Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011;6(5):e20452.
doi: 10.1371/journal.pone.0020452. Epub 2011 May 27.

Solution Structure of LC4 Transmembrane Segment of CCR5

Affiliations
Free PMC article

Solution Structure of LC4 Transmembrane Segment of CCR5

Kazuhide Miyamoto et al. PLoS One. .
Free PMC article

Abstract

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 fluorescence 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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. CD spectra of LC4.
(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.
Figure 2
Figure 2. Insertion of LC4 into SDS micelles monitored by acrylamide quenching of LC4 fluorescence.
(A) Stern–Volmer plot. (•), in phosphate buffer at pH 4.5; (▪), in phosphate buffer at pH 7.4; (○), in SDS micelles at pH 4.5; (□), in SDS micelles at pH 7.4. Peptide-coupled SDS micelles were preincubated for 15 min before measurements. [Q] is the quencher concentration (5–85 mM). The SDS micelles concentration was 10 mM, and the peptide concentration was 50 µM. (B) Stern–Volmer constants calculated from the acrylamide quenching experiments in (A). Each error bar shows the SEM from six measurements.
Figure 3
Figure 3. Two-dimensional clean-TOCSY spectrum (mixing time, 80 ms) of LC4 in d25-SDS micelles.
The assignment of each residue is marked on the spectrum. The concentration of the LC4 peptide is 2 mM. The spectrum was recorded at 20°C in 80 mM phosphate buffer (pH 4.5) containing 200 mM d25-SDS micelles.
Figure 4
Figure 4. Chemical shift differences of Hα between the experimental values and random coil values.
The experimental values were observed as the Hα 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 . The negative Δδ values showed the presence of the helical conformation.
Figure 5
Figure 5. Patterns of sequential and medium range NOE cross-peaks of LC4 in SDS micelles.
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 .
Figure 6
Figure 6. Overall structure of LC4 in SDS micelles at pH 4.5 by 1H-NMR.
(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.
Figure 7
Figure 7. Possible docking positions of LC4 and LC5 calculated using the program ZDOCK.
The NMR structure of LC5 was determined in the previous study . 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.

Similar articles

See all similar articles

Cited by 8 articles

See all "Cited by" articles

References

    1. Agrawal L, VanHorn-Ali Z, Berger EA, Alkhatib G. Specific inhibition of HIV-1 coreceptor activity by synthetic peptides corresponding to the predicted extracellular loops of CCR5. Blood. 2004;103:1211–1217. - PubMed
    1. Schlecht HP, Schellhorn S, Dezube BJ, Jacobson JM. New approaches in the treatment of HIV/AIDS - focus on maraviroc and other CCR5 antagonists. Ther Clin Risk Manag. 2008;4:473–485. - PMC - PubMed
    1. Berger EA, Murphy PM, Farber JM. Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu Rev Immunol. 1999;17:657–700. - PubMed
    1. Moore JP, Trkola A, Dragic T. Co-receptors for HIV-1 entry. Curr Opin Immunol. 1997;9:551–562. - PubMed
    1. Wyatt R, Sodroski J. The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens. Science. 1998;280:1884–1888. - PubMed

LinkOut - more resources

Feedback