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Immunoglobulin E (IgE) plays a central role in allergic reactions. IgE is a dynamic molecule that is capable of undergoing large conformational changes. X-ray crystal structures of the Fc region of IgE in complex with various ligands have shown that IgE-Fc can exist in extended and various bent conformations. IgE-Fc consists of three domains: Cε2, Cε3 and Cε4. While the complete NMR backbone assignments of the Cε2 and Cε3 domains have been reported previously, the Cε4 domain has not been assigned. Here, we report the complete backbone assignment of the Cε4 homodimer. Cε4 can be used as a model system to study dynamics and allostery in IgE, as both molecules exist as homodimers and exhibit similar binding properties to a number of ligands.

RNA-binding protein Musashi-2 (MSI2) is a key regulator in stem cells, it is over-expressed in a variety of cancers and its higher expression is associated with poor prognosis. Like Musashi-1, it contains two N-terminal RRMs (RNA-recognition Motifs, also called RBDs (RNA-binding Domains)), RRM1 and RRM2, which mediate the binding to their target mRNAs. Previous studies have obtained the three-dimensional structures of the RBDs of Musashi-1 and the RBD1:RNA complex. Here we show the binding of MSI2-RRM1 to a 15nt Numb RNA in Fluorescence Polarization assay and time resolved Fluorescence Resonance Energy Transfer assay. Using nuclear magnetic resonance (NMR) spectroscopy we assigned the backbone resonances of MSI2-RRM1, and characterized the direct interaction of RRM1 to Numb RNA r(GUAGU). Our NMR titration and structure modeling studies showed that MSI2-RRM1 and MSI1-RBD1 have similar RNA binding events and binding pockets. This work adds significant information to MSI2-RRM1 structure and RNA binding pocket, and contributes to the development of MSI2 specific and MSI1/MSI2 dual inhibitors.

We are grateful to Gottfried Otting for his helpful comments.; International audience; Despite their roles in controlling many cellular processes, weak and transient interactions between large structured macromolecules and disordered protein segments cannot currently be characterized at atomic resolution by Xray crystallography or solution NMR. Solid-state NMR does not suffer from the molecular size limitations affecting solution NMR, and it can be applied to molecules in different aggregation states, including non-crystalline precipitates and sediments. A solid-state NMR approach based on high magnetic fields, fast magic-angle sample spinning, and deuteration provides chemical-shift and relaxation mapping that enabled the characterization of the structure and dynamics of the transient association between two regions in an 80 kDa protein assembly. This led to direct verification of a mechanism of regulation of E. coli DNA metabolism.

Hop/STI1 (Hsp-organizing protein/stress-induced-phosphoprotein 1) is a molecular co-chaperone, which coordinates Hsp70 and Hsp90 activity during client protein folding through interactions with its TPR1 and TPR2A domains. Hsp90 substrates include a diverse set of proteins, many of which have been implicated in tumorigenesis. Over-expression of Hsp90 in cancer cells stabilizes mutant oncoproteins promoting cancer cell survival. Disruption of Hsp90 and its co-chaperone machinery has become a promising strategy for the treatment of cancer. STI1 has also been described as a neurotrophic signaling molecule through its interactions with the prion protein (PrP(C)). Here, we report the (1)H, (13)C and (15)N backbone assignments of the TPR1 and TPR2A domains of mouse STI1, which interact with Hsp70 and Hsp90, respectively. (1)H-(15)N HSQC spectra of TPR2A domain in the presence of a peptide encoding the C-terminal Hsp90 binding site revealed significant chemical shift changes indicating complex formation. These results will facilitate the screening of potential molecules that inhibit STI1 complex formation with Hsp70 and/or Hsp90 for the treatment of cancer and detailed structural studies of the STI1-PrP(C) complex.

Four novel 5D (HACA(N)CONH, HNCOCACB, (HACA)CON(CA)CONH, (H)NCO(NCA)CONH), and one 6D ((H)NCO(N)CACONH) NMR pulse sequences are proposed. The new experiments employ non-uniform sampling that enables achieving high resolution in indirectly detected dimensions. The experiments facilitate resonance assignment of intrinsically disordered proteins. The novel pulse sequences were successfully tested using δ subunit (20 kDa) of Bacillus subtilis RNA polymerase that has an 81-amino acid disordered part containing various repetitive sequences.

Acinetobacter baumannii is a multidrug-resistant opportunistic pathogen which induces the cytotoxicity of host cells by nosocomial infections. Peptidoglycan-associated lipoprotein (Pal) is one component of Tol-Pal system, which is involved in maintaining the integrity and stability of the outer membrane (OM) as well as the virulence of bacterial pathogens. Pal is composed of N-terminal hydrophobic domain anchoring to the OM and C-terminal OmpA-like domain interacting with peptidoglycan. Herein, we report the preparation of protein sample and the backbone assignment of recombinant OmpA-like domain of Pal protein from Acinetobacter baumannii (AbPal-71). All the backbone chemical shifts of AbPal-71 (Cα, Cβ, CO, HN, and N) were completely assigned and the secondary structure was estimated from the assigned chemical shifts. This result shows that AbPal-71 is a typical OmpA-like fold, which consists of three α-helices and four β-strands. Based on this NMR spectroscopic results, the three dimensional structural study and the interaction study between AbPal-71 and peptidoglycan are in progress.

MJ0754 is an unknown hypothetical protein from hyperthermophilic archaeon Methanococcus jannaschii DSM 2661. Here we report the protein purification and NMR spectroscopic study of the N-terminal deleted truncated form of recombinant MJ0754 protein (Δ10-MJ0754). We aquired 3D NMR spectra and assigned all the backbone chemical shifts including Cα, Cβ, CO, HN, and N of Δ10-MJ0754. The secondary structure of Δ10-MJ0754 was estimated from the assigned backbone chemical shifts. This NMR result is very useful for further structural and functional study of MJ0754 protein.

Mst1 and Mst2 (Mammalian Sterile 20-like kinase 1 and 2) are pro-apoptotic protein kinases and involved in cell proliferation and survival. The C-termini of Mst1 and Mst2 contain a common protein-protein interaction domain, named SARAH (Sav/Rassf/Hpo), which is found in three classes of eukaryotic tumour suppressors, Salvador, Rassf, and Hippo. The interaction of these SARAH domains controls apoptosis and cell cycle arrest. Moreover, Mst2 SARAH domain is known to interact with Raf, resulting in the suppression of apoptosis. In this study, we describe the sample preparation and NMR study of SARAH domain from Mst2 kinase. The gel-filtration chromatography shows that the SARAH domain of Mst2 forms a homodimer in solution. The NMR spectrum of Mst2 SARAH domain exhibit well-dispersed and homogeneous signals, which enable us to assign the backbone signals completely. These results provide a useful information for the structural and functional study of Mst2 SARAH domain.

We would like to thank Matthias Hiller and Sebastian Wegner for assistance installing and calibrating the probes in Berlin. A.J.N. was supported by fellowships from the Fulbright Program and the Alexander von Humboldt Foundation. This work was also supported by the CNRS (TGIR-RMN-THC FR3050) and from a Joint Research Activity in the 7th Framework program of the EC (BioNMR No. 261863).; International audience; The use of small rotors capable of very fast magic-angle spinning (MAS) in conjunction with proton dilution by perdeuteration and partial reprotonation at exchangeable sites has enabled the acquisition of resolved, proton detected, solid-state NMR spectra on samples of biological macromolecules. The ability to detect the high-gamma protons, instead of carbons or nitrogens, increases sensitivity. In order to achieve sufficient resolution of the amide proton signals, rotors must be spun at the maximum rate possible given their size and the proton back-exchange percentage tuned. Here we investigate the optimal proton back-exchange ratio for triply labeled SH3 at 40 kHz MAS. We find that spectra acquired on 60 % back-exchanged samples in 1.9 mm rotors have similar resolution at 40 kHz MAS as spectra of 100 % back-exchanged samples in 1.3 mm rotors spinning at 60 kHz MAS, and for (H)NH 2D and (H)CNH 3D spectra, show 10-20 % higher sensitivity. For 100 % back-exchanged samples, the sensitivity in 1.9 mm rotors is superior by a factor of 1.9 in (H)NH and 1.8 in (H)CNH spectra but at lower resolution. For (H)C(C)NH experiments with a carbon-carbon mixing period, this sensitivity gain is lost due to shorter relaxation times and less efficient transfer steps. We present a detailed study on the sensitivity of these types of experiments for both types of rotors, which should enable experimentalists to make an informed decision about which type of rotor is best for specific applications.

The backbone assignment of medium-sized proteins is rarely as straightforward as that of small proteins, and thus often requires creative solutions. Here, we describe the application of a combination of standard 3D heteronuclear methods with CC(CO)NH and a variety of MUltiplicity Selective In-phase Coherence transfer (MUSIC) experiments. Both CC(CO)NH and MUSIC are, in theory, very powerful methods for the backbone assignment of proteins. Due to low sensitivity, their use has usually been linked to small proteins only. However, we found that combining CC(CO)NH and MUSIC experiments simplified the assignment of two challenging medium-sized proteins of 13 and 19.5 kDa, respectively. These methods are to some extent complementary to each other: CC(CO)NH acquired with a long isotropic mixing time can identify amino acids with large aliphatic side chains. Whereas the most sensitive MUSIC experiments identify amino acid types that cannot be detected by CC(CO)NH, comprising the residues with acid and amide groups, and aromatic rings in their side chains. Together these methods provide a means of identifying the majority of peaks in the 2D 15N HSQC spectrum which simplifies the backbone assignment work even for proteins, e.g., small kinases, whose standard spectra resulted in little spectral resolution and low signal intensities. publishedVersion