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

Article abstract of DOI:10.1038/s41467-018-07700-1

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 phlorizi

Full text of DOI:10.1038/s41467-018-07700-1

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https://doi.org/10.1038/s41467-018-07700-1
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1 Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158, USA. 2 Cardiovascular Research Institute, University of
California, San Francisco, CA 94158, USA. 3 Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1751, USA.
4 Department of Chemistry and Molecular Biology, Centre for Antibiotic Resistance (CARe) at University of Gothenburg, SE-40530 Gothenburg, Sweden.
Correspondence and requests for materials should be addressed to E.M.W. (email: EWright@mednet.ucla.edu) or to M.G. (email: michael.grabe@ucsf.edu)
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mong the six human sodium-dependent glucose trans- (Supplementary Methods and Supplementary Fig. 2), and we then
porter (SGLT) subtypes widely expressed in the small used the alignment to create outward-facing SGLT models based
intestine, kidney, lung, muscle, and brain1, hSGLT1 is the on the SiaT structure with Modeller17. Initial glucose docking
A
primary transporter in the intestine, while glucose reabsorption in studies were carried out on these models to confirm the role of
the kidney is accomplished mostly by hSGLT2. Patients with known residues in the glucose binding site based on published
mutations in the hSGLT2 gene develop a benign disorder called mutagenesis data and interactions observed in the inward-facing
familial renal glucosuria (FRG), in which sugar reabsorption in vSGLT co-crystal (see Methods).
the kidneys is impaired; however, they do not suffer any long-
Next, we docked phlorizin into the outward-facing model of
term consequences2. Thus, hSGLT2 inhibition has been a primary hSGLT1 using a flexible docking procedure, which allows for
focus of type 2 diabetes (T2DM) research in the past decade3. motion in the small molecule around its rotatable bonds while
Currently approved hSGLT2 inhibitors—glucosides containing a keeping the protein fixed. We then rescored each of the top 10
sugar moiety connected to an aromatic tail referred to as an binding poses, allowing the protein side chains within 4 Å of
aglycon—were also shown to reduce heart failure hospitalization phlorizin to relax with the MM/GBSA18 protocol. The top-ranked
rates by 35% compared to other diabetes treatments while also pose (green/red) occupies the same vestibule found for the MTS-
cutting deaths from any cause by 32%4. While very promising, TAMRA (2-((5(6)-tetramethyl-rhodamine)carboxylamino)ethyl
these drugs are not free from side effects5, and the lack of methanethiosulfonate) reagent19 and shows overlap of the
knowledge concerning the molecular determinants of action phlorizin sugar moiety with the redocked glucose (transparent
poses a barrier to developing new chemotypes with an improved yellow/red), while the aglycon tail occupies the outer vestibule
therapeutic window. Structurally, SGLTs fall into the large pointing towards the extracellular space (Fig. 1a, b). The sugar
leucine-transporter (LeuT) family6, and they work by means of an moiety makes hydrogen bond interactions with residues N78,
alternating access mechanism in which they first bind Na+ and H83, E102, Y290, W291, and K321, which have all been shown to
sugar from the extracellular side in a so-called outward-facing affect both phlorizin-dependent inhibition and sugar transport20.
conformation and then transition to an inward-facing con- Y290 has stacking interactions with both glucose and phlorizin,
formation to release their cargo to the cytoplasm. The structural but its hydroxyl group also hydrogen bonds directly to the
basis of how inhibitors block transport is not known.
hydroxyl at the C1 position of phlorizin. Meanwhile, Y290’s
While recent studies have attempted to dock phlorizin-derived ability to hydrogen bond to transmembrane segment 1 (TM1) is
compounds into inward-facing models of hSGLTs7, the Wright thought to play a role in sugar transport in vSGLT9. We tested the
lab has demonstrated that SGLT2 inhibitors bind from the importance of hydrogen bonding by mutating Y290 to F in
extracellular solution likely stabilizing a Na+-bound, outward- hSGLT1 and measuring the glucose transport and the inhibition
facing conformation8. Thus, a detailed molecular view of this of steady-state, glucose-induced Na+ current as a function of
interaction is not possible without a reliable outward-facing external inhibitor concentration. The inhibitory constant (Ki) for
model of SGLTs. While the closely related bacterial homolog phlorizin to the wild-type transporter was 0.22 0.04 μM under
from Vibrio parahaemolyticus (vSGLT) has been solved in the these conditions, and the value increased eightfold in the Y290F
apo-9 and sugar-bound10 conformations, both structures are mutant (1.8 0.4 μM) consistent with the loss of a single
inward facing, and although suitable for modeling hydrogen bond to the aglycon tail. Glucose transport exhibited
hSGLT–glucose complexes, our attempts to dock phlorizin into a decrease in glucose apparent affinity (K0.5) from 0.9 0.1 mM
hSGLT models built upon these templates failed. Previously, for wild type to 35 10 mM21—a nearly 35-fold change, thus
members of the superfamily have been solved in the outward- confirming the importance of the chemistry at this site for both
facing state11–13, but the sequence identities are far too low to molecules. Glucose transport may be more impacted due to its
make reliable SGLT homology models (<8%). The outward-facing smaller size and the polar nature of the interactions with the
structure of the N-acetylneuraminic acid transporter from Proteus transporter, while the mostly hydrophobic nature of the
mirabilis (SiaT) was determined at 1.95 Å resolution14, and this interactions between the aglycon tail and the transporter are
LeuT-fold transporter shares moderate sequence identity with not impacted by the Y290F mutation. Meanwhile, F101 in TM2
SGLTs (~24% identity/~46% similarity), a value that is sig- exhibits π–π stacking with aromatic elements in the aglycon tail,
nificantly higher than any other LeuT-fold transporter of known but no interactions with the sugar moiety (Fig. 1b) corroborating
structure except for vSGLT, which has similar sequence the experimental finding that F101C increases the phlorizin Ki
identity to the human SGLTs as SiaT. Here we show with a 170-fold while having no effect on sugar transport20. To assess
combination of computational and experimental approaches that the robustness of the predicted pose, we docked the aglycon tail
this outward-facing SiaT structure serves as a good template for alone, which is called phloretin, into hSGLT1. The smaller
understanding inhibitor binding and the subtype specificity of phloretin molecule (yellow/red) perfectly overlaps with the
human SGLTs.
aglycon tail of phlorizin providing additional confidence in the
pose (Fig. 1c).
Results
The hSGLT1–phlorizin complex explains mutagenesis data. Structural determinants of inhibitor subtype selectivity. Next,
Our initial attempts to dock small molecules into inward-facing we wanted to explore the differences in inhibitor binding between
models of hSGLT built on the available vSGLT structures pro- hSGLT1 and hSGLT2 in an attempt to understand why hSGLT2
vided mixed results. Glucose adopts a binding pose similar to the is more easily inhibited by phlorizin-derived molecules. We
one observed for galactose in vSGLT. However, phlorizin fails to started by docking phlorizin into an outward-facing model of
adopt a reasonable pose as the glucose moiety does not occupy hSGLT2 constructed as described for hSGLT1. The inhibitor-
the sugar binding site nor does the molecule contact any of the binding mode is conserved between both transporters (Fig. 1d).
protein residues known to influence inhibition (Supplementary The sugar moiety recapitulates the same interaction landscape
Methods and Supplementary Fig. 1). Therefore, we turned to SiaT observed for hSGLT1, which was expected since the glucose
as a potential outward-facing template. We first used a combi- binding sites are conserved. In addition, many aromatic interac-
nation of sequence15 and structure-based16 methods to reach a tions with the aglycon tail are preserved (H80/H83 and F98/F101,
consensus alignment between the SGLTs and SiaT hSGLT2/hSGLT1 numbering, respectively); however, a notable
2
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sequence difference exists between both transporters in the currents (Fig. 2 and Table 1) elicited by α-methyl-D-glucopyrano-
C-terminal end of the long extracellular loop 5 (EL5C) that side (αMDG)—a glucose analog, which we interchangeably refer
connects TM5 to TM6 (Supplementary Fig. 2). hSGLT2 has a to as glucose throughout. Unfortunately, the R267C mutant was
third aromatic residue (H268) that stacks in a parallel displace- not expressed in oocytes, but D268H exhibited an apparent Na+
ment fashion with F98 to create an aromatic cage (H80, F98, affinity (17 1.2 mM) and apparent K0.5 for glucose (1.2 0.1
H268) around the central ring of the aglycon tail. hSGLT1 does mM) both similar to wild type (36 1 and 0.9 0.1 mM,
not form this cage because 268 is an aspartic acid, and D268 respectively). Moreover, hSGLT1 D268H is inhibited by phlorizin
creates a salt bridge with the conserved R267 redirecting the to the same degree as wild type (Ki = 0.3 0.1 and 0.22 0.04
guanidinium group towards a cation–π interaction with the μM, respectively). However, the Ki for dapagliflozin is 13-fold
inhibitor. The introduction of the histidine in position 268 in lower (0.035 0.011 μM) than the wild-type Ki (0.45 0.02 μM),
hSGLT2 confers additional packing interactions absent in indicating that D268H in EL5C contributes to the selectivity of
hSGLT1.
dapagliflozin but not phlorizin. The docked structures may
Analysis of the phlorizin-docked complexes revealed only rationalize this difference. Phlorizin possesses a central aromatic
subtle structural differences in the binding poses, which we ring which protrudes towards EL5C making either a cation–π
hesitate to attribute to the increased inhibition of hSGLT2 over interaction with R267 in hSGLT1 (Fig. 1b) or π–π and Van der
hSGLT1 without further analysis since these are homology Waals packing with the imidazole of H268 in hSGLT2 (Fig. 1d).
models and phlorizin has rather low subtype selectivity (eightfold Meanwhile, dapagliflozin lies deeper in the binding pocket, and
increase in inhibition of hSGLT2 over hSGLT1). To gain greater its central ring is too far to make a strong cation–π interaction
insight into subtype specificity, we next docked the T2DM drug with R267 in hSGLT1 (Fig. 2a), but it is still able to establish
dapagliflozin into both outward-facing models to identify excellent interactions with H268 in hSGLT2 (Fig. 2b). Thus, the
structural differences between the proteins that may explain introduction of D268H in hSGLT1 may impact dapagliflozin
why this drug has a >100-fold higher potency for hSGLT222. binding more than phlorizin due to deeper binding of
Dapagliflozin docks into both transporters in a very similar dapagliflozin.
manner with the sugar moiety occupying the glucose binding site
and the aglycon tail occupying the extracellular vestibule (Fig. 2).
Additionally, overlap with the phlorizin structures (transparent)
reveals that the two molecules engage with the transporters
similarly. The aromatic cage around the aglycon tail persists in
hSGLT2, and again R267 has a cation–π interaction with the tail
in hSGLT1. Again, as for phlorizin, the introduction of the
histidine in position 268 in hSGLT2 confers additional packing
interactions absent in hSGLT1. This difference might provide
some degree of selectivity, and we tested this possibility
experimentally.
To do so, we focused on two mutants: hSGLT1 D268H, which
mimics EL5C of hSGLT2, and hSGLT1 R267C, which although
conserved, only interacts with phlorizin in our hSGLT1-docked
model, but not in hSGLT2 (Fig. 2a, b). We expressed both
mutants in Xenopus laevis oocytes and recorded sugar-induced
The outer gate partially closes over bound inhibitors. Our
analysis has focused on residues in direct contact with the inhi-
bitors, and now we turn our attention to mutations in hSGLT1
that impact glucose and phlorizin binding, but do not establish
direct contact based on the model. The first two positions are
F453 and Q457, which are on the same face of helix TM10 in the
extracellular vestibule, are both solvent exposed, and more than
10 Å away from phlorizin (Fig. 3a). Nonetheless, mutations to
cysteine at these positions increase the K0.5 of glucose 2- and 14-
fold20, respectively, while they increase the Ki of phlorizin inhi-
bition 4.5- and 27-fold20, respectively. We believe that their
influence can be understood through an induced-fit hypothesis
since the extracellular half of TM9 and TM10 and the small
intervening loop form an outer gate in the inward-facing states of
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hSGLT1 D268H
hSGLT1 WT
hSGLT2 WT
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hSGLT1 WT
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K321
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[Pz] (μM)
[Dapa] (nM)
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Table 1 Measured Ki and K0.5 values for hSGLT2, hSGLT1 and hSGLT1 mutants
Transporter
K0.5 αMDG (mM) K0.5 Na+ (mM)
Ki phlorizin (μM)
Ki phloretin
(μM)
Ki dapagliflozin (μM) Ki dapa-aglycon
(μM)
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vSGLT10, Mhp123, BetP24, and LeuT13 that closes over the sub- hSGLT1–glucose modeled complex and the inward-facing
strate to occlude it from the extracellular solution. If TM9-10 is occluded vSGLT structure10 (Supplementary Figs. 1a and 3).
dynamic and phlorizin binding results in partial closure of the
To test our induced-fit hypothesis, we carried out 1 μs of fully
gate, it may bring the extracellular end of TM10 into contact with atomistic molecular dynamics (MD) simulations on the
the substrates (sugar and inhibitors) as in the inward-facing hSGLT1–phlorizin bound complex to determine how elements
4
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decided to explore the primary difference between hSGLT1 and
hSGLT2—the latter binds only a single Na+, while hSGLT1 binds
2 Na+. There is little information regarding the structural
influence that binding 1 vs. 2 Na+ imparts to SGLTs; however,
our experiments suggest that hSGLT1 binds glucose five times
more tightly than hSGLT2 (Table 1), which may or may not be
related to differences in stoichiometry27. Structural insight into
ion binding comes from spectroscopic studies on LeuT13, which
has a 2-to-1 stoichiometry, and Mhp128 and vSGLT29, which
bind a single Na+ at the Na2 site like hSGLT2. Interestingly,
under saturating Na+ levels, presumably resulting in the binding
of 2 Na+ ions, LeuT adopts an outward-open conformation with
the TM9-10 gate wide open. However, saturating Na+ does not
result in an outward-open conformation for Mhp128 and
vSGLT29. Thus, we hypothesize that the second Na+ binding
event stabilizes the outer gate in an open conformation, while a
single Na+ binding at the Na2 site does not efficiently hold the
outer gate open. Since our model and simulation suggest an
induced-fit mechanism of binding, the corollary of our hypothesis
is that hSGLT2 may bind phlorizin-like molecules more tightly
because the gate closes more easily over the inhibitors to make
several contacts with residues belonging to the extracellular
portion of TM10. To test this hypothesis, we attempted to abolish
binding of Na+ to the additional Na site in hSGLT1 and measure
the influence on inhibitors and substrate.
a
b
t = 0 ns
t = 1 μs
EL5C
Phlorizin
TM9
Q457
TM10
Starting model
Partially occluded state
Fig. 3
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s h o w n f o r c l a r it y
Unfortunately, the crystallographic position of the Na1 site in
hSGLT1 is unknown. The LeuT X-ray structures revealed that a
non-conserved Na+ binding site (termed Na1) directly coordi-
nates the substrate and the protein30. Initial functional work on
hSGLT1 seemed to suggest that Na+ also binds to a Na1-like
site31 (termed Na1), but our attempts to simulate Na+ at this
position in either single or double occupancy (at Na1 and
Na2 sites, Supplementary Table 1) failed to provide a stable
configuration (Supplementary Fig. 4a, b). Next, we searched for
potential Na+ binding sites in the vicinity, and we identified a
region in hSGLT1 that provides excellent Na+ coordination from
two well-conserved polar groups (S396 and T395), an acidic
of the outer gate behave. During the simulation (Supplementary
Movie 1), we observe substantial movement in the TM9-10 outer
gate (ice blue) bringing Q457 into close proximity with the
inhibitor while at the same time heading toward an occluded state
of the transporter in which both the outer gate and inner gate are
closed (Fig. 3). Partial gate closure occurs via a rigid rotation of
TM9 and a bending of TM10 around a central kink, but EL5C
(red) also plays a role in closure by forming a cap over the
aglycon tail of the inhibitor in a lid-like fashion (Fig. 3b).
Disruption of the Na3 site enhances aglycon potency. Similar to residue (D204), and coordinating backbone carbonyl from S392,
sites in the extracellular gate, the mutation S392A impacts glucose which also forms part of the Na2 site (Fig. 4a). This site is the
transport and phlorizin potency20, but it does not directly contact proposed second Na+ binding site (termed Na3) which was
the molecules. Unlike the outer gate positions, S392 fails to make resolved in the SiaT X-ray structure14. When we simulated
direct contact in both the inward-facing and outward-facing hSGLT1 with Na+ in Na3, in single and double (Na2 and Na3)
models, and it is located more than 10 Å away from the substrate occupancy, the Na3 ion remained stable (Supplementary Fig. 4c,
in the outward-facing model (Fig. 4a). It forms part of the Na2 d). As one might expect, Na3 is present in hSGLT1, but not
Na+ binding site based on the vSGLT structures9, 10, and it is hSGLT2 or vSGLT (Supplementary Fig. 2). The critical difference
conserved throughout the solute sodium symporter family (see with hSGLT2 is the hydrophobic substitution at T395 by alanine
Supplementary Fig. 2 for partial list). Thus, we hypothesize that (Fig. 4).
S392A disrupts Na+ binding and subsequently reduces substrate
To further assess the likelihood that Na3 is a suitable Na+ site,
binding through allosteric means. Experimentally, it is known we carried out a comprehensive analysis of all known Na+
that Na+ binding precedes substrate binding from the extra- binding sites in the RCSB database32. While such analysis has
cellular face25, 26, as ion binding opens access to the substrate and been performed previously33, the dataset analyzed here is twice as
inhibitor binding sites19 while also pre-organizing the sites for large. Sodium sites have a five-point coordination arising from
increased binding affinity. Our recent simulations of SiaT support interactions with both side chain elements and main chain
the claim that ions aid in pre-organizing residues in the substrate carbonyls. The putative Na3 site in hSGLT1 (Fig. 4a and
binding site, as we showed that the bound sialic acid was far more Supplementary Fig. 5) is coordinated by Asp, Ser, and Thr side
stable in the binding site when Na+ was bound at Na2 and/or chains, which are the first, fourth, and fifth most common
Na3, despite no direct contact between the substrate and ions14. A coordinating residues, as well as a Ser main chain interaction,
network of interactions exists in all LeuT transporters extending which is the fourth most common main chain motif (Supple-
from the Na2 site to the unwound section of TM1 that directly mentary Fig. 6). Structurally similar coordination sites involving
contacts the transported substrate (Fig. 4a, b), and when Na+ either a Ser/Thr at positions i and i + 1 along the primary
binding at Na2 is impaired, we hypothesize that TM1 does not sequence, as observed for both hSGLT1 and SiaT, returned the
adopt a conformation compatible with tight sugar and inhibitor structurally unrelated sodium-calcium exchanger, which none-
binding.
theless binds Na+ with a similar coordination and also contains
Motivated by the impact that Na+ binding to the Na2 site has an acidic group. Lastly, D204 interacts with the Na+ in a
on substrate and inhibitor binding, coupled with the fact that bidentate fashion, which represents nearly half of all Asp residues
EL5C explains only part of the selectivity towards hSGLT2, we in Na+ binding sites, several of which are pictured in
N
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G77
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Phloretin
Glucose
Phloretin
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Na2
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hSGLT1
S396
Glucose
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hSGLT2
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hSGLT1 WT:
hSGLT1 WT:
40
Dapa-aglycon
Phloretin
Dapagliflozin
Phlorizin
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n = 1.0 0.07
20
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hSGLT1 T395A:
hSGLT1 T395A:
Dapagliflozin
Dapa-aglycon
Phloretin
Phlorizin
n = 2.2 0.03
–20
–40
–60
hSGLT1 T395A
hSGLT1 WT
hSGLT2 WT
hSGLT1 T395A
hSGLT1 WT
0
25
50
75
100 125
0
25
50
75
100
0.0
2.5
5.0
7.5
10.0
0
250
500
750 1000
RT/F in ([αMDG]o)
[αMDG] (mM)
[Inhibitor] (μM)
[Inhibitor] (μM)
Fig. 4
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the hallmarks of an excellent Na+ coordination site.
compared to the wild-type transporter (350 100 vs. 220 nM,
To determine if this putative position is a true sodium binding respectively). We believe that the decrease in affinity for sugar
site, we expressed the hSGLT1 T395A mutant in Xenopus oocytes and the increase of Ki for phlorizin is explained by the lack of full
and recorded sugar-induced Na+ currents. The mutant displays Na+ occupancy at the Na2 site under these conditions, which
markedly decreased apparent affinity for Na+ (104 50 mM) results in failure to pre-organize the sugar binding site and
compared to wild type (36 1 mM), a loss of pre-steady-state suboptimal binding of glucose and the glucose moiety. We
currents that looks more hSGLT2-like than hSGLT1-like, and a therefore tested the potency of the aglycon tail against this
decreased Na+-to-glucose coupling ratio from 2.2 0.03 to 1.1
mutant. As we predicted, phloretin potency is increased 2.7-fold
0.08 as measured via reversal potentials (Fig. 4c), and confirmed in the Na3-defective mutant (Ki = 20 7 μM compared to 55
by estimates from Na+ Hill coefficients that were reduced from 12 μM for the wild type), which is marginally better than
1.7 0.03 to 1.1 0.3 (Supplementary Table 2). Thus, experi- phloretin inhibition of hSGLT2 (Ki = 27 3 μM). We also tested
ments suggest that the Na3 site is the additional Na+ binding site, dapagliflozin inhibition of the hSGLT1 T395A mutant and
and T395A has hSGLT2-like properties with an apparent 1-to-1 determined that it was unchanged from wild type, while the dapa-
stoichiometry. Next, we measured the apparent affinity for aglycon, the aglycon tail of dapagliflozin, inhibited the mutant
glucose to hSGLT1 T395A in non-saturating 100 mM external three times better (Ki = 187 80 and 425 50 μM, respectively).
Na+ and found a 38-fold decrease from 0.9 0.1 mM in wild type Finally, we attempted to introduce the Na3 site into hSGLT2 by
to 34 4 mM (Table 1). This mutation has a dramatic effect on mutating A395 to Thr (Supplementary Table 2). Unfortunately,
glucose affinity that is comparable to Na2 binding site mutations this construct exhibited poor expression (about 10% of wild type),
(S392A/C >100 mM and S393A/C ~4 mM)20. Interestingly, the and it was not possible to determine the stoichiometry. None-
mutant sugar K0.5 is eightfold lower than the value for wild-type theless, the mutant appears to have similar sugar and sodium
hSGLT2, despite position 395 being an alanine in both proteins. affinity (4 0.6 and 30 10 mM, respectively) to wild type (4.4
Next, we measured the inhibition constant of phlorizin to 0.1 and 22 1 mM, respectively), while inhibition by phloretin,
hSGLT1 T395A, and rather than increasing the inhibition dapa-aglycon, and phlorizin (Ki = 129 70, >600, and 0.04 0.01
6
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μM, respectively) are all decreased when compared to wild type hSGLT1 that may contribute to its greater inhibition by phlorizin
(Ki = 27 3, 110 30, and 0.03 0.01 μM, respectively). The (eightfold) and dapagliflozin (>100-fold) is the presence of H268
reduced inhibition by phlorizin-like molecules is what we expect in EL5c, which gives rise to an aromatic cage around the central
if Na+ at the Na3 site stabilizes the transporter in an outward- ring of the aglycon composed of three residues rather than two
facing conformation. Due to large standard error, we cannot (Fig. 1b, d). D268 is the equivalent residue in hSGLT1, and it
comment on inhibition by dapagliflozin.
forms a salt bridge with the adjacent conserved residue R267.
Simply mutating D268 to histidine in hSGLT1, so that it
resembles EL5C in hSGLT2, leads to a 13-fold increase in dapa-
gliflozin potency arising from improved packing interactions
Discussion
In this study, our goal was to provide a structural basis for the based on our docked models (Table 1). However, this single point
inhibition of SGLTs by drugs approved by the Food and Drug mutation in EL5C cannot explain the >100-fold selectivity of
Administration for the treatment of T2DM. A hallmark of these dapagliflozin for hSGLT2 over hSGLT1.
drugs is their high potency and specificity for the hSGLT2 iso-
One discrepancy between the structural models and functional
form uniquely expressed in the kidney, where they lower blood data concerns the mutation of two residues on the solvent-
glucose levels by reducing glucose reabsorption. Our approach exposed face of helix TM10, Q457, and F453 in hSGLT1, that
was to carefully construct structural models of hSGLT1 and decrease glucose transport and phlorizin-dependent inhibition
hSGLT2 based on the crystal structures of two bacterial homo- despite no interactions in the models (Table 1). Our fully ato-
logs, vSGLT and SiaT, dock the SGLT inhibitors into the mistic molecular dynamic simulations reveal substantial rotation
homology models, and compare the docking poses with func- of TM9 and bending in the external half of TM10 upon phlorizin
tional studies on SGLT mutant transporters. Our results reveal binding bringing Q457 close to the inhibitor, while EL5C forms a
that the inhibitors bind to the Na+-bound, outward-facing con- cap over the inhibitor (see Figs. 3 and 5 and Supplementary
formation of hSGLT1 and hSGLT2 with the glucose moiety of the Movie 1). Thus, our simulations together with mutagenesis stu-
inhibitor in the sugar binding site and the aglycon tail in the dies suggest that the inhibited state of the transporters involves
external vestibule. We predict that subtype selectivity arises from rearrangements in the extracellular gate.
two sources: (1) differences in the C-terminal end of EL5, and (2)
Another major difference between hSGLT2 and hSGLT1 is that
allosteric control of the outer gate by Na+ binding.
glucose transport is driven by one Na+ ion, not two, and so we
The validity of the hSGLT1 and hSGLT2 structural models was turned to Na+ stoichiometry as another potential determinant of
demonstrated by: (1) the conservation of the hSGLT1–glucose inhibitor specificity. We identified a putative Na+ binding site in
interaction landscape in the inward- and the outward-facing hSGLT1 (Na3), not present in hSGLT2 (Fig. 4), which is below
states with respect to the sugar-bound vSGLT structure (Sup- the conserved Na2 site (see Supplementary Fig. 5 and ref. 14). The
plementary Figs. 1a and 3), and (2) phlorizin and dapagliflozin Na3 site is also present in SiaT14, but is distinct from the LeuT
binding to the external surface—but not the cytoplasmic surface Na1 and BetP Na1’ sites, and while having the same name, it is
—consistent with functional studies8 and data generated here not the same site identified as Na3 in GlyT2 (see Supplementary
(Figs. 1 and 2 and Supplementary Fig. 1b and Table 1). The Fig. 7 for an overview of LeuT-fold Na+ binding sites). Mutation
inhibitor binding site is remarkably similar for hSGLT1 and of a Na3 coordinating residue, T365A, in hSGLT1 dramatically
hSGLT2 in that the glucose moiety of the inhibitor, phlorizin and reduces Na+ affinity, and impacts glucose transport and inhibitor
dapagliflozin, overlaps the glucose binding site (Figs. 1 and 2). potency. Most importantly, the T365A mutant is more easily
This is not surprising since the coordinating residues are con- inhibited by the aglycon tail of phlorizin and dapagliflozin
served between the two transporters and mutation of these resi- (Table 1), supporting our hypothesis that inhibitors bind via an
dues disrupts sugar transport and inhibition by phlorizin (Table induced-fit mechanism as shown in the cartoon in Fig. 5. In this
1). Second, the aglycon tails, phloretin and dapa-aglycon, fit into a model, sites in EL5C (red) provide some of the energetics of
large vestibule leading from the external solution to the glucose inhibitor binding, while also providing some of the subtype
binding site (Figs. 1 and 2, and ref. 19). Aglycon/vestibule inter- selectivity. A synergistic factor comes from partial closure of the
actions are dominated by π–π interactions and hydrophobic outer gate, which is more energetically favorable with only a
contact with F101 and H83 in hSGLT1 and F98 and H80 in single Na+ at the Na2 site (hSGLT2) than it is with 2 Na+ ions
hSGLT2. Mutation of F101 in hSGLT1 results in a 170-fold bound (hSGLT1).
decrease in phlorizin inhibition with no change in glucose
There are several lines of evidence supporting the hypothesis
transport (Table 1). A significant difference between hSGLT2 and that Na+ allosterically controls the outer gate. Extensive
Phlorizin-like molecules
EL5C
Induced fit
with outer gate
a
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hSGLT1
hSGLT2
Na2
Na3
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Na2
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biochemical data show that external Na+ is required for the production. Hydrogen atoms were constrained with LINCS47, long-range electro-
statics were computed with particle mesh Ewald, and van der Waals and Cou-
lombic interactions were switched between 10 and 12 Å.
alkylation of cysteine residues in the glucose binding site in
hSGLT120, and active site voltage clamp demonstrates that
The best hSGLT1–phlorizin model from the docking run was simulated with
external Na+ controls the opening and closing of the external gate
in real time (1–10 ms)19. In addition, mutation of Na2 site resi-
dues blocks the voltage-dependent opening and closing of the
outer gates31. As already discussed, vSGLT29 and Mhp128, both of
which have 1-to-1 stoichiometries, fail to fully open their outer
gates in high Na+, while cysteine accessibility studies of LeuT and
SERT, which have 2-to-1 stoichiometries, both require Na+
binding to open the outer gate34. Similarly, double
electron–electron resonance studies on LeuT reveal that high Na+
levels cause the outer gate to open35. Thus, we wondered whether
we could engineer in a second Na+ site to hSGLT2 by mutating
the residue A395 to T (the Na3 binding residue present in
hSGLT1). Although hampered by very low expression levels in
oocytes, the results partially corroborate the gating hypothesis in
Na+ bound to the Na2 and Na3 sites. The system was again prepared with the
CHARMM-GUI webserver in an identical manner to the Na+ simulations and
converted with in-house scripts into the AMBER format. Simulations were carried
out using the ff14SB AMBER48 parameter set for the protein, the
Joung–Cheatham49 parameters for the monovalent ions, LIPID1750 for the lipids,
and the gaff51 force field for phlorizin. The TIP3P model was used to simulate the
water. Phlorizin geometry was first optimized and its electrostatic potential was
calculated with Gaussian v9-E0152 with the 6–31 G* basis set. Next, the RESP53
charges were fitted into the electrostatic potential with antechamber54 and the force
field parameters, topology, and starting coordinates were generated with
AmberTools. Simulations were carried out with the pmemd55 MD engine on
GPUs. Minimization consisted of 10,000 steps, switching from the steepest descent
algorithm to conjugated gradient after 5000 steps. The system was then gradually
heated from 0 to 303.15 K over 15 ps, and harmonic restraints with a spring
constant of 10 kcal/mol/Å2 were applied to all heavy atoms except water oxygens.
After reaching 100 K, we switched from NVT to NPT. After 105 ps, force constants
of 10, 5, and 2.5 kcal/mol/Å2 were applied to the substrates and nearby residues
that aglycon tails and phlorizin showed lower levels of inhibition (within 5 Å), protein, and lipid headgroups, respectively. Restraints were gently
removed over the next ~8 ns, and the system was simulated for 1 μs. Pressure (1
bar) was maintained using a semi-isotropic pressure tensor and the Berendsen
to the mutant hSGLT2 than the wild type (Supplementary
Table 2).
barostat. Temperature was maintained with the Langevin thermostat with a friction
In summary, we propose a structural model that explains inhi-
bitor binding to the Na+-bound, outward-facing conformation of
hSGLTs that largely accounts for their functional activity. While we
have identified two independent components of subtype selectivity,
it still remains to be shown that these elements, together with
saturated Na2 occupancy, fully account for the higher potency of
the drugs to the renal hSGLT2 isoform. The first component is the
formation of a hydrophobic cage surrounding the central ring of
coefficient of 1 ps−1. The SHAKE56 algorithm was used with a 2-fs time step. A
non-bonded cutoff of 10 Å was used and electrostatics were calculated using the
particle mesh Ewald method.
Virtual screening. The Small Molecule Drug Discovery Suite 2016-1 (Schrödinger
LLC)57 was used for all the docking calculations. Protein isoforms were prepared in
a ready-to-dock-format with the Protein Preparation Wizard. The hydroxyl group
orientations and protonation states were assigned at pH 7 using PROPKA58 in Epik
v3.5. Small molecules were drawn with Maestro v10.5, and their protonation states
the aglycon in hSGLT2, but only partially in hSGLT1. The addi- and tautomers assigned at pH 7.0 2 in Epik v3.5. Conformers were assigned using
LigPrep v3.7 with the OPLS force field v359. Docking was performed as follows.
Side chain rotamers in the sugar binding site in both hSGLT1 and hSGLT2 were
tional aromatic residue in hSGLT2 is H268 on an extracellular loop
that forms a cap over the bound inhibitor. The second component
is differences in sodium binding, where the lower potency of
inhibitor binding to hSGLT1 is due to the second sodium stabi-
lizing the transporter in an open conformation that lacks stabi-
lizing interactions between the inhibitor and elements of the
transporter found on the mobile extracellular gate. Further vali-
dation of our predictions awaits the crystal structures of human
SGLTs, molecular simulations, and functional assay on mutants;
together, these studies will lead to improved drugs.
optimized with induced-fit docking60. The models were first aligned to the
galactose-bound vSGLT structure (PDB ID 3DH4), and restraints were applied to
the heavy atoms of the redocked sugar allowing 1.5 Å deviation from the binding
mode observed in the X-ray structure. The Standard Precision61 scoring function
was used for the first docking stage followed by the more accurate XP62 function
for refinement. Next, small molecules (phlorizin, phloretin, dapagliflozin, dapa-
aglycon) were docked into both hSGLT1 and hSGLT2 allowing for small molecule
flexibility while keeping the protein rigid to obtain a maximum of 10 optimal
binding poses. Prior to docking, a 6 × 6 × 8Å3 docking grid was erected on the
glucose-optimized binding sites. The Glide XP scoring function was employed, and
the strain energy was included in the final docking score. Planarity of aromatic
groups was enforced and only trans conformations of amide groups were allowed.
These 10 poses for each molecule were then rescored with the MM/GBSA protocol
to allow side chain relaxation around the small molecule, using the VSGB solvent
model63 and an implicit membrane. Membrane boundaries were determined based
on results from MD simulations, and protein atoms up to 4 Å from the molecule
were allowed to move. Final binding poses were picked based on a combination of
visual inspection, chemical intuition, and score.
Methods
Sequence alignment and homology modeling. Both a primary sequence align-
ment (EMBOSS stretcher15) and a structural alignment (MatchMaker16 within
Chimera v1.10.136) were determined for vSGLT (PDB ID 3DH4) and SiaT (PDB
ID 5NV9 throughout) followed by hand adjustments to arrive at a consensus
alignment used for model building. hSGLT1 and hSGLT2 were added to the
alignment based on previously published alignments to vSGLT10. Outward-facing
models were then created with this alignment using Modeller v9.1517 and the
transmembrane stretches of TM1-10 from SiaT and the extracellular loops from
vSGLT (PDB ID 3DH4) as templates. Outward-facing vSGLT models also included
TM0 and a C-terminal tag from the apo vSGLT structure (PDB ID 2XQ2). In all
cases, one hundred models were generated using the variable target function
method37, and the best model was selected based on DOPE38 score and visual
inspection.
Chemistry. Dapa-aglycon22 was synthesized by copper-catalyzed hydroxylation of
the corresponding aryl iodide64. A mixture of 1-chloro-2-(4-ethoxybenzyl)-4-
iodobenzene (1 mmol), CuI (0.3 mmol), 1,10-phenanthroline (0.6 mmol), and
CsOH (3.3 mmol) in dimethyl sulfoxide/water (3:1, 4 ml) was stirred at room
temperature for 1 h and then heated to 100 °C for 2 h. After cooling, liquid was
transferred to a separatory funnel and diluted with ethyl acetate and washed with 1
M HCl. The organic layer was dried over Na2SO4 and concentrated under reduced
pressure. The crude product was purified by flash column chromatography using
hexanes-ethyl acetate as eluents. Proton nuclear magnetic resonance (1H NMR)
(300 MHz, CDCl3) δ 7.21 (1H, d, J = 8.5 Hz), 7.09 (2H, d, J = 8.4 Hz), 6.83 (2H, d,
J = 8.4 Hz), 6.62 (1H, dd, J = 8.5, 2.7 Hz), 6.55 (1H, d, J = 2.6 Hz), 4.76 (1H, s), 4.01
(2H, dd, J = 13.9, 6.9 Hz), 3.96 (2H, s), 1.40 (3H, dd, J = 6.9, 6.9 Hz). The activity of
MD simulations. Models were oriented in the membrane using the OPM server39
or superposition to previously oriented models followed by embedding with
CHARMM-GUI40 in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine mem-
brane. Final systems 112,000 atoms in size were neutralized in 150 mM NaCl. The
CHARMM3641 parameter set with the TIP3P water model42 was used with
GROMACS v2016.143 for the MD engine. Equilibration consisted of 10,000 steps of
minimization, or until maximum forces dropped below 5000 kJ/mol/nm, followed
by 75 ps of NVT (constant-temperature, constant-volume) dynamics and then 0.3
ns of NPT (constant-temperature, constant-pressure) dynamics. During equili-
bration, restraints on the bound ions, backbone, side chain, lipid heavy atoms, and
dihedral angles of the lipid tails were reduced from 2–10 kcal/mol/Å2 to zero.
Production runs ranged from 7 to 30 ns. Temperature (303.15 K) was maintained
using the Berendsen44 algorithm during equilibration followed by Nosé–Hoover45
for production using a 1 ps coupling time constant. Pressure (1 bar) was main-
tained with a Berendsen44 barostat followed by a Parrinello–Rahman46 barostat
with a 5 ps period. The time step was 1 fs during equilibration and 2 fs during
this dapa-aglycon was consistent with that observed previously22
.
Molecular biology. Mutants of hSGLT1 in pBluescript65 and hSGLT2 in
pT7TS66, 67 were prepared using the QuickChange site-directed mutagenesis kit
(Statagene La Jolla, CA, USA), using the primers provided in Supplementary
Table 3. Capped wild-type and mutant cRNA was synthesized from linearized
plasmids using T3 and T7 mMessage mMachine kits (Ambion).
Functional expression of hSGLT1 in oocytes. Xenopus laevis (Nasco) oocytes
(V–VI) were prepared as previously described20 and injected with 50 nl of cRNA
(1 μg/μl). In the case of hSGLT2, co-injection of MAP17 cRNA was required for
8
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A R T I C L E
transport activity67, 68. hSGLT1 and hSGLT2 activity was confirmed by the mea-
surement of sodium-dependent uptake of 50 μM α-methyl-D-glucopyranoside as
described previously20. Briefly, oocytes were incubated for 30 min in the presence
of 50 μM αMDG (5 μM [14C]αMDG) in a buffer containing (in mM) 100 NaCl, 2
KCl, 1 MgCl2, 1 CaCl2, and 10 N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic
acid) (HEPES)/Tris (pH 7.5). After incubation, oocytes were rinsed thoroughly
with ice-cold buffer containing (mM) 100 choline Cl–, 2 KCl, 1 MgCl2, 1 CaCl2,
and 10 HEPES/Tris (pH 7.5), individually lysed with 5% sodium dodecyl sulfate,
and assayed for radioactivity.
8. Ghezzi, C. et al. SGLT2 inhibitors act from the extracellular surface of the cell
membrane. Physiol. Rep. 2, https://doi.org/10.14814/phy2.12058 (2014).
9. Watanabe, A. et al. The mechanism of sodium and substrate release from the
binding pocket of vSGLT. Nature 468, 988–991 (2010).
10. Faham, S. et al. The crystal structure of a sodium galactose transporter reveals
mechanistic insights into Na+/sugar symport. Science 321, 810–814 (2008).
11. Weyand, S. et al. Structure and molecular mechanism of a nucleobase-cation-
symport-1 family transporter. Science 322, 709–713 (2008).
12. Perez, C., Koshy, C., Yildiz, O. & Ziegler, C. Alternating-access mechanism in
conformationally asymmetric trimers of the betaine transporter BetP. Nature
490, 126–130 (2012).
13. Krishnamurthy, H. & Gouaux, E. X-ray structures of LeuT in substrate-free
outward-open and apo inward-open states. Nature 481, 469–474 (2012).
14. Wahlgren, W. Y. et al. Substrate-bound outward-open structure of a Na
(+)-coupled sialic acid symporter reveals a new Na(+) site. Nat. Commun. 9,
1753 (2018).
15. Myers, E. W. & Miller, W. Optimal alignments in linear space. Comput. Appl.
Biosci. 4, 11–17 (1988).
16. Meng, E. C., Pettersen, E. F., Couch, G. S., Huang, C. C. & Ferrin, T. E. Tools
for integrated sequence-structure analysis with UCSF chimera. BMC
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17. Eswar, N. et al. Comparative protein structure modeling using Modeller. Curr.
Protoc. Bioinformatics Chapter 5, Unit-56 (2006).
18. Massova, I. & Kollman, P. A. Combined molecular mechanical and
continuum solvent approach (MM-PBSA/GBSA) to predict ligand binding.
Perspect. Drug Discov. 18, 113–135 (2000).
Electrophysiology. Injected oocytes were incubated at 18 °C, and after 3–4 days,
two electrode voltage clamp experiments were performed as previously described20
.
Briefly, SGLT inward glucose currents were measured as a function of the external
Na+ (0–100 mM NaCl), sugar (0–100 mM αMDG), and inhibitors (0.001–100 μM).
Steady-state and hSGLT1 capacitive currents were recorded using voltage steps
from a holding voltage, Vh of −60 mV, to voltages in the range of −180 to +50 mV
in 20 mV increments. The apparent affinity of αMDG, K0.5, was estimated from
plots of the inward current as a function of the external αMDG concentration.
Under our experimental conditions, K0.5 is a close approximation of the αMDG
binding constant to the Na+-bound outward conformation of SGLT. The apparent
affinity for Na+, K0.5, was estimated from plots of the αMDG inward current as a
function of external Na+ concentration. For hSGLT1 the interpretation of K0.5 is
more complex owing to the presence of two Na+ binding sites. In this case, the K0.5
is a lumped coefficient for Na+ binding to the two sites in the outward-facing
hSGLT1 conformation. The inhibitor constants for phlorizin and derivatives were
determined by: (i) measuring the effect of increasing concentrations of inhibitor on
the αMDG inward currents measured in presence of NaCl (100 mM) and a αMDG
equivalent to K0.5 of the specific mutant or wild type22, and (ii) from the inhibitor
effect on hSGLT1 capacitive currents in the absence of sugar20. The Na+-to-αMDG
stoichiometry was determined from the reversal potential measurements using the
Gibbs free energy as previously described67. The stoichiometry determinations
were reinforced by estimates of αMDG and Na+ Hill coefficients, and in some
19. Gorraitz, E., Hirayama, B. A., Paz, A., Wright, E. M. & Loo, D. D. F. Active site
voltage clamp fluorometry of the sodium glucose cotransporter hSGLT1. Proc.
Natl Acad. Sci. USA 114, E9980–E9988 (2017).
20. Sala-Rabanal, M. et al. Bridging the gap between structure and kinetics of
human SGLT1. Am. J. Physiol. Cell. Physiol. 302, C1293–C1305 (2012).
21. Jiang, X., Loo, D. D., Hirayama, B. A. & Wright, E. M. The importance of
being aromatic: pi interactions in sodium symporters. Biochemistry 51,
9480–9487 (2012).
cases, by measurement of 22Na and [14C]αMDG uptakes under voltage clamp20, 21
.
Na+ binding site analysis. We compiled a non-redundant protein structural
database via the Advanced Search dialog at rcsb.org with the following search
criteria (3 June 2018): {Chemical ID(s): NA and; Polymeric type is Free and;
Experimental Method is X-RAY and; Resolution is between 0.0 and 2.5 and;
Sequence Length is between 40 and 100000 and; Chain Type: there is a Protein
chain but not any DNA or RNA and; XrayRefinementQuery: refine.ls_R_fac-
tor_obs.comparator = between refine.ls_R_factor_obs.min = 0 refine.ls_R_fac-
tor_obs.max = .3 and; Representative Structures at 50% Sequence Identity}. The
resulting database was comprised of 1750 proteins containing sodium ions, filtered
at 50% sequence similarity and with X-ray resolution ≤2.5 Å and Robs ≤0.3. The
PDB files were downloaded from the RCSB website, and biological assemblies were
created via the python program Prody69. Labels of unique chains (by sequence
similarity) were saved via a custom table of the RCSB search results. The protein
structures were protonated by the program Reduce70 with N/Q/H residues allowed
to flip.
22. Hummel, C. S. et al. Structural selectivity of human SGLT inhibitors. Am. J.
Physiol. Cell. Physiol. 302, C373–C382 (2012).
23. Shimamura, T. et al. Molecular basis of alternating access membrane transport
by the sodium-hydantoin transporter Mhp1. Science 328, 470–473 (2010).
24. Koshy, C. et al. Structural evidence for functional lipid interactions in the
betaine transporter BetP. EMBO J. 32, 3096–3105 (2013).
25. Loo, D. D. F., Hirayama, B. A., Karakossian, M. H., Meinild, A. K. & Wright,
E. M. Conformational dynamics of hSGLT1 during Na+/glucose cotransport.
J. Gen. Physiol. 128, 701–720 (2006).
26. Adelman, J. L. et al. Stochastic steps in secondary active sugar transport. Proc.
Natl. Acad. Sci. USA 113, E3960–E3966 (2016).
27. Hummel, C. S. et al. Glucose transport by human renal Na+/D-glucose
cotransporters SGLT1 and SGLT2. Am. J. Physiol. Cell. Physiol. 300, C14–C21
(2011).
28. Kazmier, K., Sharma, S., Islam, S. M., Roux, B. & Mchaourab, H. S.
Conformational cycle and ion-coupling mechanism of the Na+/hydantoin
transporter Mhp1. Proc. Natl Acad. Sci. USA 111, 14752–14757 (2014).
29. Paz, A. et al. Conformational transitions of the sodium-dependent sugar
transporter, vSGLT. Proc. Natl. Acad. Sci. USA 115, E2742–E2751 (2018).
30. Yamashita, A., Singh, S. K., Kawate, T., Jin, Y. & Gouaux, E. Crystal structure
of a bacterial homologue of Na+/Cl−-dependent neurotransmitter
transporters. Nature 437, 215–223 (2005).
31. Loo, D. D. F., Jiang, X., Gorraitz, E., Hirayama, B. A. & Wright, E. M.
Functional identification and characterization of sodium binding sites in Na
symporters. Proc. Natl Acad. Sci. USA 110, E4557–E4566 (2013).
32. Berman, H. M. et al. The Protein Data Bank. Nucleic Acids Res. 28, 235–242
(2000).
Data availability
Data supporting the findings of this manuscript are available from the corre-
sponding authors upon reasonable request. A reporting summary for this Article is
available as a Supplementary Information file.
Received: 9 October 2018 Accepted: 14 November 2018
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Acknowledgements
This work was supported by NIH grant GM089740 (to M.G.), DK19567 (to E.M.W.),
T32HL7731-25 (supporting N.P.), and GM122603 (supporting H.J.). P.B. was supported
by American Heart Association Post-doctoral Fellowship 18POST33960587 and T.A. by
American Diabetes Association Post-doctoral Fellowship 1-16-PDF-005. Simulations
were performed at the San Diego Supercomputer Center through the support of Grant
MCB-80011 from the Extreme Science and Engineering Discovery Environment. We
wish to acknowledge the generosity of Professor Jean-Yves Lapointe in providing the
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Author contributions
P.B., M.G., and E.M.W. designed the research study. P.B. performed the modeling, virtual
screening, and MD simulations. C.G. performed the electrophysiology. C.G. and T.A.
performed the molecular biology. H.J. performed the chemistry. N.P. carried out the
bioinformatics on sodium sites. R.F. solved the template structures. C.K. and M.P.J.
provided useful insights on the modeled complexes. P.B. and M.G. led the manuscript
writing and all authors approved the final version.
Additional information
Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467-
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