![]() ![]() Using bioinformatics, we identified GltPh gain-of-function mutations in the flexible helical hairpin domain HP2 and applied linear free energy relationship analysis to infer that the transition state structurally resembles the inward-facing conformation. ![]() Here, we provide insight into the structure of the high-energy transition state of GltPh that limits the rate of the substrate translocation process. ![]() Earlier single-molecule fluorescence imaging of the archaeal model glutamate transporter homologue GltPh from Pyrococcus horikoshii suggested that the slow conformational transition from the outward- to the inward-facing state, when the bound substrate is translocated from the extracellular to the cytoplasmic side of the membrane, is rate limiting to transport. Their overall mechanisms are often well understood, but the structural features setting their rates are mostly unknown. Membrane transporters mediate cellular uptake of nutrients, signaling molecules, and drugs. Our findings provide insight into the mechanism by which glutamate transporters support their dual function, and add information that will assist in mapping the complete transport cycle shared by the solute carrier 1A transporter family. The functional properties of this cavity, combined with molecular dynamics simulations, reveal it to be an aqueous-accessible chloride permeation pathway that is gated by two hydrophobic regions and is conserved across mammalian and archaeal glutamate transporters. Here we report the cryo-electron microscopy structure of a glutamate transporter homologue in an open-channel state, which reveals an aqueous cavity that is formed during the glutamate transport cycle. However, the molecular mechanisms that enable these dual-function transporters to carry out two seemingly contradictory roles are unknown. Glutamate transporters also conduct chloride ions by means of a channel-like process that is thermodynamically uncoupled from transport6,7,8. The removal of extracellular glutamate is achieved by plasma-membrane-bound transporters, which couple glutamate transport to sodium, potassium and pH gradients using an elevator mechanism. Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, and its precise control is vital to maintain normal brain function and to prevent excitotoxicity. We hypothesize that the rigid cytoplasmic half of the domain mediates substrate and ion recognition and coupling, whereas the extracellular labile half sets the affinity and dynamic properties. The structure at 2.2-Å resolution details a pattern of waters in the intracellular half of the domain and resolves classes with subtle differences in the substrate-binding site. By contrast, an equilibrated structure does not show such classes. Consistently, cryo-EM on samples frozen within seconds after substrate addition reveals the presence of structural classes with perturbed helical packing of the extracellular half of the transport domain in regions adjacent to the binding site. The low-affinity substate of the mutant is transient following substrate binding. Wild type GltPh shows similar binding properties, and modulation of the substate equilibrium correlates with transport rates. Here, we demonstrate that in a mutant GltPh, which exclusively populates the outward-facing state, at least two substates coexist in slow equilibrium, binding the substrate with different apparent affinities. Recent studies of an archaeal homologue, GltPh, revealed transport rate heterogeneity, which is inconsistent with simple kinetic models however, its structural and mechanistic determinants remain undefined. There is a wealth of structural and mechanistic information about this protein family. The secret world does not have a challenge counterpart.Integral membrane glutamate transporters couple the concentrative substrate transport to ion gradients. They are accessed by either pressing the "Challenge Levels" checkbox on the top left of the Worlds Menu or by pressing the triangle button to the right of the Worlds button on the Main Menu. The Secret World is hidden from view until the normal mode for Worlds 1-5 are beaten Unbreaking and Under Budget.Ĭhallenge Worlds are unlocked when the corresponding normal mode version of each level is completed Unbreaking and Under Budget. World 1: Pine Mountains World 2: Glowing Gorge World 3: Tranquil Oasis World 4: Sanguine Gulch World 5: Serenity Valley World 6 (Secret World): Steamtown The main campaign of Poly Bridge 2 consists of 5 normal worlds, 5 challenge worlds, and 1 secret world. ![]()
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