Capturing the inherent structural dynamics of the HIV-1 envelope glycoprotein fusion peptide

Abstract

The N-terminal fusion peptide (FP) of the human immunodeficiency virus (HIV)-1 envelope glycoprotein (Env) gp41 subunit plays a critical role in cell entry. However, capturing the structural flexibility in the unbound FP is challenging in the native Env trimer. Here, FP conformational isomerism is observed in two crystal structures of a soluble clade B transmitted/founder virus B41 SOSIP.664 Env with broadly neutralizing antibodies (bNAbs) PGT124 and 35O22 to aid in crystallization and that are not specific for binding to the FP. Large rearrangements in the FP and fusion peptide proximal region occur around M530, which remains anchored in the tryptophan clasp (gp41 W623, W628, W631) in the B41 Env prefusion state. Further, we redesigned the FP at position 518 to reinstate the bNAb VRC34.01 epitope. These findings provide further structural evidence for the dynamic nature of the FP and how a bNAb epitope can be restored during vaccine design.

Fig. 1

Crystal structures of closed prefusion structure of B41 SOSIP.664 Env trimer. a, b Views of the Fab complex down the trimer axis with gp120 in pink and gp41 in cyan. The cartoon representation is overlaid with a transparent molecular surface. Fabs PGT124 (dark gray) and 35O22 (green) are from the 3.50 and 3.80 Å structures in space groups P23 (a) and P6₃ (b), respectively. c Comparison of the protomer/trimer backbones from crystal structures of trimers of clade A (BG505 SOSIP.664, bound and unbound), B (JRFL SOSIP.664), G (X1193.c1 SOSIP.664), and the new B41 SOSIP.664 structure at 3.50 Å. d Glycosylation observed in the electron density maps in the two different B41 SOSIP.664 crystal structures. The blue, gray, and green bars along the Y-axis represent the number of N-glycan moieties of N-acetylglucosamine (NAG/GlcNAc) (maximum possible, two), β-d-mannopyranose (BMA) (maximum, one), and α-d-mannopyranose (MAN) (maximum, eight), respectively. The X-axis represents N-glycosylation sites on B41 SOSIP.664. e, f Side views of B41 SOSIP.664 protomer (gp120: light blue, gp41: light orange) in cartoon representation, overlaid with a transparent molecular surface showing two conformational states of the free N-terminus FP (pink) going away from (3.50 Å) and towards (3.80 Å) the C-terminus (i.e. proximal to the membrane)

Fig. 2

Different conformational states of the fusion peptide in B41 SOSIP.664. a The FP (red) of gp41 at 3.50 Å points away from HR2. Inset shows the 2Fo–Fc electron density composite omit maps (1.0σ) for the fusion peptide (residues 512–527, red), and FPPR (residues 528–540, green). b The FP from the 3.80 Å structure extends towards HR2. Inset shows the 2Fo–Fc electron density map (1.0σ) for the FP and FPPR. c Overlay of FP and FPPR regions of gp41 from the two crystal structures (3.50 Å: light pink, 3.80 Å: light blue). Inset illustrates the large rearrangement of FPPR and major Cα displacement for example of S534 (~11.6 Å) in B41 SOSIP.664 following the same resolution-based color-code. d Different conformational states of FP illustrated in the overlay of the FP, FPPR, and HR1N regions of PGT151-bound WT JRFL ΔCT (PDB ID: 5FUU; cyan), vFP16-bound BG505 DS-SOSIP (PDB ID: 6CDI; green), vFP20-bound BG505 DS-SOSIP (PDB ID: 6CDE; blue), and VRC34.01-bound BG505 SOSIP.664 (PDB ID: 5I8H; orange), recognized by the respective bNAbs. Inset shows the structural plasticity of the FP around F522. e F522 in FP (pink) of B41 SOSIP.664 in the 3.80 Å structure is tucked inside the gp120/gp41 interface and acts as  a pivot for the remaining FP residues. Another conformational state of FP is captured in the 3.50 Å structure where F522 exits its hydrophobic-binding pocket, causing the FP and FPPR to rotate in opposite directions without affecting the location and conformation of M530 (green), which acts an anchor in the gp41 prefusion state

Fig. 3

Restoration of the VRC34.01 epitope on B41 and delineation of Env breathing. a Amino acid conservation of the FP (X-axis: residues 512–527, Y-axis: percent conservation) in BG505 SOSIP.664 (black) and B41 SOSIP.664 (gray). The difference in conservation is shown in rectangles for BG505 SOSIP.664 and ovals for B41 SOSIP.664. For assessing conservation of FP residues, 5451 HIV-1 sequences from Los Alamos National Laboratory (LANL) HIV-1 database were analyzed. b The FP residues of both isolates are aligned to highlight the differences at positions 515, 518, and 519. Construction of the B41mut1 SOSIP.664 is illustrated in the lower panel. c No binding is observed between FP-directed Fab VRC34.01 with wild-type FP of B41 SOSIP.664 (left panel). Restoration of VRC34.01 binding to the mutated B41mut1 SOSIP.664 FP (K_d = 12 nM, three Fabs bind per trimer) is illustrated (right panel). The enthalpy and entropy are measured in kcal per mol and cal per mol per deg, respectively. Data points not included in the fit are indicated by an asterisk. All binding experiments are measured by ITC and reported values are averages from two independent measurements. d Binding kinetics of B41 FP and FP (mut1) peptide variants of VRC34.01, as determined by BLI. e Crystal structure of His-tagged B41mut1FP peptide bound to VRC34.01. f View down the three-fold trimer apex of B41 SOSIP.664 showing opening and closing of the trimer at 3.50 Å (left) and 3.80 Å (right) resolutions, respectively. Inter-V2 distances (Å; dashed lines) are measured between the Cα residues of R166 (cyan). Top views of both crystal structures showing center of mass of each protomer (gp120: green, gp41: red) overlaid on the Env cartoon (light gray) for orientation. Inter-gp41 and inter-gp120 distances (measured in Å; lines) are measured between the center of masses. The insets of the two B41 structures are shown in the side view (right). The angle is measured between center of mass of gp41 and gp120 of a protomer to reflect the movement between domains that leads to a slight opening of the trimer

Fig. 4

Comparison of open and closed prefusion structures of B41 SOSIP.664. a Top view of open B41 SOSIP.664 bound to sCD4 and 17b cryo-EM structure (PDB 5VN3). b Binding of sCD4 and Fab17b induce conformational changes in the FP and its proximal region of gp41. The FP (red) is inserted towards the trimer core and the FPPR (green) extends away from trimer interface. c Binding of sCD4 to B41 SOSIP.664 propagates structural changes to the C1 region that drive the formation of the α0 helix (residues 63–72). d Top view of B41 SOSIP.664 bound to Fabs PGT124 and 35O22 shows the closed prefusion Env structure. e The free N-terminus of the FP (red) in closed prefusion structure of B41 SOSIP.664 points away from the trimer core compared to the open prefusion trimer. f The closed prefusion B41 SOSIP.664 trimer lacks the well-formed α0 helix shown in c. g Conformational flexibility of B41 SOSIP.664 FP in closed and open prefusion trimers