Dataset Information

Nucleotide-free MalK drives the transition of the maltose transporter to the inward-facing conformation.

ABSTRACT: The complex MalFGK2 hydrolyzes ATP and alternates between inward- and outward-facing conformations during maltose transport. It has been shown that ATP promotes closure of MalK2 and opening of MalFG toward the periplasm. Yet, why the transporter rests in a conformation facing the cytosol in the absence of nucleotide and how it returns to this state after hydrolysis of ATP is unknown. The membrane domain MalFG may be naturally stable in the inward-facing conformation, or the ABC domain may catalyze the transition. We address this question by analyzing the conformation of MalFG in nanodiscs and in proteoliposomes. We find that MalFG alone exists in an intermediate state until MalK binds and converts the membrane domain to the inward-facing state. We also find that MalK, if overly-bound to MalFG, blocks the transition of the transporter, whereas suppressor mutations that weaken this association restore transport. MalK therefore exploits hydrolysis of ATP to reverse the conformation of MalFG to the inward-facing conformation, a step essential for release of maltose in the cytosol.

SUBMITTER: Bao H

PROVIDER: S-EPMC3975029 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Nucleotide-free MalK drives the transition of the maltose transporter to the inward-facing conformation.

Bao Huan H Duong Franck F

The Journal of biological chemistry 20140213 14

The complex MalFGK2 hydrolyzes ATP and alternates between inward- and outward-facing conformations during maltose transport. It has been shown that ATP promotes closure of MalK2 and opening of MalFG toward the periplasm. Yet, why the transporter rests in a conformation facing the cytosol in the absence of nucleotide and how it returns to this state after hydrolysis of ATP is unknown. The membrane domain MalFG may be naturally stable in the inward-facing conformation, or the ABC domain may cataly ...[more]

PMID: 24526688

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Project description:The dopamine transporter (DAT), a member of the neurotransmitter:Na(+) symporter (NSS) family, terminates dopaminergic neurotransmission and is a major molecular target for psychostimulants such as cocaine and amphetamine, and for the treatment of attention deficit disorder and depression. The crystal structures of the prokaryotic NSS homolog of DAT, the leucine transporter LeuT, have provided critical structural insights about the occluded and outward-facing conformations visited during the substrate transport, but only limited clues regarding mechanism. To understand the transport mechanism in DAT we have used a homology model based on the LeuT structure in a computational protocol validated previously for LeuT, in which steered molecular dynamics (SMD) simulations guide the substrate along a pathway leading from the extracellular end to the intracellular (cytoplasmic) end.Key findings are (1) a second substrate binding site in the extracellular vestibule, and (2) models of the conformational states identified as occluded, doubly occupied, and inward-facing. The transition between these states involve a spatially ordered sequence of interactions between the two substrate-binding sites, followed by rearrangements in structural elements located between the primary binding site and the cytoplasmic end. These rearrangements are facilitated by identified conserved hinge regions and a reorganization of interaction networks that had been identified as gates.Computational simulations supported by information available from experiments in DAT and other NSS transporters have produced a detailed mechanistic proposal for the dynamic changes associated with substrate transport in DAT. This allosteric mechanism is triggered by the binding of substrate in the S2 site in the presence of the substrate in the S1 site. Specific structural elements involved in this mechanism, and their roles in the conformational transitions illuminated here describe, a specific substrate-driven allosteric mechanism that is directly amenable to experiment as shown previously for LeuT.

Project description:Employing molecular dynamics (MD) simulations, the pathway and mechanism of substrate unbinding from the inward-facing state of the Na(+)-coupled galactose transporter, vSGLT, have been investigated. During a 200-ns equilibrium simulation, repeated spontaneous unbinding events of the substrate from its binding site have been observed. In contrast to the previously proposed gating role of a tyrosine residue (Y263), the unbinding mechanism captured in the present equilibrium simulation does not rely on the displacement and/or rotation of this side chain. Rather, the unbinding involves an initial lateral displacement of the substrate out of the binding site which allows the substrate to completely emerge from the region covered by the side chain of Y263 without any noticeable conformational changes of the latter. Starting with the snapshots taken from this equilibrium simulation with the substrate outside the binding site, steered MD (SMD) simulations were then used to probe the translocation of the substrate along the remaining of the release pathway within the protein's lumen and to characterize the nature of protein-substrate interactions involved in the process. Combining the results of the equilibrium and SMD simulations, we provide a description of the full translocation pathway for the substrate release from the binding site into the cytoplasm. Residues E68, N142, T431, and N267 facilitate the initial substrate's displacement out of the binding site, while the translocation of the substrate along the remainder of the exit pathway formed between TM6 and TM8 is facilitated by H-bond interactions between the substrate and a series of conserved, polar residues (Y138, N267, R273, S365, S368, N371, S372, and T375). The observed molecular events indicate that no gating is required for the release of the substrate from the crystallographically captured structure of the inward-facing state of SGLT, suggesting that this conformation might represent an open, rather than occluded, state of the transporter. This article is part of a Special Issue entitled: Membrane protein structure and function.

Dataset Information

Nucleotide-free MalK drives the transition of the maltose transporter to the inward-facing conformation.

Publications

Nucleotide-free MalK drives the transition of the maltose transporter to the inward-facing conformation.

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