The conformation and orientation of a 27-residue CCR5 peptide in a ternary complex with HIV-1 gp120 and a CD4-mimic peptide

Abstract

Interaction of CC chemokine receptor 5 (CCR5) with the human immunodeficiency virus type 1 (HIV-1) gp120/CD4 complex involves its amino-terminal domain (Nt-CCR5) and requires sulfation of two to four tyrosine residues in Nt-CCR5. The conformation of a 27-residue Nt-CCR5 peptide, sulfated at Y10 and Y14, was studied both in its free form and in a ternary complex with deglycosylated gp120 and a CD4-mimic peptide. NMR experiments revealed a helical conformation at the center of Nt-CCR5(1-27), which is induced upon gp120 binding, as well as a helical propensity for the free peptide. A well-defined structure for the bound peptide was determined for residues 7-23, increasing by 2-fold the length of Nt-CCR5's known structure. Two-dimensional saturation transfer experiments and measurement of relaxation times highlighted Nt-CCR5 residues Y3, V5, P8-T16, E18, I23 and possibly D2 as the main binding determinant. A calculated docking model for Nt-CCR5(1-27) suggests that residues 2-22 of Nt-CCR5 interact with the bases of V3 and C4, while the C-terminal segment of Nt-CCR5(1-27) points toward the target cell membrane, reflecting an Nt-CCR5 orientation that differs by 180° from that of a previous model. A gp120 site that could accommodate (CCR5)Y3 in a sulfated form has been identified. The present model attributes a structural basis for binding interactions to all gp120 residues previously implicated in Nt-CCR5 binding. Moreover, the strong interaction of sulfated (CCR5)Tyr14 with (gp120)Arg440 revealed by the model and the previously found correlation between E322 and R440 mutations shed light on the role of these residues in HIV-1 phenotype conversion, furthering our understanding of CCR5 recognition by HIV-1.

Fig. 1. NMR spectra of free Nt-CCR5(1-27)

Sections of (A) NOESY spectrum at 300 K, (B) NOESY spectrum at 274 K. Experiments were carried out on a 1.25 mM Nt-CCR5(1-27) sample (10 mM phosphate buffer, pH 7.0 with no sodium chloride added), using 250 and 200 ms mixing times for spectra at 300 and 274 K, respectively. Spectra were measured on an 800 MHz NMR spectrometer equipped with a cryo-probe. A few example interactions are labeled.

Fig. 2. Medium-range NOE interactions of free and gp120-bound Nt-CCR5(1-27)

NOE interactions of (a) derived from T_1ρ-filtered NOESY spectra of 1 mM of Nt-CCR5(1-27) in the presence of 0.1 mM deglycosylated-^trgp120/^miCD4 complex measured at 274, 288, 300 and 308 K, in order to resolve peak overlap, in 20 mM D₁₁-Tris buffer, 300 mM NaCl, pH 7.0 and (b) free Nt-CCR5(1-27) derived from a NOESY spectrum measured at 274 K in 10 mM phosphate buffer, pH 7.0 with no sodium chloride added. The observed inter-residue connectivities are marked with filled rectangles, and the connectivities that could not be unambiguously assigned due to cross peak overlaps are marked with empty rectangles. All spectra were measured on an 800 MHz NMR spectrometer equipped with a cryo-probe.

Fig. 3. NMR spectra of bound (a) and free (b) Nt-CCR5(1-27)

(a) A section of a T_1ρ-filtered NOESY spectrum of Nt-CCR5(1-27) and deglycosylated-^trgp120/^miCD4; Experiment carried out on a 1 mM Nt-CCR5(1-27) sample in the presence of 0.1 mM deglycosylated-^trgp120/^miCD4 (10:1). (b) A section of a NOESY spectrum of free Nt-CCR5(1-27); Experiment carried out on a 0.6 mM Nt-CCR5(1-27) sample. Both experiments were carried out on samples dissolved in the same conditions (20 mM D₁₁-Tris buffer, 300 mM NaCl, pH 7.0) using 100 ms mixing time and were measured at 300 K on an 800 MHz NMR spectrometer equipped with a cryo-probe. A few example interactions are labeled. *Peak assignment is better resolved at a different temperature.

Fig. 4. Ensemble of 10 lowest energy structures of Bound Nt-CCR5(1-27)

The converging structures of the segment S7-I23 of Nt-CCR5(1-27) bound to deglycosylated-^trgp120/^miCD4 are presented.

Fig. 5. 1D-STD NMR spectrum of Nt-CCR5(1-27) in the presence of deglycosylated-^trgp120/^miCD4

Reference and difference spectra are shown in black and red, respectively. (a) Aromatic region showing assignment. (b) Amidic, aromatic and aliphatic regions showing identifiable assignment of different residues. Spectra were recorded at 300 K on samples containing 0.1 mM deglycosylated-^trgp120/^miCD4 and l mM Nt-CCR5(1-27) in 20 mM D₁₁-Tris buffer, 300 mM NaCl, pH 7.0 at pH 7.0 using on-resonance irradiation at -2 ppm. Superscript S indicates sulfated Tyr.

Fig. 6. HOHAHA and 2D-STD-HOHAHA spectra

The aliphatic (a,b) and aromatic (c,d) region of a HOHAHA spectrum (a,c) and a 2D-STD-HOHAHA spectrum acquired using on-resonance irradiation at -2 ppm (b,d) of 1 mM Nt-CCR5(1-27) in the presence of 0.1 mM deglycosylated-^trgp120/^miCD4 in 20 mM D₁₁-Tris buffer, 300 mM NaCl, pH 7.0. Arrows and dotted lines show assignments. Spectra were measured at 308 K on an 800 MHz NMR spectrometer equipped with a cryo-probe.

Fig. 7. Quantification of STD enhancements

1D-STD (grey bars) and 2D-STD-HOHAHA (black bars) enhancements calculated by dividing peak intensities in the 1D-STD or 2D-STD-HOHAHA to the corresponding peak intensities in a 1D or HOHAHA spectra (respectively). Values were normalized to the largest enhancement. Bars represent the highest enhancement that could be unambiguously determined for each residue. Asterisk marks residues whose resonances could be resolved in the 1D-STD spectrum and for which quantification of the STD enhancement could be determined but in the 2D-STD, cross-peak overlap due to lower resolution in the acquisition dimension of the 2D-spectrum in comparison with the 1D-STD spectrum, prevented the quantification of the STD for each of the residues whose cross-peaks overlapped (i.e. Y3 and Y15, V5 and V25, I12 and I23).

Fig. 8. Changes in the T_1ρ relaxation times of Nt-CCR5(1-27) residues upon binding to the ^trgp120/^miCD4 complex

Changes in T_1ρ relaxation times of H_α of various residues in Nt-CCR5(1-27), presented as the ratio (percentage) between the T_1ρ relaxation time of the Nt-CCR5(1-27) residue bound to deglycosylated-^trgp120/^miCD4 and that of the same residue in the free form, as a function of residue number. Only residues which were well resolved in the HOHAHA spectra are presented. A 60% value of the ratio representing a reduction of 40% in T_1ρ, is drawn as cutoff. Residues exhibiting ratio above this cut-off are regarded as residues that do not interact with the deglycosylated-^trgp120/^miCD4 complex.

Fig. 9. Docking model of Nt-CCR5(1-27) bound to gp120

The lowest energy model of the Nt-CCR5(1-27)/gp120 complex within the highest score HADDOCK cluster is presented. The putative location of the membrane with respect to the Nt-CCR5(1-27) is drawn. The C4 and V3 regions of gp120 are depicted in blue and cyan, respectively, and Nt-CCR5(1-27) in red . Binding residues are depicted in stick representation and labeled.

Fig. 10. Electrostatic potential of the gp120 surface

(a) Vacuum electrostatic potential of gp120. The C4 and V3 regions of gp120 are depicted in cartoon representation and binding residues are depicted in stick representation and labeled. (b) A helical wheel projection obtained by looking down the axis of the P8-E18 segment of the calculated Nt-CCR5 structure. C_α atoms are depicted as balls. The projection shows a negatively charged face which interacts with the positively charged face of gp120.