Bisignano P et al. (2018), Inhibitor binding mode and allosteric regulatio...

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Nat Commun 2018 Dec 07;91:5245. doi: 10.1038/s41467-018-07700-1.

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Inhibitor binding mode and allosteric regulation of Na+-glucose symporters.

Bisignano P , Ghezzi C , Jo H , Polizzi NF , Althoff T , Kalyanaraman C , Friemann R , Jacobson MP , Wright EM , Grabe M .

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Sodium-dependent glucose transporters (SGLTs) exploit sodium gradients to transport sugars across the plasma membrane. Due to their role in renal sugar reabsorption, SGLTs are targets for the treatment of type 2 diabetes. Current therapeutics are phlorizin derivatives that contain a sugar moiety bound to an aromatic aglycon tail. Here, we develop structural models of human SGLT1/2 in complex with inhibitors by combining computational and functional studies. Inhibitors bind with the sugar moiety in the sugar pocket and the aglycon tail in the extracellular vestibule. The binding poses corroborate mutagenesis studies and suggest a partial closure of the outer gate upon binding. The models also reveal a putative Na+ binding site in hSGLT1 whose disruption reduces the transport stoichiometry to the value observed in hSGLT2 and increases inhibition by aglycon tails. Our work demonstrates that subtype selectivity arises from Na+-regulated outer gate closure and a variable region in extracellular loop EL5.

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	Fig. 1. Predicted binding mode of phlorizin to hSGLT1 and hSGLT2. a Phlorizin bound to the outward-facing hSGLT1. The protein is gray, phlorizin is green and red, and Na+ at the putative Na2/Na3 sites are yellow. EL5c (red) is represented as cartoon. The 2D structures of phlorizin and phloretin are also shown. b Close-up view of phlorizin bound to hSGLT1. The sugar moiety occupies the sugar binding site aligning with the docked glucose (transparent yellow/red) where it makes excellent hydrogen bonding with the protein. The aglycon tail creates face-to-face aromatic π–π interactions with H83 and F101 and a cation–π interaction with R267 on EL5C. c Phloretin and glucose bound to hSGLT1. Phloretin (yellow/red) superposes over the aglycon tail from the phlorizin molecule (transparent green/red) in b, while glucose (yellow/red) aligns well with the sugar moiety. d Close-up view of phlorizin bound to hSGLT2. The binding mode is conserved in hSGLT2, although the aglycon tail is more tightly packed due to an aromatic cage that forms around the central ring made up of residues H80, F98, and H268 on EL5C
	Fig. 2. Predicted binding mode of dapagliflozin to hSGLT1 and hSGLT2. Dapagliflozin (orange/red) adopts a similar pose to phlorizin (transparent green/red) in both hSGLT1 (a) and hSGLT2 (b). Interactions in the sugar binding site and extracellular vestibule are preserved including the electrostatic interaction with R267 (hSGLT1) and the aromatic cage formed by H80, F98, and H268 (hSGLT2). c αMDG hSGLT1 D268H (black squares) sugar currents. αMDG currents were measured at Vm = −50mV. The red dashed curve is a representative, wild-type hSGLT1 response. For the D268H mutant K0.5 = 1.2 ± 0.1 mM, while wild-type hSGLT1 K0.5 = 0.9 ± 0.1 mM. For c and d, data are normalized to the current measured at 10 mM αMDG. d Na+ dependence of αMDG currents measured in 10 mM αMDG at Vm = −50mV. For hSGLT1 D268H K0.5 = 17 ± 1.2 mM and for wild-type hSGLT1 K0.5 = 36 ± 1 mM. Hill coefficients hSGLT1 D268H and wild-type were 1.62 ± 0.03 and 1.61 ± 0.1. e Phlorizin and f dapagliflozin effect on the Qmax for hSGLT1 D268H, we estimated a Ki = 0.3 ± 0.1 and 0.035 ± 0.011 µM for phlorizin and dapagliflozin, respectively, while the wild-type Ki = 0.22 ± 0.04 and 0.45 ± 0.02 µM. For c–f, each data point is the mean ± SEM of n ≥ 5 oocytes
	Fig. 3. Outer gate closure explains the impact of TM9-10 residues on substrate binding. a The outer gate residue Q457 on TM10 does not interact with phlorizin (green/red) in the outward-facing model of hSGLT1. b A molecular dynamics simulation of the hSGLT1–phlorizin complex reveals significant movement in the TM9-10 outer gate bringing Q457 into direct contact with phlorizin. Additionally, EL5C (red) closes over the top of the inhibitor. This figure is a snapshot from the trajectory at 1 μs. Lipids and water are not shown for clarity
	Fig. 4. Allosteric communication between sodium and substrate sites. a The hSGLT1–phlorizin model shows the location of Na+ (transparent yellow) in the Na2 site over 8 Å from the inhibitor and the putative Na3 site 14 Å away. The Na2 Na+ interacts with the side chains of S392 and S393 and the carbonyl backbone of I79, while the ion at Na3 is stabilized by backbone carbonyls, side chains of T395 and S396, and bidentate interaction with D204. b The hSGLT2–phlorizin model shows the location of the conserved Na2 binding site (transparent yellow), but the putative Na3 site is lost by the hydrophobic substitution A395. c hSGLT1 T395A and wild-type hSGLT1 and hSGLT2 stoichiometries were determined from the reversal potential (Vrev). The inverse of the slope, Na+-to-glucose coupling ratio, n, is 1-to-1 Na+-to-substrate stoichiometry for the hSGLT2 wild-type and the T395A hSGLT1 mutant and 2-to-1 for wild-type hSGLT1. d αMDG dependence of hSGLT1 T395A (black squares) sugar current in injected oocytes. K0.5 = 34 ± 4 mM compared to K0.5 = 0.9 ± 0.1 mM for wild type. Data in d–f are normalized to the current measured in 10 mM αMDG without inhibitors, and the red dashed curves are representative, wild-type hSGLT1 responses. e Phlorizin (black squares) and dapagliflozin (open squares) effect on αMDG currents for hSGLT1 T395A in 30 mM αMDG. For hSGLT1 T395A Ki = 0.35 ± 0.10 and 0.4 ± 0.1 µM for phlorizin and dapagliflozin, respectively, while wild type estimated Ki = 0.22 ± 0.04 and 0.45 ± 0.02 µM for phlorizin and dapagliflozin, respectively. f Phloretin (black circles) and dapa-aglycon (open circles) effect on αMDG currents for hSGLT1 T395A in 30 mM αMDG. For hSGLT1 T395A Ki = 20 ± 7 and 187 ± 80 µM for phloretin and dapa-aglycon, respectively, while the wild-type Ki = 55 ± 12 and 425 ± 50 µM for phloretin and dapa-aglycon, respectively. Each data point is the mean ± SEM of n = 10 oocytes (c), n ≥ 5 oocytes (d), and n ≥ 7 oocytes (e, f)
	Fig. 5. Allosteric model of inhibitor binding to SGLTs. The binding of phlorizin-like inhibitors to the outward-facing state of SGLTs leads to a partial closure of the TM9-10 outer gate in an induced fit mechanism. a, c Binding of 2 Na+ to hSGLT1 stabilizes the outer gate in an open conformation making it less favorable for the gate to close over the aglycon tails of inhibitors. b, d The absence of the Na3 site in hSGLT2 makes partial closure and inhibitor binding more favorable and therefore the inhibitors more potent. Additionally, elements of EL5C (red) contribute to better binding to hSGLT2

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