Solution structure of the 5'-terminal hairpin of the 7SK small nuclear RNA.

The small nuclear 7SK RNA regulates RNA polymerase II (RNA Pol II) transcription, by sequestering and inhibiting the positive transcription elongation factor b (P-TEFb). P-TEFb is stored in the 7SK ribonucleoprotein (RNP) that contains the three nuclear proteins Hexim1, LaRP7, and MePCE. P-TEFb interacts with the protein Hexim1 and the 7SK RNA. Once P-TEFb is released from the 7SK RNP, it activates transcription by phosphorylating the C-terminal domain of RNA Pol II. P-TEFb also plays a crucial role in the replication of the human immunodeficiency virus HIV-1, through its recruitment by the viral transactivator Tat. Previous work demonstrated that the protein Tat promotes the release of P-TEFb from the 7SK RNP through direct binding to the 7SK RNA. Hexim1 and Tat proteins both comprise conserved and similar arginine-rich motifs that were identified to bind the 7SK RNA at a repeated GAUC site located at the top of the 5'-terminal hairpin (HPI). Here, we report the solution structure of this region as determined by nuclear magnetic resonance, to identify HPI structural features recognized by Hexim1 and Tat. The HPI solution structure displays an elongated shape featuring four helical segments interrupted by one internal loop and three bulges with distinct folds. In particular, the repeated GAUC motif adopts a pre-organized geometry. Our results suggest that the binding of Hexim1 and Tat to the 7SK RNA could originate from a conformational selection of this motif, highlighting how RNA local structure could lead to an adaptive recognition of their partners.

Rapid acquisition of 1H and 19F NMR experiments for direct and competition ligand-based screening.

Direct and competition ligand-based NMR experiments are often used in the screening of chemical fragment libraries against a protein target due to the high relative sensitivity of NMR for protein-binding events. A plethora of NMR methods has been proposed for this purpose. Two of these techniques are the (19)F T(2) filter and the (1)H selective T(2) filter experiments. Modifications of the pulse sequences of these experiments have resulted in a ∼2-fold reduction in the experiment time thus allowing an increase in the screening throughput and making NMR an attractive technique for screening large compound collections.

Both lipid environment and pH are critical for determining physiological solution structure of 3-D-conserved epitopes of the HIV-1 gp41-MPER peptide P1.

In terms of background, the solution structure of monomeric peptide P1 (residues 649-683), located in the conserved membrane proximal region (MPER) of HIV-1 envelope glycoprotein gp41, is first reported here in dodecylphosphocholine (DPC) micelles. P1 is the minimal MPER region that permits interaction with the mucosal galactosyl ceramide HIV-receptor; it also contains epitopes recognized by major gp41-specific, broadly neutralizing immunoglobulin Gs (IgGs), 2F5 and 4E10, determinant in HIV fusion/infection. Our principal findings were as follows: the structural stability of P1 is pH dependent, as the alpha-helix comprising Q653 I682, present at pH 3.3, is destabilized at higher pH values. At pH 6, the E-rich N-terminal half of P1 (residues 650-666), partially overlapping the 2F5-specific epitope, becomes fully disordered, while the W-rich C-terminal half conserves two shorter helices (W666-W670 and W672-W680), separated by a well-defined bend overlapped by the 4E10-specific epitope. The two IgGs bind to P1 on DPC micelles with binding parameters (K(d)) in the nanomolar range. Next, P1 was derivatized with phosphatidylethanolamine at its C terminal and inserted into liposomes of varied lipid composition, thereby enabling P1 to move laterally. Alternatively, an infectious virus-binding assay was established. The K(d) of both 2F5 and 4E10 IgGs measured on viral liposome and virus are similar and much lower than for the binding of the free peptide. In conclusion, P1, in a lipid environment, is an optimized MPER-derived peptide suitable for designing an immunogen inducing broadly neutralizing antibodies to HIV.

The yeast C/D box snoRNA U14 adopts a "weak" K-turn like conformation recognized by the Snu13 core protein in solution.

Non-coding RNAs associate with proteins to form ribonucleoproteins (RNPs), such as ribosome, box C/D snoRNPs, H/ACA snoRNPs, ribonuclease P, telomerase and spliceosome to ensure cell viability. The assembly of these RNA-protein complexes relies on the ability of the RNA to adopt the correct bound conformation. K-turn motifs represent ubiquitous binding platform for proteins found in several cellular environment. This structural motif has an internal three-nucleotide bulge flanked on its 3' side by a G•A/A•G tandem pairs followed by one or two non-Watson-Crick pairs, and on its 5' side by a classical RNA helix. This peculiar arrangement induces a strong curvature of the phosphodiester backbone, which makes it conducive to multiple tertiary interactions. SNU13/Snu13p (Human/Yeast) binds specifically the U14 C/D box snoRNA K-turn sequence motif. This event is the prerequisite to promote the assembly of the RNP, which contains NOP58/Nop58 and NOP56/Nop56 core proteins and the 2'-O-methyl-transferase, Fibrillarin/Nop1p. The U14 small nucleolar RNA is a conserved non-coding RNA found in yeast and vertebrates required for the pre-rRNA maturation and ribose methylation. Here, we report the solution structure of the free U14 snoRNA K-turn motif (kt-U14) as determined by Nuclear Magnetic Resonance. We demonstrate that a major fraction of free kt-U14 adopts a pre-folded conformation similar to protein bound K-turn, even in the absence of divalent ions. In contrast to the kt-U4 or tyrS RNA, kt-U14 displays a sharp bent in the phosphodiester backbone. The U•U and G•A tandem base pairs are formed through weak hydrogen bonds. Finally, we show that the structure of kt-U14 is stabilized upon Snu13p binding. The structure of the free U14 RNA is the first reference example for the canonical motifs of the C/D box snoRNA family.

Intermolecular interactions between the neurotensin and the third extracellular loop of human neurotensin 1 receptor.

Neurotensin (NT) is a tridecapeptide hormone in the periphery and neurotransmitter in the brain that principally activates three receptor subtypes, named NTS1, NTS2, and NTS3. Since little is known about its structure in the presence of its principal receptor NTS1, we determined it using the key domain of the receptor, i.e. the third extracellular loop. We conclude the following: (i) for the receptor fragment, NT binding modifies its central part, underlying the great flexibility and adaptability of this region; (ii) for bound NT, the extended conformation of its C-terminus is confirmed for the first time in experimental conditions and in the presence of a part of the receptor; and (iii) despite some substitutions, the human receptor residues that are involved in the interaction with NT could be similar to those of the rat receptor which play an important role in NT binding.

Solution structure of a truncated anti-MUC1 DNA aptamer determined by mesoscale modeling and NMR.

Mucin 1 is a well-established target for the early diagnosis of epithelial cancers. The nucleotides of the S1.3/S2.2 DNA aptamer involved in binding to variable number tandem repeat mucin 1 peptides have been identified using footprinting experiments. The majority of these binding nucleotides are located in the 25-nucleotide variable region of the total aptamer. Imino proton and 2D NMR spectra of truncated and total aptamers in supercooled water reveal common hydrogen-bonding networks and point to a similar secondary structure for this 25-mer sequence alone or embedded within the total aptamer. NMR titration experiments confirm that the TTT triloop structure is the primary binding site and show that the initial structure of the truncated aptamers is conserved upon interaction with variable number tandem repeat peptides. The thermal dependence of the NMR chemical shift data shows that the base-paired nucleotides melt cooperatively at 47 ± 4°C. The structure of the 25-mer oligonucleotide was determined using a new combined mesoscale molecular modeling, molecular dynamics and NMR spectroscopy investigation. It contains three Watson-Crick pairs, three consecutive mispairs and four Watson-Crick pairs capped by a TTT triloop motif. The 3D model structures (PDB 2L5K) and biopolymer chain elasticity molecular models are consistent with both NMR and long unconstrained molecular dynamics (10 ns) in explicit water, respectively. Database Structural data are available in the Protein Data Bank and BioMagResBank databases under the accession numbers 2L5K and 17129, respectively.

NMR assignment of PN2-3, a tubulin interaction subdomain of the CPAP protein.

We report the NMR assignment of the PN2-3 subdomain of the CPAP protein. It has been previously shown that this motif interacts with tubulin, inhibits microtubule nucleation from the centrosome and depolymerizes taxol-stabilized microtubules.

NMR solution structure of neurotensin in membrane-mimetic environments: molecular basis for neurotensin receptor recognition.

Neurotensin (NT) is a 13-residue neuropeptide that exerts multiple biological functions in the central and peripheral nervous system. Little is known about the structure of this neuropeptide, and what is known only concerns its C-terminal part. We determined here for the first time the structure of the full-length NT in membrane-mimicking environments by means of classical proton-proton distance constraints derived from solution-state NMR spectroscopy. NT was found to have a structure at both its N and C termini, whereas the central region of NT remains highly flexible. In TFE and HFIP solutions, the NT C-terminus presents an extended slightly incurved structure, whereas in DPC it has a beta turn. The N-terminal region of NT possesses great adaptability and accessibility to the microenvironment in the three media studied. Altogether, our work demonstrates a structure of NT fully compatible with its NTR-bound state.

The beta-thymosin/WH2 domain; structural basis for the switch from inhibition to promotion of actin assembly.

The widespread beta-thymosin/WH2 actin binding domain has versatile regulatory properties in actin dynamics and motility. beta-thymosins (isolated WH2 domain) maintain monomeric actin in a "sequestered" nonpolymerizable form. In contrast, when repeated in tandem or inserted in modular proteins, the beta-thymosin/WH2 domain promotes actin assembly at filament barbed ends, like profilin. The structural basis for these opposite functions is addressed using ciboulot, a three beta-thymosin repeat protein. Only the first repeat binds actin and possesses the function of ciboulot. The region that shows the strongest interaction with actin is an amphipathic N-terminal alpha helix, present in all beta-thymosin/WH2 domains, which recognizes the ATP bound actin structure and uses the shear motion of actin linked to ATP hydrolysis to control polymerization. Crystallographic ((1)H, (15)N), NMR, and mutagenetic data reveal that the weaker interaction of the C-terminal region of beta-thymosin/WH2 domain with actin accounts for the switch in function from inhibition to promotion of actin assembly.

Coupling of folding and binding of thymosin beta4 upon interaction with monomeric actin monitored by nuclear magnetic resonance.

Thymosin beta4 is a major actin-sequestering protein, yet the structural basis for its biological function is still unknown. This study provides insight regarding the way this 43-amino acid peptide, mostly unstructured in solution, binds to monomeric actin and prevents its assembly in filaments. We show here that the whole backbone of thymosin beta4 is highly affected upon binding to G-actin. The assignment of all amide protons and nitrogens of thymosin in the bound state, obtained using a combination of NMR experiments and selective labelings, shows that thymosin folds completely upon binding and displays a central extended region flanked by two N- and C-terminal helices. The cleavage of actin by subtilisin in the DNase I binding loop does not modify the structure of thymosin beta4 in the complex, showing that the backbone of the peptide is not in close proximity to segment 42-47 of actin. The combination of our NMR results and previously published mutation and cross-link data allows a better characterization of the binding mode of thymosins on G-actin.

A structural genomics initiative on yeast proteins.

A canonical structural genomics programme is being conducted at the Paris-Sud campus area on baker's yeast proteins. Experimental strategies, first results and identified bottlenecks are presented. The actual or potential contributions to the structural genomics of several experimental structure-determination methods are discussed.