Tilted, Uninterrupted, Monomeric HIV-1 gp41 Transmembrane Helix from Residual Dipolar Couplings

Abstract

Cryo-electron microscopy and X-ray crystallography have shown that the pre- and postfusion states of the HIV-1 gp41 viral coat protein, although very different from one another, each adopt C₃ symmetric structures. A stable homotrimeric structure for the transmembrane domain (TM) also was modeled and supported by experimental data. For a C₃ symmetric structure, alignment in an anisotropic medium must be axially symmetric, with the unique axis of the alignment tensor coinciding with the C₃ axis. However, NMR residual dipolar couplings (RDCs) measured under three different alignment conditions were found to be incompatible with C₃ symmetry. Subsequent measurements by paramagnetic relaxation enhancement, analytical ultracentrifugation, and DEER EPR, indicate that the transmembrane domain is monomeric. ¹⁵N NMR relaxation data and RDCs show that TM is highly ordered and uninterrupted for a total length of 32 residues, extending well into the membrane proximal external region.

Figure 1.

NMR study of HIV-1 clade D gp41-TM. (A) Domain architecture of gp41. The peptide sequence used in this study comprises residues N677 to S716. (B) Assigned ¹H-N TROSY-HSQC spectrum of TM in the presence of 150 mM DMPC/DHPC (q=0.4) bicelles. Resonances from MPER, TM and CT region are color coded in blue, red and olive, respectively.

Figure 2.

Backbone dynamics of gp41 TM from N relaxation rates, measured at 900 MHz ¹H frequency. (A) R₁; (B) R₂; and (C) N-{¹H} NOE values were subjected to standard Lipari-Szabo analysis^, to yield the generalized order parameters, S² (D).

Figure 3.

RDC analysis of gp41-TM. (A) ¹H-N residual dipolar couplings obtained by aligning TM with DOTA M8-thulium (black), stretched neutral (green) and positively charged (blue) polyacrylamide gel. Superimposed are the “dipolar wave” oscillations, calculated for an idealized perfectly straight helix, using parameters δ=15.8° and ρ=2πn/3.6. Near axial symmetry of the thulium alignment tensor, with its unique axis within 10° of the helix axis, cause sin(θ) oscillations to be small in amplitude. (B) Correlation plot between ¹D_NH RDCs collected in neutral gel (green) or by thulium alignment (black) against values measured in positively charged gel. (C) Ribbon diagram for the ordered region of the TM helix, derived from the RDCs (PDB entry 6B3U; Figure S4).

Figure 4.

Evaluation of the oligomerization state of HIV-1 clade D TM. (A) ¹H^N{−N} R₂ rates in U-N, ²H gp41-TM in the presence of 2 molar equivalents of paramagnetic ¹⁴N TM R707C-MTSL (red) and reduced nitroxide (blue) spin label. The difference in ¹H R₂ rates induced by the paramagnetic spin label is shown in black. Rates are listed in Table S7. (B) Sedimentation equilibrium data on the GB1-TM fusion construct (see SI). Absorbance is shown as a function of radial position at three different rotor speeds, 10000 (yellow), 20000 (red) and 35000 rpm (blue). Residuals are shown in the bottom panel.