Highlights d Transmembrane helix of death receptor 5 oligomerizes to drive downstream signaling d The transmembrane helix in lipid bilayer forms dimer-trimer interaction network d Receptor ectodomain in pre-ligand state inhibits receptor clustering and activation d Ligand binding overcomes the pre-ligand autoinhibition

The membrane-proximal external region (MPER) of the HIV-1 envelope glycoprotein (Env) bears epitopes of broadly neutralizing antibodies (bnAbs) from infected individuals; it is thus a potential vaccine target. We report an NMR structure of the MPER and its adjacent transmembrane domain in bicelles that mimic a lipid-bilayer membrane. The MPER lies largely outside the lipid bilayer. It folds into a threefold cluster, stabilized mainly by conserved hydrophobic residues and potentially by interaction with phospholipid headgroups. Antigenic analysis and comparison with published images from electron cryotomography of HIV-1 Env on the virion surface suggest that the structure may represent a prefusion conformation of the MPER, distinct from the fusion-intermediate state targeted by several well-studied bnAbs. Very slow bnAb binding indicates that infrequent fluctuations of the MPER structure give these antibodies occasional access to alternative conformations of MPER epitopes. Mutations in the MPER not only impede membrane fusion but also influence presentation of bnAb epitopes in other regions. These results suggest strategies for developing MPER-based vaccine candidates.

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Expansions of polyglutamine (polyQ) tracts in nine different proteins cause a family of neurodegenerative disorders called polyQ diseases. Because polyQ tracts are potential therapeutic targets for these pathologies there is great interest in characterizing the conformations that they adopt and in understanding how their aggregation behavior is influenced by the sequences flanking them. We used solution NMR to study at single-residue resolution a 156-residue proteolytic fragment of the androgen receptor that contains a polyQ tract associated with the disease spinobulbar muscular atrophy, also known as Kennedy disease. Our findings indicate that a Leu-rich region preceding the polyQ tract causes it to become α-helical and appears to protect the protein against aggregation, which represents a new, to our knowledge, mechanism by which sequence context can minimize the deleterious properties of these repetitive regions. Our results have implications for drug discovery for polyQ diseases because they suggest that the residues flanking these repetitive sequences may represent viable therapeutic targets.

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Structural characterization of transmembrane proteins in isotropic bicelles has become an increasingly popular application of solution NMR spectroscopy, as the fast-tumbling bicelles are membrane-like yet can often yield spectral quality comparable to those of detergent micelles. While larger bicelles are closer to the true lipid bilayer, it remains unclear how large the bicelles need to be to allow accurate assessment of protein transmembrane partition in lipid bilayer. Here, we address the above question from the perspective of protein residing in the bicelles, through systematic measurement of protein chemical shift and transmembrane partition at different lipid:detergent ratios (q), ranging from 0.3 to 0.7, using the transmembrane domain of human Fas receptor as model system. We found that the lipid environment of the bicelles, as reflected by the protein chemical shift, begins to be perturbed when the q is reduced to below 0.6. We also implemented a solvent paramagnetic relaxation enhancement (PRE) approach for bicelles to show that the protein transmembrane partition in bicelles with q = 0.5 and 0.7 are very similar, but at q = 0.3 the solvent PRE profile is significantly different. Our data indicate that q values between 0.5 and 0.6 are good compromise between high resolution NMR and closeness to the membrane environment, and allow accurate characterization of protein position in lipid bilayer.

The characterization of intrinsically disordered proteins (IDPs) by NMR spectroscopy is made difficult by the extensive spectral overlaps. To overcome the intrinsic low-resolution of the spectra the introduction of high-dimensionality experiments is essential. We present here a set of high-resolution experiments based on direct (13)C-detection which proved useful in the assignment of α-synuclein, a paradigmatic IDP. In particular, we describe the implementation of 4D HCBCACON, HCCCON, HCBCANCO, 4/5D HNCACON and HNCANCO and 3/4D HCANCACO experiments, specifically tailored for spin system identification and backbone resonances sequential assignment. The use of non-uniform-sampling in the indirect dimension and of the H-flip approach to achieve longitudinal relaxation enhancement rendered the experiments very practical.

Here, we present a structural and dynamic description of CBP-ID4 at atomic resolution. ID4 is the fourth intrinsically disordered linker of CREB-binding protein (CBP). In spite of the largely disordered nature of CBP-ID4, NMR chemical shifts and relaxation measurements show a significant degree of α-helix sampling in the protein regions encompassing residues 2-25 and 101-128 (1852-1875 and 1951-1978 in full-length CBP). Proline residues are uniformly distributed along the polypeptide, except for the two α-helical regions, indicating that they play an active role in modulating the structural features of this CBP fragment. The two helical regions are lacking known functional motifs, suggesting that they represent thus-far uncharacterized functional modules of CBP. This work provides insights into the functions of this protein linker that may exploit its plasticity to modulate the relative orientations of neighboring folded domains of CBP and fine-tune its interactions with a multitude of partners.