Principles of Alternating Access in LeuT-fold Transporters: Commonalities and Divergences

Alamo, Diego del; Meiler, Jens; Mchaourab, Hassane S.

doi:10.26434/chemrxiv-2022-nk6h3

Cited by 5 publications

(4 citation statements)

References 137 publications

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“…Physiologically, we naïvely expect outward-open, rather than occluded, to be the preferred substrate-free conformation. However, the occluded or inward-open states of other LeuT-fold importers are often more stable, with changes in environmental conditions or the presence of substrate stabilizing specific states and lowering barriers to conformational transitions 51 . For example, SGLT1 and DraNramp require a negative membrane potential to transport substrates and this negative membrane potential stabilizes the outward-open state of SGLT1 21, 52 .…”

Section: Discussionmentioning

confidence: 99%

High-resolution structures map the metal import pathway in an Nramp transporter

Ray

Berry

Wilson

et al. 2022

Preprint

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Transporters of the Nramp (Natural resistance-associated macrophage protein) family import divalent transition metal ions into cells of most organisms. By supporting metal homeostasis, Nramps prevent disorders related to metal insufficiency or overload. Previous studies revealed that Nramps take on a LeuT fold and identified the metal-binding site. We present high-resolution structures of Deinococcus radiodurans Nramp in three stable conformations of the transport cycle revealing that global conformational changes are supported by distinct coordination geometries of its physiological substrate, Mn2+, across conformations and conserved networks of polar residues lining the inner and outer gates. A Cd2+-bound structure highlights differences in coordination geometry for Mn2+ and Cd2+. Measurements of metal binding using isothermal titration calorimetry indicate that the thermodynamic landscape for binding and transporting physiological metals like Mn2+ is different and more robust to perturbation than for transporting the toxic Cd2+ metal.

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Section: Discussionmentioning

confidence: 99%

High-resolution structures map the metal import pathway in an Nramp transporter

Ray

Berry

Wilson

et al. 2022

Preprint

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“…The bundle domain of LeuT-fold transporters such as Mhp1 and vSGLT1 is reportedly fixed, while the hash domain and gating helices rotate to transport substrates via an alternating-access mechanism 12 . When the bundle domains are superimposed between the inward and outward structures, TM13 and MAP17 are also well superimposed, while the other parts of the transporter change their location and conformation (Fig.…”

Section: Structural Rearrangement From the Outward To Inward Conforma...mentioning

confidence: 99%

“…2e) are currently being developed for the treatment of diabetes 9,11 . SGLT1 and SGLT2 belong to the LeuT transporter family and are conserved in all bacterial and animal taxa, with six isoforms in humans 12,13 . Structural homology modeling studies have been conducted using SGLT from Vibrio parahaemolyticus (vSGLT) and similar protein structures, such as SiaT 14,15 , because of the difficulties in protein preparation and structural analysis.…”

Section: Introductionmentioning

confidence: 99%

“…A functional model of SGLT2 glucose uptake has been proposed: SGLT2 undergoes a conformational change to an outward-facing conformation by binding to a single Na + ion (at Na2 site 16 ) before binding to the substrate, depending on Na + concentration gradient across the plasma membrane, which then promotes glucose binding. Subsequently, Na + and sugars are incorporated into the cell in an inward-open conformation 3,12,13 . In contrast, SGLT1 requires two Na + ions (at Na2 and Na3 sites) for glucose transport, which probably affects its glucose affinity 16 .…”

Section: Introductionmentioning

confidence: 99%

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Structural insights into the mechanism of the human SGLT2–MAP17 glucose transporter

Hiraizumi¹,

Akashi²,

Murasaki³

et al. 2023

Preprint

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Selective sodium-glucose cotransporter 2 (SGLT2) plays an important role in glucose reabsorption. SGLT2 inhibitors suppress glucose reabsorption from the kidneys, thus reducing blood glucose levels in type 2 diabetes patients. We and other groups have developed several SGLT2 inhibitors starting from a natural product, phlorizin, but their action mechanisms remain unknown. Here, we elucidated the physiological hSGLT2-MAP17 complex structures bound to five SGLT2 inhibitors using single-particle cryo-electron microscopy. Canagliflozin, dapagliflozin, TA-1887, and sotagliflozin were bound in the outward-facing structure, whereas phlorizin was bound in the inward-open structure. The phlorizin-hSGLT2 interaction biochemically exhibited biphasic binding. Phlorizin weakly binds, via the phloretin motif, from its intracellular side near the Na+-binding site, while strongly interacts from its extracellular side. Unexpectedly, bound Na+ stabilizes the outward-open conformation, while its release allows the transporter to adopt inward-open state. Our results first visualized the Na+-binding and inward-open conformation of hSGLT2-MAP17, clarifying the unprecedented Na+-dependent sugar transport mechanism with MAP17 acting as a scaffold, and may pave the way for development of next-generation SGLT inhibitors.

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Alternating Access of a Neurotransmitter:Sodium Symporter Bacterial Homolog Determined from AlphaFold2 Ensembles and DEER Spectroscopy

Schwartz,

Stein,

Gil-Iturbe

et al. 2024

Preprint

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Neurotransmitter:sodium symporters (NSSs) play critical roles in neural signaling by regulating neurotransmitter uptake into cells powered by sodium electrochemical gradients. Bacterial NSSs orthologs, including MhsT fromBacillus halodurans, have emerged as model systems to understands the structural motifs of alternating access in NSSs and the extent of conservation of these motifs across the family. Here, we apply a novel computational/experimental methodology to illuminate the energy landscape of MhsT alternating access. Capitalizing on our recently developed method, Sampling Protein Ensembles and Conformational Heterogeneity with AlphaFold2 (SPEACH_AF), we derived clusters of MhsT models spanning the transition from inward-facing to outward-facing conformations. Systematic application of double electron-electron resonance (DEER) spectroscopy revealed ligand-dependent movement of multiple structural motifs that underpins MhsT’s conformational cycle. Remarkably, comparative DEER analysis in detergent micelles and lipid nanodiscs highlight the profound effect of the environment on the energetics of conformational changes. Through experimentally-derived selection of collective variables, we present a model of ion and substrate powered transport by MhsT consistent with the conformational cycle derived from DEER. Our findings not only advance the understanding of MhsT’s function but also uncover motifs of conformational dynamics conserved within the broader context of the NSS family and within the LeuT-fold class of transporters. Importantly, our methodological blueprint introduces a novel approach that can be applied across a diverse spectrum of transporters to describe their energy landscapes.Significance StatementThe neurotransmitter:sodium symporter (NSS) family plays a crucial role in neurotransmitter reuptake, a sodium-dependent process that transports neurotransmitters from the synapse back into the neuron. This study investigates the bacterial tryptophan transporter MhsT, a homolog of human NSSs, using the deep learning method AlphaFold2 in conjunction with double electron-electron resonance spectroscopy. This combined approach enables us to map the energy landscape that dictates the conformational shifts crucial for MhsT’s function. Furthermore, we reveal how the environment modulates the transporter’s dynamics. From our research, we develop a model of MhsT transport that highlights the extent of mechanistic conservation across the NSS family. Additionally, we introduce a comprehensive framework for exploring the energetic landscapes of transporters, effectively integrating computational and experimental methods.

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Principles of Alternating Access in LeuT-fold Transporters: Commonalities and Divergences

Cited by 5 publications

References 137 publications

High-resolution structures map the metal import pathway in an Nramp transporter

High-resolution structures map the metal import pathway in an Nramp transporter

Structural insights into the mechanism of the human SGLT2–MAP17 glucose transporter

Alternating Access of a Neurotransmitter:Sodium Symporter Bacterial Homolog Determined from AlphaFold2 Ensembles and DEER Spectroscopy

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