Simulation of Spontaneous Substrate Binding Revealing the Binding Pathway and Mechanism and Initial Conformational Response of GlpT

摘要: Transport of glycerol-3-phosphate (G3P) across the inner membrane E. coli is mediated by transporter (GlpT). GlpT belongs to organophosphate:phosphate antiporter family major facilitator superfamily (MFS), largest known secondary active transporters (1–11). MFS are present in all three kingdoms life and include several medically pharmaceutically relevant proteins, e.g., efflux pumps conferring resistance antibiotics bacteria, or chemotherapeutics cancer cells (1, 8, 10–16). Apart from its role nutrient uptake, also associated with uptake antibiotic fosfomycin, (a G3P-analog), such that bacterial strains dysfunctional mutants exhibit fosfomycin (13, 17–19). Moreover, highlighted as a structural functional model for other has been used template homology modeling eucaryotic transporters, including human homologs, hexose (GLUT1) glucose-6-phosphate (G6PT) (9, 20). Structurally, organized into two six-transmembrane-helix bundles, N- C-terminal halves, exhibiting pseudo-twofold symmetry weak sequence (Figure 1A) (8, 13, 21). The cytoplasmic-open structure captured crystal (8) revealed lumen formed between which closed on periplasmic side open cytoplasmic side. It was proposed this provides substrate translocation pathway, apex implicated putative substrate-binding site 1A B). includes pseudo-symmetrically positioned, highly conserved arginines (R45 R269), respectively, well histidine (H165) located them 1B; see Figure S1 Supporting Information alignment phosphate antiporters). binding constituting chains shows high degree similarity hexose-6-phosphate (UhpT), more extensively studied homolog (8). Indeed, UhpT residues corresponding R45 R269 functionally indispensable, whereas can be mutated lysines without significant loss function 21–24). significance confirmed mutagenesis experiments directly performed (25). Figure 1 Structure, simulation system, schematic mechanism GlpT An “alternating-access” / “rocker-switch mechanism” transport cycle GlpT, appears monomer under physiological conditions 22). “rocker-switch” term refers inversion protein's conformation during cycle, while describes change accessibility sides membrane, i.e., transition cytoplasmic- periplasmic-open states. According model, Pi results closure vestibule opening (formation state). Replacement G3P state induces returning initial state, through reverse set protein conformational changes 1C). These suggested involve substrate-induced weakening interactions halves initially along formation new opposite 13). Kinetic studies have shown interconversion states rate limiting temperature-dependent, indicating involvement large changes, found temperature-independent rapid. Accordingly, accelerate lowering activation energy (10). Starting structure, 10 degrees rigid-body rotation each half sufficient close 11, Also, recent computational study 9–10 generates maximal electrostatic interaction (26). Based available biochemical (10, 25), put forward. In combined experimental (27) focusing molecular basis selectivity using dynamics (MD) simulations, experiments, we identified exclusively bind not Pi. process pathway binding, effect titration driving force particularly rocker-switch yet described. Here report equilibrium MD simulations membrane-embedded natural substrates possible states, address some these questions. describe key involved recruitment site. They capture large-scale excited result partial protein. Other events perturbation network salt bridges, likely necessary