Editorial: Structure and Mechanism of Microbial Membrane Active Transporters

Xiaoxu Jiang^1*Chuck Randall Smallwood²Phillip E Klebba³Maria Carmela Bonaccorsi di Patti⁴

• ¹Department of Chemistry and Biochemistry, California State University, San Bernardino, San Bernardino, United States
• ²Special Projects Department, Sandia National Laboratories, Albuquerque, United States
• ³Department of Biochemistry & Molecular Biophysics, Kansas State University, Manhattan, United States
• ⁴Department of Biochemical Sciences 'A. Rossi Fanelli', Universita degli Studi di Roma La Sapienza, Rome, Italy

The process of xylose uptake in yeast is critical for the yeast-based biofuel production. In this collection, Taveira et al. (2024) present a comprehensive review of MFS and SWEET xylose transporters, integrating crystallography, cryo-EM, and molecular dynamics to reveal how rockerswitch motions and gating kinetics determine substrate specificity. By pairing structural data with machine-learning-guided sequence analysis and directed evolution, the authors identify conserved features that underlie transport efficiency and regulatory diversity. Their synthesis illustrates how iterative cycles of modeling and experiment can illuminate long-standing questions in sugar transport, from substrate coupling to allosteric control.Artificial Intelligence (AI) technology is reshaping the field of structural biology by allowing researchers to quickly and accurately predict three-dimensional protein structures. This is particularly meaningful for the structure determination of membrane transporters, which remains challenging using traditional experimental approaches. Using AlphaFold2-Multimer, Mahendran and Orlando (2024) performed structural predictions of all potential ABC transporter complexes encoded in the genome of B. subtilis, providing not only a comprehensive structural gallery of ABC transporters in this model organism but also suggesting novel folds and topologies not observed in previously solved structures. Comparisons between the predicted architecture and experimentally determined structures indicate that AlphaFold harnesses high accuracy and reliability. Their study demonstrates the enormous potential of AI in the structural determination of membrane proteins. Compared to X-ray crystallography and cryo-EM, AI offers significant advantages in both efficiency and cost-effectiveness. On the other hand, the authors also point out that some predicted structures contain noticeable errors, suggesting that AI algorithms could be further improved, particularly in considering the effects of protein-lipid interactions on transporter structure. Therefore, experimental methods are still needed for data confirmation.In another endeavor, Zakharzhevskaya et al. ( 2025) uncover a vesicle-associated Type VI Secretion System (T6SS) in B. fragilis, showing that its structural and regulatory components active even without direct cell contact. Proteogenomic mapping identifies effectorimmunity pairs outer-membrane vesicles, suggesting a dual mode of energy-dependent secretion and passive dissemination. This finding expands the mechanistic scope of T6SS, linking molecular architecture to intercellular communication and competitive dynamics.The acquisition of nutrients is one of the defining properties of biological membranes. Bacteria acquire amino acids through two main mechanisms: synthesizing them from simpler precursors or absorbing them directly from their environment. Although prototrophic strains can produce all 20 standard amino acids de novo, when available bacteria will preferentially obtain them from the environment, because the latter route is more energy-efficient than internally synthesizing them. They do so with active transport systems for specific free amino acids, that encompass well known primary or secondary transporters in the inner or cytoplasmic membranes of Gramnegative bacterial cells. But what is the cellular response when the main uptake systems for a particular amino acid are lost? Bubnov et al. (2025), "Multiple routes for nonphysiological Lthreonine uptake in Escherichia coli K-12," explores and answers this question, by finding multicopy suppressors and chromosomal mutations that complement the loss of L-threonine uptake activity. In an E. coli strain lacking the major threonine-specific permeases, their phenotypic assays identified YhjE and SdaC as two alternative entry points for threonine. These data both illustrate the multiply redundant mechanisms for absorption of these essential solutes, as well as the potential complexity of engineering commercial strains for threonine production.The study of membrane protein structure-function relationships has hugely benefited from the combination of computational methods and site-directed mutagenesis, the latter allowing to experimentally test new hypotheses provided by the former. A good example is the paper contributed by Chen et al. on GerAB, one of the subunits of the B. subtilis spore germinant receptor GerA. This receptor is part of the germinosome, a complex that resides at the inner membrane and responds to nutrients to start B. subtilis spore germination. Employing Steered Molecular Dynamics (SMD), the authors investigate the properties of a previously predicted water channel in GerAB and identify three residues (Y97, L199 and F342) that may interfere with water passage. Site-directed mutagenesis reveals a role for these residues in the stability of GerAB; in fact, the mutant proteins are expressed at much lower level than the wild type, compromising the function of the germinant receptor. Although this result does not allow to assign a specific function of Y97, L199 and F342 in water passage, it illustrates how unanticipated roles of amino acid residues beyond those predicted by computational methods may be uncovered when experimental methods come into play.Together, the studies in this collection underscore how mechanistic biochemistry, anchored in structure, dynamics, and regulation, continues to define and expand our understanding of microbial membrane transport. We expect that the results presented here will also shed light on other research topics regarding membrane proteins and membrane transport.