Solution Structure and Membrane Interaction of the Cytoplasmic Tail of HIV-1 gp41 Protein

Abstract

The cytoplasmic tail of gp41 (gp41CT) remains the last HIV-1 domain with an unknown structure. It plays important roles in HIV-1 replication such as mediating envelope (Env) intracellular trafficking and incorporation into assembling virions, mechanisms of which are poorly understood. Here, we present the solution structure of gp41CT in a micellar environment and characterize its interaction with the membrane. We show that the N-terminal 45 residues are unstructured and not associated with the membrane. However, the C-terminal 105 residues form three membrane-bound amphipathic α helices with distinctive structural features such as variable degree of membrane penetration, hydrophobic and basic surfaces, clusters of aromatic residues, and a network of cation-π interactions. This work fills a major gap by providing the structure of the last segment of HIV-1 Env, which will provide insights into the mechanisms of Gag-mediated Env incorporation as well as the overall Env mobility and conformation on the virion surface.

Keywords: Gag polyprotein; HIV-1; NMR; bicelles; cytoplasmic tail; envelope protein; gp41; matrix protein; membrane; micelles.

Figure 1. NMR Spectra of the Two Independent gp41CT Domains

(A) Amino acid sequence of the HIV-1 gp41CT protein (pNL4-3 isolate). Sequences of gp41CT_N and gp41CT_C are highlighted in blue and black, respectively. (B) 2D ¹H-¹⁵N HSQC spectrum obtained for gp41CT_N (200 μM) at 32 °C. (C) 2D ¹H-¹⁵N HSQC spectrum obtained for gp41CT_C (300 μM) at 50 °C. Tryptophan side chain signals are colored in magenta (inset). Dashed lines indicate side chain amide signals.

Figure 2. Secondary Structure and Helical Wheel Diagrams of the gp41CT_C Protein

(A) Secondary structure representation of the gp41CT protein based on the NMR data. (B) Helical wheel diagrams of the LLP2, LLP3, and LLP1 motifs. Amino acid sequences are plotted clockwise. Hydrophobic and aromatic residues are represented by light and dark green squares, respectively. Polar residues are shown as yellow circles, while positively and negatively charged residues as blue and red pentagons, respectively. The helical wheels are oriented so that their hydrophobic moments (indicated in the centers) point upwards. Helical wheels were generated via a modified script obtained from http://rzlab.ucr.edu/scripts/wheel/.

Figure 3. Structural Features of the gp41CT_C Protein

(A) The C^α positional RMSDs between the 20 low-energy structures of gp41CT_C are represented as a sausage plot of the representative gp41CT_C structure (line thickness is proportional to the RMSD values). The RMSDs from an average structure were calculated after aligning each α-helical segment separately. For residues located at the overlapping boundaries of individual segments, an average of the two RMSD values was calculated. Bending between the helical fragments is variable in the 20 calculated structures. (B) Cartoon representation of the gp41CT_C protein showing the extensive hydrophobic interface formed by Leu, Ile, Val, Ala, Trp and Phe residues (green sticks). (C) Surface representation of the gp41CT_C colored according to electrostatic surface potential. A basic patch is formed in the arginine-rich LLP1 motif. (D) Cartoon representation of the gp41CT_C protein showing Tyr and Trp residues as green sticks. Notice the cluster of six aromatic residues at the beginning of LLP3. The majority of aromatic residues are deeply buried in the interior of DPC micelle. (E) Surface representation of the gp41CT_C protein showing Arg and Lys residues (blue spheres).

Figure 4. Intermolecular Contacts Between gp41CT_C and Micelles

(A) 3D ¹³C-half-filtered/¹³C-edited NOESY spectrum for ¹³C-labeled gp41CT_C showing intermolecular NOEs between DPC and gp41CT_C residues. Colored intermolecular NOE cross-peaks correspond to colored atoms on the DPC structure. (B) A selected region of 2D ¹H-¹H NOESY spectrum for gp41CT_C showing NOEs between side chains of aromatic residues and DPC acyl chain methylene groups.

Figure 5. Membrane Interaction of gp41CT_C and Overall Env Organization on the Virion Surface

(A) A model of gp41CT_C bound to a membrane bilayer constructed based on the NMR data using the representative structure of gp41CT_C with only minor modifications of the dihedral angles in the hinge regions to create an extended molecule. Length of the extended gp41CT_C domain shown here is 160 Å. Top and bottom panels show side and top views of the protein, respectively. Residues indicated as red spheres interact extensively with the interior of the membrane while those in blue are mostly exposed and interact with the polar head. Membrane bilayer was generated in VMD membrane builder plug-in (Humphrey et al., 1996). (B) Top panel: A model depicting the gp120 and gp41 proteins on the surface of HIV-1 particles. The gp41CT_C domain is penetrating deeply in the inner leaflet of the membrane. Lower panel: An expanded view of the inner leaflet of the PM showing gp41CT penetrating the bilayer.