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Cryo-EM structure of ACE2-SIT1 in complex with tiagabine.
Bröer A
,
Hu Z
,
Kukułowicz J
,
Yadav A
,
Zhang T
,
Dai L
,
Bajda M
,
Yan R
,
Bröer S
.
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The pharmacology of amino acid transporters in the SLC6 family is poorly developed compared to that of the neurotransmitter transporters. To identify new inhibitors of the proline transporter SIT1 (SLC6A20), its expression in Xenopus laevis oocytes was optimized. Trafficking of SIT1 was augmented by co-expression of angiotensin-converting enzyme 2 (ACE2) in oocytes but there was no strict requirement for co-expression of ACE2. A pharmacophore-guided screen identified tiagabine as a potent non-competitive inhibitor of SIT1. To understand its binding mode, we determined the cryo-electron microscopy (cryo-EM) structure of ACE2-SIT1 bound with tiagabine. The inhibitor binds close to the orthosteric proline binding site, but due to its size extends into the cytosolic vestibule. This causes the transporter to adopt an inward-open conformation, in which the intracellular gate is blocked. This study provides the first structural insight into inhibition of SIT1 and generates tools for a better understanding of the ACE2-SIT1 complex. These findings may have significance for SARS-CoV-2 binding to its receptor ACE2 in human lung alveolar cells where SIT1 and ACE2 are functionally expressed.
Figure 1. Modulation of SIT1 activity by ACE2.Xenopus laevis oocytes were injected with cRNA for SIT1 (5 ng) B0AT1 (5 ng), B0AT2 (5 ng) and ACE2 (20 ng) alone or in combination or remained uninjected (n.i.) and were incubated for 3–6 days. A, uptake of [14C] leucine in oocytes expressing B0AT1 and/or ACE2 on day 3 and day 4 (n = 11–12 biological replicates). B, uptake of [14C] proline in oocytes expressing SIT1 and/or ACE2 on day 3 and day 4 (n = 12 biological replicates). C, detection of SIT1 and ACE2 in oocytes after 6 days of expression of SIT1 alone or in combination with ACE2 (n = 3 biological replicates, quantification in side panel, effect of ACE2 p = 0.01 paired t test). D, uptake of [14C]proline in oocytes expressing B0AT2 and/or ACE2 on day 3 and day 4 (n = 7–12 biological replicates). Distribution of data (median, 25 and 75 percentile and SD) shown, one-way ANOVA p-values shown as ∗<0.05 and ∗∗∗<0.001 (Scheffé post hoc test).
Figure 2. Inhibitors of SIT1.A, structures of GABA transport inhibitors. B, oocytes were injected with 15 ng SIT1 cRNA and/or 15 ng ACE2 cRNA or remained uninjected (ni). After incubation for 4 days proline uptake activity (100 μM [14C]proline) was determined in the presence and absence of GABA transport inhibitors (100 μM) (n = 8–12 biological replicates). C, dose-response curve for tiagabine at 100 μM and 500 μM [14C]proline in oocytes expressing SIT1-ACE2 (n = 9–15 biological replicates, error bars represent S.D.). D, kinetic parameters of proline transport in oocytes expressing SIT1-ACE2 (n = 8–14 biological replicates, error bars represent S.D.). E, dose-response curve for tiagabine at 100 μM and 500 μM [14C]proline in oocytes expressing SIT1 alone (n = 9–15 biological replicates, error bars represent S.D.). F, oocytes were injected with cRNA encoding SIT1 (15 ng), B0AT1 (10 ng) B0AT2 (12.5 or 25 ng) and ACE2 (1:1 ratio to SIT1 and B0AT1) or remained uninjected (ni). Activity was measured by uptake of 100μM labelled substrate (leucine for B0AT2, proline for SIT1) in the absence and presence of tiagabine (100 μM) (n = 11–14 biological replicates). Distribution of data (median, 25 and 75 percentile and SD) shown, one-way ANOVA p-values shown as ∗∗∗<0.001 (Scheffé post hoc test).
Figure 3. Overall structure of the ACE2-SIT1 bound with tiagabine (TGB).A, Cryo-EM map of the full length of ACE2-SIT1 complex with tiagabine (TGB). ACE2 and SIT1 bound with tiagabine (royal blue) are represented in light coral and light sky blue, respectively. In the insert of the Cryo-EM map of the human ACE2-STI1 complex, the density corresponding to tiagabine is color-coded in royal blue. B, the hydrogen bond network at the tiagabine binding pocket of the SIT1 structure. Hydrogen bonds clusters are indicated by yellow dashed lines. C, hydrophobic interactions formed among Phe250, Leu252, Phe256, Phe262, Val279, Ala22 and Tyr21.
Figure 4. The conformational movement of SIT1 with different states.A, a structural comparison is presented between the apo structure of SIT1 (PDB: 7Y75) and SIT1 bound with tiagabine. The apo structure is depicted in pink, while SIT1 with tiagabine is shown in light sky blue. The alternation in the positions of TM1 and TM6 are illustrated in the middle and right panels. B, structural comparison is provided for SIT1 bound with proline and tiagabine, respectively. The superimposed structures of SIT1 bound with proline are represented in yellow green, SIT1 with tiagabine in light sky blue, and tiagabine itself colored in royal blue.
Figure 5. Structural comparisonofSIT1 and GAT1 bound with tiagabine.A, the superimposed structures of SIT1 with tiagabine (light sky blue), GAT1 bound with tiagabine (PDB: 7Y7Z, bright yellow), and GAT1 with tiagabine (PDB: 7SK2, light purple). B, structural comparison between SIT1 and GAT1 (PDB: 7Y7Z), with tiagabine colored in royal blue and orange, respectively. C, structural comparison between SIT1 and GAT1 (PDB: 7SK2), with tiagabine presented in royal blue and purple, respectively. The gating residues Phe250 and Tyr21 in SIT1, corresponding to Phe294 and Tyr60 in GAT1, are depicted in ball-stick representation.
Figure 6. Expression of SIT1 and ACE2 in human alveolar cells.A, schematic histology of lung alveoli. B, detection of RNA expression of markers of type 1 and type 2 alveolar cells and of SIT1 and ACE2 in primary human alveolar cells by RT-PCR (n = 3 biological replicates). C, transport of 100 μM [14C]proline was measured in the presence and absence of 10 mM pipecolate (Pip), 10 mM MeAIB and their combination to delineate different transport activities (n = 8–10 biological replicates). D, SIT1 and ACE2 were detected by western blotting in human alveolar cells. Membrane proteins were enriched by surface biotinylation and binding to streptavidin beads (samples are separated by an empty lane). Na,K-ATPase was used as a loading control (n = 2).
