Structural principles of RNA catalysis in a 2'-5' lariat-forming ribozyme.

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.

Multi-phosphorylation of the intrinsically disordered unique domain of c-Src studied by in-cell and real-time NMR spectroscopy.

Intrinsically disordered regions (IDRs) are preferred sites for post-translational modifications essential for regulating protein function. The enhanced local mobility of IDRs facilitates their observation by NMR spectroscopy in vivo. Phosphorylation events can occur at multiple sites and respond dynamically to changes in kinase-phosphatase networks. Here we used real-time NMR spectroscopy to study the effect of kinases and phosphatases present in Xenopus oocytes and egg extracts on the phosphorylation state of the "unique domain" of c-Src. We followed the phosphorylation of S17 in oocytes, and of S17, S69, and S75 in egg extracts by NMR spectroscopy, MS, and western blotting. Addition of specific kinase inhibitors showed that S75 and S69 are phosphorylated by CDKs (cyclin-dependent kinases) differently from Cdk1. Moreover, although PKA (cAMP-dependent protein kinase) can phosphorylate S17 in vitro, this was not the major S17 kinase in egg extracts. Changes in PKA activity affected the phosphorylation levels of CDK-dependent sites, thus suggesting indirect effects of kinase-phosphatase networks. This study provides a proof-of-concept of the use of real-time in vivo NMR spectroscopy to characterize kinase/phosphatase effects on intrinsically disordered regulatory domains.

Structure of the K-turn U4 RNA: a combined NMR and SANS study.

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°. 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.

A new member of the ribbon-helix-helix transcription factor superfamily from the plant pathogen Xanthomonas axonopodis pv.citri.

XACb0070 is an uncharacterized protein coded by the two large plasmids isolated from Xanthomonas axonopodis pv. citri, the agent of citrus canker and responsible for important economical losses in citrus world production. XACb0070 presents sequence homology only with other hypothetical proteins belonging to plant pathogens, none of which have their structure determined. The NMR-derived solution structure reveals this protein is a homodimer in which each monomer presents two domains with different structural and dynamic properties: a folded N-terminal domain with beta alpha alpha topology which mediates dimerization and a long disordered C-terminal tail. The folded domain shows high structural similarity to the ribbon-helix-helix transcriptional repressors, a family of DNA-binding proteins of conserved 3D fold but low sequence homology: indeed XACb0070 binds DNA. Primary sequence and fold comparison of XACb0070 with other proteins of the ribbon-helix-helix family together with examination of the genes in the vicinity of xacb0070 suggest the protein might be the component of a toxin-antitoxin system.

N-lobe dynamics of myosin light chain dictates its mode of interaction with myosin V IQ1.

Myosin V motors regulate secretion and cell division in eukaryotes. How MyoV activity is differentially regulated by essential and calmodulin light chain binding remains unclear. We have used NMR spectroscopy to compare the dynamic behavior of Mlc1p, a budding yeast essential light chain, with that of the Xenopus laevis calmodulin. Both proteins have a similar structure and bind similar target proteins but differ in the mechanism by which they interact with the myosin V IQ1. This interaction is essential for MyoV activity. Here, we show that the rigid conformation of the loop connecting the two EF-hand motifs of the Mlc1p N-lobe explains its differential ability to interact with myosin V IQ1. Moreover, we show that the maintenance of the N-lobe structure is required for the essential function of Mlc1p in vivo. These data show that the core characteristics of myosin light chain N-lobes differentiate Mlc1p and calmodulin binding capability.

13C-detection in RNA bases: revealing structure-chemical shift relationships.

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.

An oxygen-sensitive toxin-antitoxin system.

The Hha and TomB proteins from Escherichia coli form an oxygen-dependent toxin-antitoxin (TA) system. Here we show that YmoB, the Yersinia orthologue of TomB, and its single cysteine variant [C117S]YmoB can replace TomB as antitoxins in E. coli. In contrast to other TA systems, [C117S]YmoB transiently interacts with Hha (rather than forming a stable complex) and enhances the spontaneous oxidation of the Hha conserved cysteine residue to a -SOxH-containing species (sulfenic, sulfinic or sulfonic acid), which destabilizes the toxin. The nuclear magnetic resonance structure of [C117S]YmoB and the homology model of TomB show that the two proteins form a four-helix bundle with a conserved buried cysteine connected to the exterior by a channel with a diameter comparable to that of an oxygen molecule. The Hha interaction site is located on the opposite side of the helix bundle.

The SH3 Domain Acts as a Scaffold for the N-Terminal Intrinsically Disordered Regions of c-Src.

Regulation of c-Src activity by the intrinsically disordered Unique domain has recently been demonstrated. However, its connection with the classical regulatory mechanisms is still missing. Here we show that the Unique domain is part of a long loop closed by the interaction of the SH4 and SH3 domains. The conformational freedom of the Unique domain is further restricted through direct contacts with SH3 that are allosterically modulated by binding of a poly-proline ligand in the presence and in the absence of lipids. Our results highlight the scaffolding role of the SH3 domain for the c-Src N-terminal intrinsically disordered regions and suggest a connection between the regulatory mechanisms involving the SH3 and Unique domains.

Phosphorylation of unique domains of Src family kinases.

Members of the Src family of kinases (SFKs) are non-receptor tyrosine kinases involved in numerous signal transduction pathways. The catalytic, SH3 and SH2 domains are attached to the membrane-anchoring SH4 domain through the intrinsically disordered "Unique" domains, which exhibit strong sequence divergence among SFK members. In the last decade, structural and biochemical studies have begun to uncover the crucial role of the Unique domain in the regulation of SFK activity. This mini-review discusses what is known about the phosphorylation events taking place on the SFK Unique domains, and their biological relevance. The modulation by phosphorylation of biologically relevant inter- and intra- molecular interactions of Src, as well as the existence of complex phosphorylation/dephosphorylation patterns observed for the Unique domain of Src, reinforces the important functional role of the Unique domain in the regulation mechanisms of the Src kinases and, in a wider context, of intrinsically disordered regions in cellular processes.

Lipid binding by the Unique and SH3 domains of c-Src suggests a new regulatory mechanism.

c-Src is a non-receptor tyrosine kinase involved in numerous signal transduction pathways. The kinase, SH3 and SH2 domains of c-Src are attached to the membrane-anchoring SH4 domain through the flexible Unique domain. Here we show intra- and intermolecular interactions involving the Unique and SH3 domains suggesting the presence of a previously unrecognized additional regulation layer in c-Src. We have characterized lipid binding by the Unique and SH3 domains, their intramolecular interaction and its allosteric modulation by a SH3-binding peptide or by Calcium-loaded calmodulin binding to the Unique domain. We also show reduced lipid binding following phosphorylation at conserved sites of the Unique domain. Finally, we show that injection of full-length c-Src with mutations that abolish lipid binding by the Unique domain causes a strong in vivo phenotype distinct from that of wild-type c-Src in a Xenopus oocyte model system, confirming the functional role of the Unique domain in c-Src regulation.

NMR assignments of HIV-2 TAR RNA.

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.