The envelope spike of HIV-1, which consists of three external gp120 and three transmembrane gp41 glycoproteins, recognizes its target cells by successively binding to its primary CD4 receptor and a coreceptor molecule. Until recently, atomic-resolution structures were available primarily for monomeric HIV-1 gp120, in which the V1, V2 and V3 variable loops were omitted (gp120 core ), in complex with soluble CD4 (sCD4). Differences between the structure of HIV gp120 core in complex with sCD4 and the structure of unliganded simian immunodeficiency virus gp120 core led to the hypothesis that gp120 undergoes a major conformational change upon sCD4 binding. To investigate the conformational flexibility of gp120, we generated two forms of mutated gp120 amenable for NMR studies: one with V1, V2 and V3 omitted ( mut gp120 core ) and the other containing the V3 region N]Ile-labeled unliganded mut gp120 core showed many fewer crosspeaks than the expected number, and also many fewer crosspeaks in comparison with the labeled mut gp120 core bound to the CD4-mimic peptide, CD4M33. This finding suggests that in the unliganded form, mut gp120 core shows considerable flexibility and motions on the millisecond time scale. In contrast, most of the expected crosspeaks were observed for the unliganded mut gp120 core (+V3), and only a few changes in chemical shift were observed upon CD4M33 binding. These results indicate that mut gp120 core (+V3) does not show any significant conformational flexibility in its unliganded form and does not undergo any significant conformational change upon CD4M33 binding, underlining the importance of V3 in stabilizing the gp120 core conformation.
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). Due to the TRNOE phenomenon, intermolecular NOE cross peaks are characterized by both the chemical shifts (CSs) of the protein protons and the average CSs of the peptide protons, which are dominated by the CSs of the protons of the free peptide. Previously, the TRNOE phenomenon was used almost exclusively to investigate the conformation of small ligands bound to large biomolecules. Here, we demonstrate that TRNOE can be extended to enable the study of intermolecular interactions in small- and medium-sized protein complexes. We used the C-edited/ C-filtered TRNOE experiment to study the interactions of the chemokine regulated upon activation, normal T cell, expressed and secreted (RANTES) with a 27-residue peptide, containing two sulfotyrosine residues, representing the N-terminal segment of the CCR5 receptor ((Nt-CCR5(1-27). The TRNOE phenomenon led to more than doubling of the signal-to-noise ratios (SNRs) for the intermolecular NOEs observed in the C-edited/ C-filtered experiment for the 11.5-kDa monomeric RANTES/Nt-CCR5(1-27) complex. An even better improvement in the SNR was achieved with dimeric Nt-CCR5(1-27)/RANTES (23 kDa), especially in comparison with the spectra measured with a 1 : 1 protein to peptide ratio. In principle, the isotope-edited/isotope-filtered TRNOE spectrum can discern all intermolecular interactions involving nonexchangeable protons in the complex.
In a wide spectrum of neurodegenerative diseases, self-assembly of pathogenic proteins to cytotoxic intermediates is accelerated by the presence of metal ions such as Cu2+. Only low concentrations of these...
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