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

Abstract

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.

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

“…Moreover, equivalent residues of CCL5 V49 in CXC chemokines, CXCL12 V49 and CXCL8 L49, form hydrophobic contacts with the aromatic moiety of tyrosine residues that may be sulfated in the receptor. Together with the structures of Nt-CCR5(1-27):CCL5 (P9S) and Nt-CCR3 (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23):CCL11, these observations support and help rationalize the general promiscuity between receptor and chemokine ligands, in particular among the CC family.…”

“…These observations underline the importance of measuring intermolecular NOEs when calculating the structure of protein complexes. It is our contention that, for the majority of proteins where chemical shift perturbations are measurable, intermolecular NOEs can be detected using the transferred NOE approach . Such pairwise interactions clarify the underlying basis for the chemical shift perturbation and whether it is due to a direct or indirect effect.…”

“…Using 15 N and 13 C uniformly labeled (U‐ 15 N and U‐ 13 C) CCL5(P9S) in the presence of fivefold molar concentration of unlabeled doubly sulfated Nt‐CCR5(1‐27), hereafter referred to as Nt‐CCR5(1‐27), we performed 3D NMR experiments to obtain complete backbone and side‐chain assignments for CCL5(P9S), excluding the N‐terminal amine. Under the measurement conditions, ~ 90% of CCL5(P9S) is in the bound state enabling us to use the chemical shift data to assess the secondary structure of CCL5(P9S) in the bound state. We used the chemical shifts of HN, Hα, Cα, Cβ, and CO to predict a total of 96 backbone (φ,ψ) and 30 side‐chain (χ 1 ) torsion angle values with the program TALOS‐N .…”

“…The differences in conformation between both peptides may be ascribed to the sulfation of different tyrosine residues. In Nt-CCR3 (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23), the sulfation of consecutive residues, CCR3-NT sY16 and CCR3-NT sY17, likely leads to steric and electrostatic repulsion between the side-chains and forces the backbone in this region to adopt a nonstandard turn, while in Nt-CCR5 , the spacing between NT sY10 and NT sY14 allows the formation of a standard helical turn so that both tyrosine residues face the chemokine surface.…”

“…Recently, we applied transferred NOE to detect intermolecular interactions between an N‐terminal CCR5 peptide and CCL5 and identified a CCL5 double mutant (CCL5(E66S, P9S)) that is mostly monomeric in solution . In the present study, we use this construct to assign 33 pairwise intermolecular NOE interactions to the corresponding protons of doubly sulfated Nt‐CCR5(1‐27) and monomeric CCL5.…”