As part of a program for the development of synthetic vaccines against the pathogen Shigella flexneri, we used a combination of NMR and molecular modeling methods to study the conformations of the O-specific polysaccharide (O-SP) of S. flexneri 5a and of four related synthetic pentasaccharide fragments. The NMR study, based on the analysis of 1 H and 13 C chemical shifts, the evaluation of inter-residue distances, and the measurement of oneand three-bond heteronuclear coupling constants, showed that the conformation of one of the four pentasaccharides is similar to that of the native O-SP in solution. Interestingly, inhibition enzyme-linked immunosorbent assay demonstrated that a protective monoclonal antibody specific for S. flexneri 5a has a greater affinity for this pentasaccharide than for the others. We carried out a complete conformational search on the pentasaccharides using the CICADA algorithm interfaced with MM3 force field. We calculated Boltzmann-averaged inter-residue distances and 3 J C,H coupling constants for the different conformational families and compared the results with NMR data for all pentasaccharides. Our experimental data are consistent with only one conformational family. We also used molecular modeling data to build models of the O-SP with the molecular builder program POLYS. The models that are in agreement with NMR data adopt right-handed 3-fold helical structures in which the branched glucosyl residue points outwards.

Microtubules are cytoskeletal components involved in multiple cell functions such as mitosis, motility, or intracellular traffic. In vivo, these polymers made of ␣␤-tubulin nucleate mostly from the centrosome to establish the interphasic microtubule network or, during mitosis, the mitotic spindle. Centrosomal P4.1-associated protein (CPAP; also named CENPJ) is a centrosomal protein involved in the assembly of centrioles and important for the centrosome function. This protein contains a microtubule-destabilizing region referred to as PN2-3. Here we decrypt the microtubule destabilization activity of PN2-3 at the molecular level and show that it results from the sequestration of tubulin by PN2-3 in a non-polymerizable 1:1 complex. We also map the tubulin/PN2-3 interaction both on the PN2-3 sequence and on the tubulin surface. NMR and CD data on free PN2-3 in solution show that this is an intrinsically unstructured protein that comprises a 23-amino acid residue ␣-helix. This helix is embedded in a 76-residue region that interacts strongly with tubulin. The interference of PN2-3 with well characterized tubulin properties, namely GTPase activity, nucleotide exchange, vinblastine-induced self-assembly, and stathmin family protein binding, highlights the ␤ subunit surface located at the intermolecular longitudinal interface when tubulin is embedded in a microtubule as a tubulin/PN2-3 interaction area. These findings characterize the PN2-3 fragment of CPAP as a protein with an unprecedented tubulin sequestering mechanism distinct from that of stathmin family proteins. Microtubules (MTs)6 are major components of the cytoskeleton shaped as tubes whose walls are composed of ␣␤-tubulin heterodimers (tubulin). During interphase, they form a dynamic network that participates in the organization of the cell architecture and serves as tracks for molecular motors that convey intracellular cargos and organelles throughout the cytoplasm. In dividing cells, during the M phase, this network is fully reorganized to form spindle MTs on which chromosome pairs align and segregate. To fulfill this wide range of functions, MTs alternate phases of growth and shrinkage in a process known as dynamic instability. The architecture of the MT array results from the action of endogenous proteins that affect MT nucleation and dynamics. MTs are nucleated by the ␥-tubulin ring complex and the ␥-tubulin small complex that, together with proteins that regulate their effects, are localized at the centrosome (1); in differentiated cells this is the primary MT organizing center. Endogenous proteins that regulate MT dynamics include MT-associated proteins that stabilize MTs and proteins that destabilize them like depolymerizing kinesins and tubulin-sequestering proteins. One of the best characterized families of tubulin-sequestering proteins is that of stathmin.

Connexins are structurally related transmembrane proteins that assemble to form gap junction channels involved in the mediation of intercellular communication. It has been shown that the intracellular tail of connexin43 (Cx43) interacts with tubulin and microtubules with putative impacts on its own intracellular trafficking, its activity in channel communication, and its interference with specific growth factor signal transduction cascades. We demonstrate here that the microtubule binding of Cx43 is mainly driven by a short region of 26 amino acid residues located within the intracellular tail of Cx43. The nuclear magnetic resonance structural analysis of a peptide (K26D) corresponding to this region shows that this peptide is unstructured when free in solution and adopts a helix conformation upon binding with tubulin. In addition, the resulting K26D-tubulin molecular complex defines a new structural organization that could be shared by other microtubule partners. Interestingly, the K26D-tubulin interaction is prevented by the phosphorylation of K26D at a src kinase specific site. Altogether, the results elucidate the mechanism of the interaction of Cx43 with the microtubule cytoskeleton and propose a pathway for understanding the microtubule-dependent regulation of Cx43 gap junctional communications and the involvement of Cx43 in TGF-β signal transduction.

Microtubules are major cytoskeletal components involved in numerous cellular functions such as mitosis, cell motility, or intracellular traffic. These cylindrical polymers of alphabeta-tubulin assemble in a closely regulated dynamic manner. We have shown that the stathmin family proteins sequester tubulin in a nonpolymerizable ternary complex, through their stathmin-like domains (SLD) and thus contribute to the regulation of microtubule dynamics. We demonstrate here that short peptides derived from the N-terminal part of SLDs impede tubulin polymerization with various efficiencies and that phosphorylation of the most potent of these peptides reduces its efficiency as in full-length stathmin. To understand the mechanism of action of these peptides, we undertook a NMR-based structural analysis of the peptide-tubulin interaction with the most efficient peptide (I19L). Our results show that, while disordered when free in solution, I19L folds into a beta-hairpin upon binding to tubulin. We further identified, by means of saturation transfer difference NMR, hydrophobic residues located on the beta2-strand of I19L that are involved in its tubulin binding. These structural data were used together with tubulin atomic coordinates from the tubulin/RB3-SLD crystal structure to model the I19L/tubulin interaction. The model agrees with I19L acting through an autonomous tubulin capping capability to impede tubulin polymerization and provides information to help understand the variation of efficiency against tubulin polymerization among the peptides tested. Altogether these results enlighten the mechanism of tubulin sequestration by SLDs, while they pave the way for the development of protein-based compounds aimed at interfering with tubulin polymerization.

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 structural rearrangements accompanying mRNA during translation in mammalian cells remain poorly understood. Here, we discovered that YB-1 (YBX1), a major partner of mRNAs in the cytoplasm, forms a linear nucleoprotein filament with mRNA, when part of the YB-1 unstructured C-terminus has been truncated. YB-1 possesses a cold-shock domain (CSD), a remnant of bacterial cold shock proteins that have the ability to stimulate translation under the low temperatures through an RNA chaperone activity. The structure of the nucleoprotein filament indicates that the CSD of YB-1 preserved its chaperone activity also in eukaryotes and shows that mRNA is channeled between consecutive CSDs. The energy benefit needed for the formation of stable nucleoprotein filament relies on an electrostatic zipper mediated by positively charged amino acid residues in the YB-1 C-terminus. Thus, YB-1 displays a structural plasticity to unfold structured mRNAs into extended linear filaments. We anticipate that our findings will shed the light on the scanning of mRNAs by ribosomes during the initiation and elongation steps of mRNA translation.

TDP-43 is a nuclear RNA-binding protein that forms neuronal cytoplasmic inclusions in two major neurodegenerative diseases, ALS and FTLD. While the self-assembly of TDP-43 by its structured N-terminal and intrinsically disordered C-terminal domains has been widely studied, the mechanism by which mRNA preserves TDP-43 solubility in the nucleus has not been addressed. Here, we demonstrate that tandem RNA Recognition Motifs of TDP-43 bind to long GU-repeats in a cooperative manner through intermolecular interactions. Moreover, using mutants whose cooperativity is impaired, we found that the cooperative binding of TDP-43 to mRNA may be critical to maintain the solubility of TDP-43 in the nucleus and the miscibility of TDP-43 in cytoplasmic stress granules. We anticipate that the knowledge of a higher order assembly of TDP-43 on mRNA may clarify its role in intron processing and provide a means of interfering with the cytoplasmic aggregation of TDP-43.

Fucoidan is a potent inhibitor of the human complement system whose activity is mediated through interactions with certain proteins belonging to the classical pathway, particularly the protein C4. Branched fucoidan oligosaccharides displayed a higher anticomplementary activity as compared to linear structures. Nuclear magnetic resonance (NMR) characterization of the branched oligosaccharides and saturation transfer difference-NMR experiment of the interaction with the protein C4 allowed the identification of the glycan residues in close contact with the target protein. Transferred nuclear Overhauser effect spectroscopy experiment and molecular modeling of fucoidan oligosaccharides indicated that the presence of side chains reduces the flexibility of the oligosaccharide backbone, which thus adopts a conformation which is very close to the one recognized by the protein C4. Together, these results suggest that branching of fucoidan oligosaccharides, determining their conformational state, has a major impact on their anticomplementary activity.