due to small couplings to phosphorus, the extreme overlap of the sugar protons and the fast relaxation of the spins involved in the magnetization transfer. Here we present a new methodology that relies on heteronuclear Constant Time Correlation Spectroscopy (CT-COSY). The three vicinal 3JPH3', 3JPHS' and 3JPHS" scalar couplings can be obtained by monitoring the intensity decay of the P1-H3'(i-1) peak as a function of the constant time T in a 2D correlation map. The advantage of the new method resides in the possibility of measuring the two 3JPH5' and 3JPH5" scalar couplings even in the presence of overlapped H5'/H5" resonances, since the quantitative information is extracted from the intensity decay of the P-H3' peak. Moreover, the relaxation of the H3' proton is considerably slower than that of the H5'/H5" geminal protons and the commonly populated conformations of the phosphate backbone are associated with large 3JPH3' couplings and relatively small 3JPH5'/H5". These two facts lead to optimal signal-to-noise ratio for the P-H3' correlation compared to the P-H5'/H5" correlation. The heteronuclear CT-COSY experiment is suitable for oligonucleotides in the 10-15 kDa molecular mass range and has been applied to the 30mer HIV-2 TAR RNA. The methodology presented here can be used to measure P-H dipolar couplings (DPH) as well. We will present qualitative results for the measurement of P-Hbase and P-H2' dipolar couplings in the HIV-2 TAR RNA and will discuss the reasons that so far precluded the quantification of the DPHS for the 30mer RNA.
Determination of aliphatic side-chain conformation using cross-correlated relaxation: application to an extraordinarily stable 2'-aminoethoxy-modified oligonucleotide triplex.
Carlomagno, T., Blommers, M.J., Meiler, J., Cuenoud, B. & Griesinger, C.
J Am Chem Soc. 2001 Aug 1;123(30):7364-70.
The structural basis for the extraordinary stability of a triple-stranded oligonucleotide in which the third strand contains 2'-aminoethoxy-substituted riboses is investigated by NMR spectroscopy. The enhanced stability of the modified triplex in comparison to the unmodified DNA triplex of the same sequence can be attributed to strong interactions of the aminoethoxy groups of the third strand with the phosphate groups of the purine strand. In molecular dynamics calculations the aminoethoxy side chain was found to be rather flexible, allowing for the presence of hydrogen bonds between the aminoethoxy group of the third strand and two different phosphates of the backbone of the second strand. To investigate the conformational preference of the aminoethoxy side chain a new NMR method has been developed which relies on CH-CH dipolar-dipolar cross-correlated relaxation rates. The results indicate that the aminoethoxy side chains adopt mainly a gauche(+) conformation, for which only one of the two hydrogen bonds inferred by NMR and molecular dynamics simulations is possible. This demonstrates a highly specific interaction between the amino group of the third strand and one of the phosphate groups of the purine strand.
Residual dipolar coupling TOCSY for direct through space correlations of base protons and phosphorus nuclei in RNA.
Hennig, M., Carlomagno, T. & Williamson, J.R.
J Am Chem Soc. 2001 Apr 11;123(14):3395-6. Europe PMC
Cross-correlated relaxation for measurement of angles between tensorial interactions.
Schwalbe, H., Carlomagno, T., Hennig, M., Junker, J., Reif, B., Richter, C. & Griesinger, C.
Methods Enzymol. 2001;338:35-81. Europe PMC
COIN TACSY, a novel approach to tailored correlation spectroscopy.
Carlomagno, T., Prasch, T. & Glaser, S.N.J.
JOURNAL OF MAGNETIC RESONANCE 2001 149(1) 52-57
A new method for the simultaneous measurement of magnitude and sign of 1DCH and 1DHH dipolar couplings in methylene groups.
Carlomagno, T., Peti, W. & Griesinger, C.
J Biomol NMR. 2000 Jun;17(2):99-109.
Heteronuclear dipolar couplings of the protein backbone have proven to have a big impact on the accuracy of protein NMR structures. H,H dipolar couplings might have the same impact on side chains. Here we present a method that combines both heteronuclear and homonuclear dipolar couplings to investigate the local conformation of methylene groups. A new pulse sequence (SPITZE-HSQC) is presented, that allows to measure the two C,H and the H,H dipolar couplings at the same time, using spin state selective transfers. The new method has been applied to the methylene groups of glycines in the protein ubiquitin. The C,H and the H,H dipolar couplings might have a key role in fast stereospecific assignment of protons in CH2 groups.
Errors in the measurement of cross-correlated relaxation rates and how to avoid them.
Carlomagno, T. & Griesinger, C.
J Magn Reson. 2000 Jun;144(2):280-7.
Cross-correlated relaxation rates Gamma are commonly obtained from constant time experiments by measuring the effect of the desired cross-correlated relaxation on an appropriate coherence during the constant time T. These measurements are affected by systematic errors, which derive from undesired cross-correlated relaxation effects taking place before and after the constant time period T. In this paper we discuss the sources and the size of these errors in an example of two pulse sequences. Higher accuracy of the measured data can be obtained by recording a set of experiments with different T values. Cross-correlated relaxation rates are measured in constant time experiments either from the differential relaxation of multiple components (J-resolved Gamma experiments) or from the efficiency of magnetization transfer between two coherences (quantitative Gamma experiments). In this paper we calculate analytically the statistical errors in both J-resolved and quantitative Gamma experiments. These formulae provide the basis for the choice of the most efficient experimental approach and parameters for a given measurement time and size of the rate. The optimal constant time T for each method can be calculated and depends on the relaxation properties of the molecule under investigation. Moreover, we will show how to optimize the relative duration of cross and reference experiments in a quantitative Gamma approach.
Ubiquitin backbone motion studied via NHN-C ' C-alpha dipolar-dipolar and C '-C ' C-alpha/NHN CSA-dipolar cross-correlated relaxation.
Carlomagno, T., Maurer, M., Hennig, M. & Griesinger, C.
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 2000 122(21) 5105-5113
Transferred cross-correlated relaxation: Application to the determination of sugar pucker in an aminoacylated tRNA-mimetic weakly bound to EF-Tu.
Carlomagno, T., Felli, I.C., Czech, M., Fischer, R., Sprinzl, M. & Griesinger, C.
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 1999 121(9) 1945-1948
New methylene specific experiments for the measurement of scalar spin-spin coupling constants between protons attached to 13C.
Carlomagno, T., Schwalbe, H., Rexroth, A., Sorensen, O.W. & Griesinger, C.
J Magn Reson. 1998 Nov;135(1):216-26.
New two- and three-dimensional NMR methods are proposed for the measurement of 3J(H, H) coupling constants between two adjacent methylene moieties. The new experiment, which is based on a combination of the E.COSY principle and double/zero quantum heteronuclear spectroscopy, has been applied to diaceton-glucose and to the protein rhodniin. The coupling constants of CH-CH2 groups have been compared with those obtained from a HCCH-E.COSY experiment to check the reliability of the results. An analysis of the coupling constants derived by comparison between experimental and simulated spectra is presented. Simulations were done with the program wtest considering fully correlated dipolar relaxation. Side-chain conformations in amino acids with adjacent methylene groups can be determined by the new experiment.
"Kin " HEHAHA Sequences, Heteronuclear Hartmann-Hahn Transfer with Different Bandwidths for Spins I and S
Carlomagno, T., Luy, B. & Glaser, S.J.
J Magn Reson. 1997 May;126(1):110-9.
A new class of heteronuclear Hartmann-Hahn experiments that is based on the simultaneous irradiation of two different multiple-pulse sequences is introduced. For these "kin " HEHAHA sequences, the scaling properties of the effective heteronuclear coupling constants are analyzed. Four kin sequences are presented with a ratio of the active bandwidths DeltanuI /DeltanuS ranging between 1/2 and 1/10. The offset dependence of the polarization-transfer efficiency is examined experimentally and with the help of numerical simulations.
Resonance assignment and secondary structure determination and stability of the recombinant human uteroglobin with heteronuclear multidimensional NMR.
Carlomagno, T., Mantile, G., Bazzo, R., Miele, L., Paolillo, L., Mukherjee, A.B. & Barbato, G.
J Biomol NMR. 1997 Jan;9(1):35-46.
Human uteroglobin (h-UG) or Clara cell 10kDa (cc10kDa) is a steroid-dependent, 17 kDa homodimeric, secretory protein with potent anti-inflammatory/immunomodulatory properties. However, the exact physiological role still remains to be determined. It has been hypothesised that its activity is exerted through the binding of a specific target represented by a small molecule (still unknown), and that the binding is regulated by the formation/disruption of two cysteine bonds. The binding properties of the reduced UG have been proved in vitro for several different molecules, but no in vivo data are available to date. However, binding has been observed between reduced rabbit UG and a protein of an apparent molecular mass of 90 kDa and, more recently, we found an h-UG-binding protein
PLUSH TACSY: Homonuclear planar TACSY with two-band selective shaped pulses applied to C(alpha),C' transfer and C (beta),C (aromatic) correlations.
Carlomagno, T., Maurer, M., Sattler, M., Schwendinger, M.G., Glaser, S.J. & Griesinger, C.
J Biomol NMR. 1996 Sep;8(2):161-70. doi: 10.1007/BF00211162.
A new homonuclear Hartmann-Hahn-type mixing scheme is introduced that effects coherence transfer between resonances in two separated frequency bands. The mixing scheme relies on the irradiation of two-band selective shaped pulses that are expanded in an MLEV-16 supercycle. Similar to heteronuclear Hartmann-Hahn experiments, a planar effective coupling tensor is created. This novel mixing scheme is applied to C(alpha),C' transfer and to the transfer between C(beta) and aromatic carbon spins.
Conformational analysis of dolastatin 10: an NMR and theoretical approach.
Benedetti, E., Carlomagno, T., Fraternali, F., Hamada, Y., Hayashi, K., Paolillo, L. & Shioiri, T.
Biopolymers. 1995 Oct;36(4):525-38.
A solution conformational analysis of dolastatin 10, a powerful antineoplastic agent, has been carried out by means of nmr techniques and theoretical calculations. 1H mono- and bidimensional nmr experiments, as well as 1H-13C heterocorrelated spectra, have been performed on CD2Cl2 solutions. The most interesting nmr data is a huge shielding of the aCH(25) proton of the Dov residue, suggesting the presence of an interaction between the N-terminal and the aromatic C-terminal ends of the molecule. The possibility of a head-to-tail intermolecular association having been discarded, the presence of a series of preferred folded conformation has been hypothesized. Conformational theoretical analysis supports the nmr hypothesis of a folded peptide-like molecule, and a series of possible conformers in good agreement with the experimental data have been analyzed.