Abstract
LeuT-like fold Na-dependent secondary active transporters form a large family of integral membrane proteins that transport various substrates against their concentration gradient across lipid membranes, using the free energy stored in the downhill concentration gradient of sodium ions. These transporters play an active role in synaptic transmission, the delivery of key nutrients, and the maintenance of osmotic pressure inside the cell. It is generally believed that binding of an ion and/or a substrate drives the conformational dynamics of the transporter. However, the exact mechanism for converting ion binding into useful work has yet to be established. Using a multi-dimensional path sampling (string-method) followed by all-atom free energy simulations, we established the principal thermodynamic and kinetic components governing the ion-dependent conformational dynamics of a LeuT-like fold transporter, the sodium/benzyl-hydantoin symporter Mhp1, for an entire conformational cycle. We found that inward-facing and outward-facing states of Mhp1 display nearly the same free energies with an ion absent from the Na2 site conserved across the LeuT-like fold transporters. The barrier separating an apo-state from inward-facing or outward-facing states of the transporter is very low, suggesting stochastic gating in the absence of ion/substrate bound. In contrast, the binding of a Na2 ion shifts the free energy stabilizing the outward-facing state and promoting substrate binding. Our results indicate that ion binding to the Na2 site may also play a key role in the intracellular thin gate dynamics modulation by altering its interactions with the transmembrane helix 5 (TM5). The Potential of Mean Force (PMF) computations for a substrate entrance displays two energy minima that correspond to the locations of the main binding site S1 and proposed allosteric S2 binding site. However, it was found that substrate's binds to the site S1 ∼5 kcal/mol more favorable than that to the site S2 for all studied bound combinations of ions and a substrate.
Highlights
Ion-coupled secondary-active transporters are integral membrane proteins involved in the cellular uptake and release of various substrates across cell membranes
Several sodium-glucose co-transporter 2 (SGLT2) inhibitors are being developed as a new class of drugs to treat type 2 diabetes [4,5], while transporters from the neurotransmitter:sodium symporter (NSS) family are common targets for anti-depressants and drug of abuse [6,7,8,9]
To show that the stabilization of the outward-facing conformations in Figure 3A is mainly caused by the binding of Na+ but not the substrate, we computed 2D Potential of Mean Force (PMF) maps (Figure 3B, left panel) for the transition from the inward-facing open (I_apo) state to the outward-facing state with a Na+ bound (O_i), for which a substrate is not involved
Summary
Ion-coupled secondary-active transporters are integral membrane proteins involved in the cellular uptake and release of various substrates across cell membranes. These transporters transport the main substrate uphill against its concentration gradient by coupling with the favorable downhill transport of ion(s). Examples of their substrates include neurotransmitters (serotonin, dopamine, and epinephrine), sugars, amino acids, and nucleobases [1,2,3]. The generally accepted mechanism for secondary-active transport is the alternating access model [10] in which the transporter changes between outward-facing and inward-facing conformations, allowing the main substrate and coupled ion(s) to bind to one side and be released from the other side. The sodium-binding site in Mhp, corresponding to the Na2 site in
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