The major facilitator superfamily (MFS) of transporters consists of three classes of membrane transporters: symporters, uniporters, and antiporters. Despite such diverse functions, MFS transporters are believed to undergo similar conformational changes within their distinct transport cycles, known as the rocker-switch mechanism. While the similarities between conformational changes are noteworthy, the differences are also important since they could potentially explain the distinct functions of symporters, uniporters, and antiporters of the MFS superfamily. We reviewed a variety of experimental and computational structural data on a select number of antiporters, symporters, and uniporters from the MFS family to compare the similarities and differences of the conformational dynamics of three different classes of transporters.

Structural dynamics of human immunodeficiency virus 1 (HIV-1) envelope (Env) glycoprotein mediate cell entry and facilitate immune evasion. Single-molecule FRET using peptides for Env labeling revealed structural dynamics of Env, but peptide use risks potential effects on structural integrity/dynamics. While incorporating noncanonical amino acids (ncAAs) into Env by amber stop-codon suppression, followed by click chemistry, offers a minimally invasive approach, this has proved to be technically challenging for HIV-1. Here, we develope an intact amber-free HIV-1 system that overcomes hurdles of preexisting viral amber codons. We achieved dual-ncAA incorporation into Env on amber-free virions, enabling single-molecule Förster resonance energy transfer (smFRET) studies of click-labeled Env that validated the previous peptide-based labeling approaches by confirming the intrinsic propensity of Env to dynamically sample multiple conformational states. Amber-free click-labeled Env also enabled real-time tracking of single virion internalization and trafficking in cells. Our system thus permits in-virus bioorthogonal labeling of proteins, compatible with studies of virus entry, trafficking, and egress from cells.

Ser195 is highly conserved as the catalytic nucleophile in trypsin-like serine proteases, yet the molecular basis for the strong preference for serine over the chemically similar threonine residue remains unclear. Here, we used well-tempered metadynamics and microsecond-long molecular dynamics (MD) simulations to characterize the conformational energetics of S195 and T195 in thrombin. Well-tempered metadynamics revealed distinct rotameric free-energy landscapes in both the apo and the ligand-bound systems. In the apo state, S195 preferentially occupied a lower-angle rotameric state, while T195 exhibited two low-energy states with similar free energies. Unbiased simulations further showed greater conformational heterogeneity and increased higher-angle sampling for T195. Evaluation of the His57–residue 195 hydrogen bond further revealed that S195 maintained close hydrogen bond distances across both rotameric regions, whereas T195 exhibited longer and more variable His57–hydroxyl distances with reduced distance-based occupancy. Analysis of the local environment showed that the additional T195 methyl group was associated with redistributed protein contacts. Additional rotamer-conditioned analyses demonstrated differences in local packing within corresponding conformational states. Furthermore, solvent accessibility and water-contact analyses revealed that the effects of the substitution on the catalytic side-chain environment depend on rotameric conformation. Together, these findings demonstrate that two complementary simulation approaches converge on similar conformational preferences and suggest that the S195T substitution alters the rotameric free-energy landscape in association with changes in catalytic geometry, local packing, and solvation. These results provide insight into the evolutionary preference for serine over threonine at the catalytic position of thrombin.

The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) plays important roles in viral entry by coordinating host-cell receptor recognition and membrane fusion. Env is synthesized as a homotrimeric precursor, gp160, which is cleaved into gp120 and gp41 subunits. Although the cleaved form of Env is required for fusion, uncleaved Env remains plentiful on the surface of HIV-1-infected cells. Previous studies have suggested that uncleaved Env is conformationally more flexible than the cleaved form making its dynamics important for understanding the broader structural landscape of HIV-1 Env. In this study, we employed microsecond-long all-atom molecular dynamics (MD) simulations to characterize the dynamics of two structures of uncleaved HIV-1 Env that have been previously assigned to the same conformational state. Our results reveal differences in their conformational behavior, including differences in collective motions, FRET distributions, and trimer symmetry. These findings provide molecular-level insight into the structural plasticity of uncleaved HIV-1 Env and highlight that different structures within the same conformational state can exhibit distinct dynamic behavior.