Molecular basis of transport and regulation in the Na+/betaine symporter BetP

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Sodium-coupled substrate transport plays a central role in many biological processes. However, despite knowledge of the structures of several sodium-coupled transporters, the location of the sodiumbinding site(s) often remains unclear. Several of these structures have the five transmembrane-helix inverted-topology repeat, LeuTlike (FIRL) fold, whose pseudosymmetry has been proposed to facilitate the alternating-access mechanism required for transport. Here, we provide biophysical, biochemical, and computational evidence for the location of the two cation-binding sites in the sodium-coupled betaine symporter BetP. A recent X-ray structure of BetP in a sodium-bound closed state revealed that one of these sites, equivalent to the Na2 site in related transporters, is located between transmembrane helices 1 and 8 of the FIRL-fold; here, we confirm the location of this site by other means. Based on the pseudosymmetry of this fold, we hypothesized that the second site is located between the equivalent helices 6 and 3. Molecular dynamics simulations of the closed-state structure suggest this second sodium site involves two threonine sidechains and a backbone carbonyl from helix 3, a phenylalanine from helix 6, and a water molecule. Mutating the residues proposed to form the two binding sites increased the apparent K m and K d for sodium, as measured by betaine uptake, tryptophan fluorescence, and 22 Na + binding, and also diminished the transient currents measured in proteoliposomes using solid supported membrane-based electrophysiology. Taken together, these results provide strong evidence for the identity of the residues forming the sodium-binding sites in BetP.secondary transport | symmetry | membrane protein | alkali metal ion | osmoregulation

Section: Mutagenesis At Pna2 Has No Effect On the Sodium Dependence Ofmentioning

confidence: 59%

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confidence: 99%

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confidence: 99%

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Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP

Khafizov¹,

Pérez²,

Koshy³

et al. 2012

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“…At the molecular level, the architecture of pmf-and smf-driven membrane proteins within the same family has consistently been found to be largely conserved (20)(21)(22)(23)(24)(25)(26)(27)(28). Thus, it appears as if the Na + or H + specificity of these systems is dictated by localized variations in their amino acid sequence, rather than by major structural or mechanistic adaptations.…”

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confidence: 95%

On the principle of ion selectivity in Na ⁺ /H ⁺ -coupled membrane proteins: Experimental and theoretical studies of an ATP synthase rotor

Leone

Pogoryelov

Meier

et al. 2015

Numerous membrane transporters and enzymes couple their mechanisms to the permeation of Na + or H + , thereby harnessing the energy stored in the form of transmembrane electrochemical potential gradients to sustain their activities. The molecular and environmental factors that control and modulate the ion specificity of most of these systems are, however, poorly understood. Here, we use isothermal titration calorimetry to determine the Na + /H + selectivity of the ion-driven membrane rotor of an F-type ATP synthase. Consistent with earlier theoretical predictions, we find that this rotor is significantly H + selective, although not sufficiently to be functionally coupled to H + , owing to the large excess of Na + in physiological settings. The functional Na + specificity of this ATP synthase thus results from two opposing factors, namely its inherent chemical selectivity and the relative availability of the coupling ion. Further theoretical studies of this membrane rotor, and of two others with a much stronger and a slightly weaker H + selectivity, indicate that, although the inherent selectivity of their ion-binding sites is largely set by the balance of polar and hydrophobic groups flanking a conserved carboxylic side chain, subtle variations in their structure and conformational dynamics, for a similar chemical makeup, can also have a significant contribution. We propose that the principle of ion selectivity outlined here may provide a rationale for the differentiation of Na + -and H + -coupled systems in other families of membrane transporters and enzymes. molecular motor | ion-coupled transport | binding thermodynamics | energy transduction | membrane bioenergetics G radients in the electrochemical potential of H + or Na + across biological membranes sustain a wide range of essential cellular process. The resulting proton or sodium motive forces (pmf, smf) are the predominant energy source for secondary-active membrane transporters, which mediate the uptake of many substances required by the cell (1-3), and also enable pathogenic bacteria to protect themselves from human-made antibiotics and other toxic compounds (4-6). Downhill membrane permeation of Na + and H + across the membrane also powers the ATP synthase (7), which produces most of cellular ATP, and energizes the rotation of bacterial flagella (8). Thus, the importance of this mode of energy transduction in cells cannot be overstated. Nevertheless, little is known about the factors that control and modulate the specificity for Na + or H + in most of these processes.It seems clear, although, that there is no correlation between function type and ion specificity; that is, the same process in different species can be coupled to either Na + or H + (2, 5-7, 9-11). Organism-specific environmental factors, such as temperature or pH, also do not provide a consistent rationale; for example, ATP synthases from thermoalkaliphilic bacteria use a H + gradient despite the scarcity of H + and the potentially greater degree of H + leakage across the membrane at hig...

“…The implied ion-to-substrate stoichiometry varies across LeuT-fold ion-coupled transporters. For instance, LeuT (17) and BetP (18) require two Na + ions that bind at two distinct sites referred to as Na1 and Na2 whereas Mhp1 (15) and vSGLT (19) appear to possess only the conserved Na2 site. Molecular dynamics (MD) Significance Na + -coupled symporters use the cellular Na + gradient to power transport of physiologically important molecules across the lipid membrane.…”

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confidence: 99%

Conformational cycle and ion-coupling mechanism of the Na ⁺ /hydantoin transporter Mhp1

Kazmier

Sharma

Islam

et al. 2014

147

Ion-dependent transporters of the LeuT-fold couple the uptake of physiologically essential molecules to transmembrane ion gradients. Defined by a conserved 5-helix inverted repeat that encodes common principles of ion and substrate binding, the LeuTfold has been captured in outward-facing, occluded, and inwardfacing conformations. However, fundamental questions relating to the structural basis of alternating access and coupling to ion gradients remain unanswered. Here, we used distance measurements between pairs of spin labels to define the conformational cycle of the Na + -coupled hydantoin symporter Mhp1 from Microbacterium liquefaciens. Our results reveal that the inward-facing and outward-facing Mhp1 crystal structures represent sampled intermediate states in solution. Here, we provide a mechanistic context for these structures, mapping them into a model of transport based on ion-and substrate-dependent conformational equilibria. In contrast to the Na + /leucine transporter LeuT, our results suggest that Na + binding at the conserved second Na + binding site does not change the energetics of the inward-and outward-facing conformations of Mhp1. Comparative analysis of ligand-dependent alternating access in LeuT and Mhp1 lead us to propose that different coupling schemes to ion gradients may define distinct conformational mechanisms within the LeuT-fold class.EPR | DEER | Na + -coupled symport | conformational dynamics | transport mechanism