Defining specific residue-to-residue interactions between the gp120 bridging sheet and the N-terminal segment of CCR5: applications of transferred NOE NMR.

Infection by HIV-1 requires protein-protein interactions involving gp120, CD4 and CCR5. We have previously demonstrated that the transferred nuclear Overhauser effect (TRNOE), in combination with asymmetric deuteration of a protein and a peptide ligand can be used to detect intermolecular interactions in large protein complexes with molecular weights up to ~ 100 kDa. Here, using this approach, we reveal interactions between tyrosine residues of a 27-residue peptide corresponding to the N-terminal segment of the CCR5 chemokine receptor, and a dimeric extended core gp120 envelope protein of HIV-1 complexed with a CD4-mimic miniprotein.

Effects of chelator lipids, paramagnetic metal ions and trehalose on liposomes by solid-state NMR.

The effects of various lipid bound paramagnetic metal ions on liposomes prepared in the presence of trehalose and chelator lipids are evaluated to observe site-specific signal changes on liposome samples with optimal resolution in solid-state NMR spectroscopy. We found that Mn, Gd and Dy have different influences on the lipid C sites depending on their penetration depths into the bilayer, which can be extracted as distance information. The trehalose-liposome mixture is efficiently packed into solid-state NMR rotors and provides optimal resolution at reasonable instrument temperatures (10-50 °C).

The solution structure of monomeric CCL5 in complex with a doubly sulfated N-terminal segment of CCR5.

Unlabelled: The inflammatory chemokine CCL5, which binds the chemokine receptor CCR5 in a two-step mechanism so as to activate signaling pathways in hematopoetic cells, plays an important role in immune surveillance, inflammation, and development as well as in several immune system pathologies. The recently published crystal structure of CCR5 bound to a high-affinity variant of CCL5 lacks the N-terminal segment of the receptor that is post-translationally sulfated and is known to be important for high-affinity binding. Here, we report the NMR solution structure of monomeric CCL5 bound to a synthetic doubly sulfated peptide corresponding to the missing first 27 residues of CCR5.

Detection of intermolecular transferred-NOE interactions in small and medium size protein complexes: RANTES complexed with a CCR5 N-terminal peptide.

NMR is a powerful tool for studying structural details of protein/peptide complexes exhibiting weak to medium binding (K > 10 μm). However, it has been assumed that intermolecular nuclear Overhauser effect (NOE) interactions are difficult to observe in such complexes. We demonstrate that intermolecular NOEs can be revealed by combining the C-edited/ C-filtered experiment with the transferred NOE effect (TRNOE).

Immunofocusing using conformationally constrained V3 peptide immunogens improves HIV-1 neutralization.

V3-directed antibodies are present in practically all HIV-1 infected patients and in individuals vaccinated with gp120. The levels of maternal V3-directed antibodies were recently shown to correlate with reduced mother to child transmission, and V3 IgGs were found to be a negative correlate of risk in the RV-144 human trial. mAb directed to the tip of the V3 are capable of broad neutralization of Tier-1 and some Tier-2 viruses.