Microsecond dynamics control the HIV-1 envelope conformation

Abstract

The HIV-1 Envelope (Env) glycoprotein facilitates host cell fusion through a complex series of receptor-induced structural changes. Although significant progress has been made in understanding the structures of various Env conformations and transition intermediates that occur within the millisecond timescale, faster transitions in the microsecond timescale have not yet been observed. In this study, we employed time-resolved, temperature-jump small angle X-ray scattering to monitor structural rearrangements in an HIV-1 Env ectodomain construct with microsecond precision. We detected a transition correlated with Env opening that occurs in the hundreds of microseconds range and another more rapid transition that preceded this opening. Model fitting indicated that the early rapid transition involved an order-to-disorder transition in the trimer apex loop contacts, suggesting that conventional conformation-locking design strategies that target the allosteric machinery may be ineffective in preventing this movement. Utilizing this information, we engineered an envelope that locks the apex loop contacts to the adjacent protomer. This modification resulted in significant angle-of-approach shifts in the interaction of a neutralizing antibody. Our findings imply that blocking the intermediate state could be crucial for inducing antibodies with the appropriate bound state orientation through vaccination.

Figure 1:. The HIV-1 Envelope Glycoprotein is Structurally Dynamic.

(A) Linear sequence of the HIV-1 Env with gp120 in blue and gp41 in light orange and the layer-1, layer-2, variable domains 1 and 2 (V1/V2), variable domain 3 (V3), b20-b21, fusion peptide (FP), heptad repeat 1 (HR1), heptad repeat 2 (HR2), membrane proximal external region (MPER), transmembrane domain (TM) and the cytoplasmic tail (CT) allosteric elements are colored in dark blue, purple, green, red, yellow, sky blue, orange, dark orange, brown, tan, and grey, respectively. Glycosylation sites as predicted by Glycosite for CH505 models are denoted by grey forks. The black dashed line represents the location of truncation in SOSIP constructs. (B) Cryo-EM structures of a closed Env trimer (left) and an occluded Env trimer (middle) and an open Env trimer (right) from the side viewpoint. Allosteric elements of the gp120 and gp41 domains are colored identically to panel A.

Figure 2:. Static Small Angle X-Ray Scattering Profiles Capture HIV-1 Env Opening.

(A) Biolayer interferometry (BLI) binding response for 17b (left), 19b (middle), and PGT145 (right) for BG505 SOSIP (blue circles) and CH505TF SOSIP (red triangles). The error bars indicate the standard deviation from the arithmetic mean aggregated for 2 experiments each with five replicates for BG505 and 7 replicates for CH505 Env SOSIPs. (B) Static SAXS scattering difference curve calculated by subtracting BG505 Env SOSIP SAXS scattering profile from the CH505 Env SOSIP with the propagated standard error (SE) shown as the shaded region. Scattering difference feature (q) peak at ~0.07 Å⁻¹ is indicated by the grey dashed line. (C) CH505 Env SOSIP temperature series binding response for interactions with 17b (blue circles), 19b (red triangles), and PGT145 (green squares). The error bars indicate the SD within the arithmetic mean for a total of three replicates for each measurement. (D) SAXS scattering difference curves calculated by subtracting CH505 Env SOSIP SAXS scattering profile at 25°C from the CH505 Env SOSIP scattering profile at 50°C. Scattering difference feature peak indicated at q = 0.07 Å⁻¹ by the grey dashed line.

Figure 3:. Time resolved, Temperature-Jump SAXS of HIV-1 Env Reveals Two Opening Transitions.

(A) TR, T-Jump SAXS scattering difference curves for 1.5 μs (red) 3 μs (orange), 5 μs (light orange), 10 μs (yellow), 50 μs (green), 100 μs (cyan), 500 μs (blue), 1 ms (indigo), 10 ms (violet), and 100 ms (magenta) time delays. Dashed grey line at q=0.07 Å⁻¹ indicates scattering difference feature peak. The shaded regions indicate the standard error of the arithmetic mean for the number of replicates indicated in the Methods section. (B) Singular value decomposition left vectors 1 (LV1, blue) and 2 (LV2, red). Dashed grey line at q=0.07Å⁻¹ indicates the location of the feature peak. (C) Deconvoluted TR, T-Jump SAXS components 1 (blue) and component 2 (red) from REGALS decomposition SAXS difference curves in panel B. Dashed grey line at q=0.07 Å⁻¹ indicates the location of the feature peak. (D) REGALS pair distance distribution for component 1 (blue) and component 2 (red). (E) The SVD right vectors 1 (RV1, blue) and 2 (RV2, red) showing the contribution of LV1 and LV2 at each time delay. The point shown as a red star was left out of the SVD fit to RV2. The arithmetic mean ± standard error of the mean (SEM) determined from bootstrapping is plotted. The error bars are smaller than the data point for most time delays. (F) The predicted concentrations of REGALS component 1 (blue) and component 2 (red). (G) The area under the curve (AUC) calculated according to Simpsons Rule for the TR, T-Jump SAXS difference curves shown in panel A and fit to a double exponential decay function. The arithmetic mean ± standard error of the mean (SEM) determined from bootstrapping is plotted. The error bars are smaller than the data point for most time delays.

Figure 4:. The fast intermediate corresponds to a loss of Env trimer apex contacts.

(A) A cartoon diagram representing the closed to open-occluded transition using the same coloring as in Figure 1 (top). The theoretical SAXS difference curve (bottom, left) and the theoretical pair distance distribution difference curve (bottom, right) for the closed to open-occluded transition. (B) A cartoon diagram representing the closed to open transition using the same coloring as in Figure 1 (top). Theoretical scattering difference curves (bottom, left) and pair distance distribution difference curve (bottom, right) for the closed to open conformational transition. (C) A cartoon diagram of the Env SOSIP transition from the open-occluded conformation to the open conformation (top). The theoretical SAXS scattering difference curve (bottom, left) and pair distance distribution difference curve (bottom, left) for the open-occluded to open transition. (D) The carton diagram depicts the V1/V2 and V3 rearrangement in a gp120 monomer (top). The RMSD (bottom) for V1/V2 (green), V3 (blue), and gp120 core (blue) b-sheet a-carbons for an aggregated 250 5 μs MD simulations. The dashed lines represent the RMSD for the given domain between the starting and end conformations depicted in the diagram. (E) A cartoon diagram depicting the release of the Env SOSIP trimer apex contacts (left). The theoretical SAXS scattering difference curve (middle) and the pair distance distribution difference curve (right).

Figure 5:. Interprotomer Disulfide Bonds Stabilize the Closed Env Trimer.

(A) Differential fluorescence thermal denaturation inflection point temperature for the stabilized parent CH505 SOSIP and the inter-protomer disulfide stapled CH505 SOSIP. (B) Binding responses for an unstabilized CH505 SOSIP, the stabilized CH505 parent SOSIP, and the inter-protomer disulfide stapled CH505 SOSIP design interacting with the co-receptor binding 17b, V3-loop binding 19b, and the closed state apex interactive, trimer specific PGT145 MAbs. (C) A gaussian filtered map of the inter-protomer disulfide stapled CH505 SOSIP design bound to the CH235.12 Fab. (D) Binding responses for PGT151 captured stabilized CH505 parent SOSIP and inter-protomer disulfide stapled CH505 SOSIP design interacting with the b12 Fab. (E) A gaussian filtered map of the inter-protomer disulfide stapled CH505 SOSIP design bound to the b12 Fab. (F) (left) Structure comparison between the closed state b12 bound inter-protomer stapled CH505 SOSIP design and the open-occluded state b12 bound B41 isolate SOSIP. A single b12 bound gp120 domain from B41 is aligned to a closed state CH505 design gp120 to highlight the shift in the b12 Fv position (only β-sheets shown for clarity; pink arrows indication difference direction). Two additional gp120 domains shown as surface to highlight potential for b12 clashes. (right) Alignment of the b12 bound apex stapled CH505 SOSIP design and B41 isolate gp120 domains highlighting differences in the HCDR3 position.