Solution Structure of LC4 Transmembrane Segment of CCR5

CC-chemokine receptor 5 (CCR5) is a specific co-receptor allowing the entry of human immunodeficiency virus type 1 (HIV-1). The LC4 region in CCR5 is required for HIV-1 entry into the cells. In this study, the solution structure of LC4 in SDS micelles was elucidated by using standard 1H two-dimensional NMR spectroscopy, circular dichroism, and fluorescdence quenching. The LC4 structure adopts two helical structures, whereas the C-terminal part remains unstructured. The positions in which LC4 binds to the HIV-1 inhibitory peptide LC5 were determined by docking calculations in addition to NMR data. The poses showed the importance of the hydrophobic interface of the assembled structures. The solution structure of LC4 elucidated in the present work provides a structural basis for further studies on the HIV-1 inhibitory function of the LC4 region

CD spectra of LC4.

<p>(1) 50 µM LC4 in phosphate buffer at pH 7.4; (2) as in (1) but at pH 4.5; (3) 50 µM LC4 in SDS micelles at pH 7.4; (4) as in (3) but at pH 4.5. The experiments were carried out at room temperature in 80 mM phosphate buffer or 80 mM phosphate buffer containing 10 mM SDS.</p

Summary of Structure Statistics of LC4 in SDS micelles^a.

a<p>Except for the number of constraints, average values given for the set of 20 conformers with the lowest CYANA target function value.</p>b<p>The values were calculated for residues 157–172.</p

Patterns of sequential and medium range NOE cross-peaks of LC4 in SDS micelles.

<p>The NOEs for 2 mM LC4 were derived from the NOESY spectrum with a mixing time of 150 ms in the presence of 200 mM d₂₅-SDS micelles. The NOE patterns are used to characterize the region of the helical structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020452#pone.0020452-Wthrich1" target="_blank">[28]</a>.</p

Possible docking positions of LC4 and LC5 calculated using the program ZDOCK.

<p>The NMR structure of LC5 was determined in the previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020452#pone.0020452-Miyamoto1" target="_blank">[10]</a>. The docking positions were calculated using the program ZDOCK and then the energy-minimization calculations were performed using the program Discovery Studio. (A) Ribbon diagrams of the 5 lowest energy structures (residues Val157–Glu172 of LC4; residues Lys229–Thr239 of LC5) (B) close-up view of the binding interface of the lowest energy structure, showing the heavy atoms of the side chains.</p

Chemical shift differences of H^α between the experimental values and random coil values.

<p>The experimental values were observed as the H^α chemical shift for 2 mM LC4 in the presence of 200 mM d₂₅-SDS micelles. The random coil values were obtained from Chemical Shift Index <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020452#pone.0020452-Wishart1" target="_blank">[30]</a>. The negative Δδ values showed the presence of the helical conformation.</p

Overall structure of LC4 in SDS micelles at pH 4.5 by ¹H-NMR.

<p>(A) Stereoview illustrating a trace of the backbone atoms for the ensemble of the 20 lowest energy structures, showing the heavy atoms of the side chains (residues Val157-Leu174). The structures in the well-ordered region (residues Val157-Glu172) was superimposed over the backbone atoms. (B) Surface representation and ribbon diagram of LC4 showing the side chains (residues Val157-Glu172). The helical regions (α1 and α2) are shown in red.</p