eLife Assessment

In this useful study, the authors conducted an impressive amount of atomistic simulations with a realistic asymmetric lipid bilayer to probe how the HIV-1 envelope glycoprotein (Env) transmembrane domain, cytoplasmic tail, and membrane environment influence ectodomain orientation and antibody epitope exposure. The simulations convincingly show that ectodomain motion is dominated by tilting relative to the membrane and explicitly demonstrate the role of membrane asymmetry in modulating the protein conformation and orientation. However, due to the qualitative nature of the conducted analyses, the evidence for the coupling between membrane-proximal regions and the antigenic surface is considered incomplete. With stronger integration of prior experimental and computational literature, this work has the potential to serve as a reference for how Env behaves in a realistic, glycosylated, membrane-embedded context.

Reviewer #1 (Public review):

Summary:

In the manuscript "Conformational Variability of HIV-1 Env Trimer and Viral Vulnerability", the authors study the fully glycosylated HIV-1 Env protein using an all-atom forcefield. It combines long all-atom simulations of Env in a realistic asymmetric bilayer with careful data analysis. This work clarifies how the CT domain modulates the overall conformation of the Env ectodomain and characterizes different MPER-TMD conformations. The authors also carefully analyze the accessibility of different antibodies to the Env protein.

Strengths:

This paper is state-of-the-art, given the scale of the system and the sophistication of the methods. The biological question is important, the methodology is rigorous, and the results will interest a broad audience.

Weaknesses:

The manuscript lacks a discussion of previous studies. The authors should consider addressing or comparing their work with the following points:

(1) Tilting of the Env ectodomain has also been reported in previous experimental and theoretical work:

https://doi.org/10.1101/2025.03.26.645577

(2) A previous all-atom simulation study has characterized the conformational heterogeneity of the MPER-TMD domain:

https://doi.org/10.1021/jacs.5c15421

(3) Experimental studies have shown that MPER-directed antibodies recognize the prehairpin intermediate rather than the prefusion state:

https://doi.org/10.1073/pnas.1807259115

(4) How does the CT domain modulate the accessibility of these antibodies studied? The authors are in a strong position to compare their results with the following experimental study:

https://doi.org/10.1126/science.aaa9804

Reviewer #2 (Public review):

(1) Summary

In this work, the authors aim to elucidate how a viral surface protein behaves in a membrane environment and how its large-scale motions influence the exposure of antibody-binding sites. Using long-timescale, all-atom molecular dynamics simulations of a fully glycosylated, full-length protein embedded in a virus-like membrane, the study systematically examines the coupling between ectodomain motion, transmembrane orientation, membrane interactions, and epitope accessibility. By comparing multiple model variants that differ in cleavage state, initial transmembrane configuration, and presence of the cytoplasmic tail, the authors aim to identify general features of protein-membrane dynamics relevant to antibody recognition.

(2) Strengths

A major strength of this study is the scope and ambition of the simulations. The authors perform multiple microsecond-scale simulations of a highly complex, biologically realistic system that includes the full ectodomain, transmembrane region, cytoplasmic tail, glycans, and a heterogeneous membrane. Such simulations remain technically challenging, and the work represents a substantial computational and methodological effort.

The analysis provides a clear and intuitive description of large-scale protein motions relative to the membrane, including ectodomain tilting and transmembrane orientation. The finding that the ectodomain explores a wide range of tilt angles while the transmembrane region remains more constrained, with limited correlation between the two, offers useful conceptual insight into how global motions may be accommodated without large rearrangements at the membrane anchor.

Another strength is the explicit consideration of membrane and glycan steric effects on antibody accessibility. By evaluating multiple classes of antibodies targeting distinct regions of the protein, the study highlights how membrane proximity and glycan dynamics can differentially influence access to different epitopes. This comparative approach helps place the results in a broader immunological context and may be useful for readers interested in antibody recognition or vaccine design.

Overall, the results are internally consistent across multiple simulations and model variants, and the conclusions are generally well aligned with the data presented.

(3) Weaknesses

The main limitations of the study relate to sampling and model dependence, which are inherent challenges for simulations of this size and complexity. Although the simulations are long by current standards, individual trajectories explore only portions of the available conformational space, and several conclusions rely on pooling data across a limited number of replicas. This makes it difficult to fully assess the robustness of some quantitative trends, particularly for rare events such as specific epitope accessibility states.

In addition, several aspects of the model construction, including the treatment of missing regions, loop rebuilding, and initial configuration choices, are necessarily approximate. While these approaches are reasonable and well motivated, the extent to which some conclusions depend on these modeling choices is not always fully clear from the current presentation.

Finally, the analysis of antibody accessibility is based on geometric and steric criteria, which provide a useful first-order approximation but do not capture potential conformational adaptations of antibodies or membrane remodeling during binding. As a result, the accessibility results should be interpreted primarily as model-based predictions rather than definitive statements about binding competence.

Despite these limitations, the study provides a valuable and carefully executed contribution, and its datasets and analytical framework are likely to be useful to others interested in protein-membrane interactions and antibody recognition.

Reviewer #3 (Public review):

Summary:

This study uses large-scale all-atom molecular dynamics simulations to examine the conformational plasticity of the HIV-1 envelope glycoprotein (Env) in a membrane context, with particular emphasis on how the transmembrane domain (TMD), cytoplasmic tail (CT), and membrane environment influence ectodomain orientation and antibody epitope exposure. By comparing Env constructs with and without the CT, explicitly modeling glycosylation, and embedding Env in an asymmetric lipid bilayer, the authors aim to provide an integrated view of how membrane-proximal regions and lipid interactions shape Env antigenicity, including epitopes targeted by MPER-directed antibodies.

Strengths:

A key strength of this work is the scope and realism of the simulation systems. The authors construct a very large, nearly complete Env-scale model that includes a glycosylated Env trimer embedded in an asymmetric bilayer, enabling analysis of membrane-protein interactions that are difficult to capture experimentally. The inclusion of specific glycans at reported sites, and the focus on constructs with and without the CT, are well motivated by existing biological and structural data.

The simulations reveal substantial tilting motions of the ectodomain relative to the membrane, with angles spanning roughly 0-30{degree sign} (and up to ~50{degree sign} in some analyses), while the ectodomain itself remains relatively rigid. This framing, that much of Env's conformational variability arises from rigid-body tilting rather than large internal rearrangements, is an important conceptual contribution. The authors also provide interesting observations regarding asymmetric bilayer deformations, including localized thinning and altered lipid headgroup interactions near the TMD and CT, which suggest a reciprocal coupling between Env and the surrounding membrane.

The analysis of antibody-relevant epitopes across the prefusion state, including the V1/V2 and V3 loops, the CD4 binding site, and the MPER, is another strength. The study makes effective use of existing experimental knowledge in this context, for example, by focusing on specific glycans known to occlude antibody binding, to motivate and interpret the simulations.

Weaknesses:

While the simulations are technically impressive, the manuscript would benefit from more explicit cross-validation against prior experimental and computational work throughout the Results and Discussion, and better framing in the introduction. Many of the reported behaviors, such as ectodomain tilting, TMD kinking, lipid interactions at helix boundaries, and aspects of membrane deformation, have been described previously in a range of MD studies of HIV Env and related constructs (e.g., PMC2730987, PMC2980712, PMC4254001, PMC4040535, PMC6035291, PMC12665260, PMID: 33882664, PMC11975376). Clearly situating the present results relative to these studies would strengthen the paper by clarifying where the simulations reproduce established behavior and where they extend it to more complete or realistic systems.

A related limitation is that the work remains largely descriptive with respect to conformational coupling. Numerous experimental studies have demonstrated functional and conformational coupling between the TMD, CT, and the antigenic surface, with effects on Env stability, infectivity, and antibody binding (e.g., PMC4701381, PMC4304640, PMC5085267). In this context, the statement that ectodomain and TMD tilting motions are independent is a strong conclusion that is not fully supported by the analyses presented, particularly given the authors' acknowledgment that multiple independent simulations are required to adequately sample conformational space. More direct analyses of coupling, rather than correlations inferred from individual trajectories, would help align the simulations with the existing experimental literature. Given the scale of these simulations, a more thorough analysis of coupling could be this paper's most seminal contribution to the field.

The choice of membrane composition also warrants deeper discussion. The manuscript states that it relies on a plasma membrane model derived from a prior simulation-based study, which itself is based on host plasma membrane (PMID: 35167752), but experimental analyses have shown that HIV virions differ substantially from host plasma membranes (e.g., PMC46679, PMC1413831, PMC10663554, PMC5039752, PMC6881329). In particular, virions are depleted in PC, PE, and PI, and enriched in phosphatidylserine, sphingomyelins, and cholesterol. These differences are likely to influence bilayer thickness, rigidity, and lipid-protein interactions and, therefore, may affect the generality of the conclusions regarding Env dynamics and antigenicity. Notably, the citation provided for membrane composition is a laboratory self-citation, a secondary source, rather than a primary experimental study on plasma membrane composition.

Finally, there are pervasive issues with citation and methodological clarity. Several structural models are referred to only by PDB ID without citation, and in at least one case, a structure described as cryo-EM is in fact an NMR-derived model. Statements regarding residue flexibility, missing regions in structures, and comparisons to prior dynamics studies are often presented without appropriate references. The Methods section also lacks sufficient detail for a system of this size and complexity, limiting readers' ability to assess robustness or reproducibility.

With stronger integration of prior experimental and computational literature, this work has the potential to serve as a valuable reference for how Env behaves in a realistic, glycosylated, membrane-embedded context. The simulation framework itself is well-suited for future studies incorporating mutations, strain variation, antibodies, inhibitors, or receptor and co-receptor engagement. In its current form, the primary contribution of the study is to consolidate and extend existing observations within a single, large-scale model, providing a useful platform for future mechanistic investigations.

Author response:

In response to the comments raised, we outline below the revisions we plan to strengthen the manuscript.

First, we will expand the Introduction and Discussion sections to provide clearer comparison with prior experimental and computational studies of ectodomain tilting, MPER–TMD conformational heterogeneity, and membrane deformation, and to discuss how our simulations reproduce and extend these earlier observations.

Second, we plan to add analyses that more directly assess the coupling between ectodomain and TMD motions. We will also revise the text to emphasize the limits imposed by sampling and model dependence and to discuss the potential benefits of enhanced sampling methods.

Third, we will clarify the rationale for the chosen membrane composition and discuss how differences in lipid content between host plasma membranes and HIV virions may influence bilayer properties and Env dynamics.

Fourth, we will supplement the Methods section to improve clarity and address issues of citation throughout the manuscript.

Finally, we intend to deposit MD trajectories to a public research data repository to the extent permitted by available storage capacity.