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

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