We present BindFlow, a Python-based software for automated absolute binding free energy (ABFE) calculations at the free energy perturbation (FEP) or at the molecular mechanics Poisson-Boltzmann/generalized Born surface area [MM(PB/GB)SA] level of theory. BindFlow is free, open-source, user-friendly, easily customizable, runs on workstations or distributed computing platforms, and provides extensive documentation and tutorials. BindFlow uses GROMACS as molecular dynamics engine and provides built-in support for the small-molecule force fields GAFF, OpenFF, and Espaloma. We test BindFlow by computing affinities for 139 ligand/target pairs, involving eight different targets including six soluble proteins, one membrane protein and one non-protein host–guest system. Quantified by Pearson, Kendall, and Spearman correlations coefficients, we find that the agreement of BindFlow predictions with experiments are overall similar to gold standards in the field. Interestingly, we find that MM(PB/GB)SA achieves correlations that, for some systems and force fields, approach those obtained with FEP, while requiring only a fraction of the computational cost. This study establishes BindFlow as a validated and accessible tool for ABFE calculations.

Monkeypox virus (MPXV) is a poxvirus endemic to Central and West Africa with high epidemic potential. Pox-viruses enter host cells via a conserved entry-fusion complex (EFC), which mediates viral fusion to the cell membrane. The EFC is a promising therapeutic target, but the absence of structural data has limited the development of fusion-inhibiting treatments. Here, we investigated A16/G9, a subcomplex of the EFC that controls fusion timing. Using cryo-electron microscopy, we showed how A16/G9 interacts with A56/K2, a viral fusion suppressor that prevents superinfection. Immunization with A16/G9 elicited a protective immune response in mice. Using X-ray crystallography, we characterized two neutralizing antibodies and engineered a chimeric antibody that cross-neutralizes several poxviruses more efficiently than 7D11, the most potent antibody targeting the EFC described to date. These findings highlight the potential of A16/G9 as a candidate for subunit vaccines and identify regions of the EFC as targets for antiviral development.

Accepted for publication, to appear October 30, 2025.

Membrane fusion is a fundamental process involved in exocytosis, fertilization, or cell entry by enveloped viruses. Membrane fusion is facilitated by fusion proteins, which are anchored in membranes by helical transmembrane domains (TMDs). Previous studies showed that TMD variations may alter the fusion efficiency, suggesting that TMDs are not merely passive anchors, however the mechanism by which TMDs drive fusion is not well understood. We used high-throughput coarse-grained molecular dynamics simulations and free energy calculations to quantify effects of TMDs on the formation of the first fusion intermediate, that is, of a fusion stalk. We analyzed five physiologically relevant TMDs derived from viral fusion proteins and the SNARE complex embedded in various lipid environments. We find that the addition of TMDs favors stalk formation by typically 10 to 30 kJ/mol in a concentration-dependent manner. Using helices with sequences R₂L_nR₂ (n=6,…, 26), we find that negative hydrophobic mismatch between the TMD and the membrane core strongly promotes fusion. Analysis of the lipid tail order parameters of annular lipids revealed a strong correlation between stalk stabilization and induced lipid disorder. Together, our findings suggest that TMDs actively contribute to membrane fusogenicity by locally perturbing the membrane order.