Pendrin forms symmetric homodimer in the presence of Cl−
The full-length mouse pendrin with a N-terminal affinity tag were expressed in HEK293E cells and purified using glycol-diosgenin (GDN) in the presence of 150 mM NaCl. The purified protein was concentrated to 1.7 mg/ml, and immediately used for cryo-EM grids freezing and subsequent data collection. The overall dimer structure was determined to 3.4 Å without symmetry, showing two protomers are almost identical. C2 symmetry was then applied for further refinement, which improved the reconstruction map to 3.3 Å resolution, allowing the unambiguous interpretation of the cryo-EM density map to atomic model. During protein purification and cryo-EM data processing, no other oligomers was observed, indicating pendrin forms a dimer as other family members 20–23 (Extended Data Fig. 1).
Pendrin purified in 150 mM NaCl (Pendrin-Cl−) forms a domain-swapped homodimer (Fig. 1C and 1D). Each protomer is composed of a N-terminal domain (NTD) (residues 1–79), a transmembrane domain (TMD) (residues 80–514), a sulfate transporter and anti-sigma factor antagonist domain (STAS) (residues 535–735) containing the intervening sequence (IVS) (residues 596–650) and a C-terminal domain (CTD) (residues 736–780). Residues 1–17, 596–650 and 738–780 are missing in the model due to the flexibility. The NTD and STAS domains from each protomers interchange and form a dimeric knob at the cytoplasmic side of plasma membrane. On the other side, a spiky loop from each protomer sticks out from the membrane. The TMD follows the so-called UraA fold 24,25, with 14 TMDs divided into two inverted repeats representing the core (helix TM 1–4 and 8–11) and gate regions (helix TM 5–7 and 12–14) (Fig. 1b and 2a). As with human prestin (PDB ID: 7LGU), significant cholesterol density was observed between the two TMDs (Fig. 1c,d).
The anion-binding site between the core and gate regions within the TMD
In pendrin-Cl− structure, an unambiguous Cl− density is observed and defines the anion-binding site (Fig. 2c,d). The Cl− is embraced in the cavity between two short transmembrane helices TM3 and TM10, which is positioned at the apex of the inward-open intracellular vestibule (Fig. 2f,g).
The Cl− is mainly coordinated by the TM3-TM10 dipoles (Fig. 2g). S408 and Y105 directly interact with Cl− by forming ionic bonds (Fig. 2c). The side chains of Y105 and Q101 form the partially positive charged binding pocket, which is quite common across the family 20–23. Additionally, the positively charged residue R409 indirectly stabilize Cl− by interacting with the nearby residue Q101, which in turn interacts with S408. Mutation of R409 would alter anion binding and impair anion entrance and translocation 26, consequently R409H 27 leads to reduction of Cl− and I− transport. A406, L407 and three sequential hydrophobic residues P140, F141, P142 hallmark the partial hydrophobic feature of the pocket (Fig. 2b). Mutations of P140H 27 and P142R 27, though contributing positive charging, would cause the loss of anion transport or exchange activity, which may result from local structure disruption. Mutations of F141S 26 and P142R 27 were supposed to loss pi-pi stacking between F141 and P142 which will result into hearing problems. Additionally, residue N457 from the gate region also helps to stabilize the pocket by forming a hydrogen bond with Q101 (Fig. 2d).
Taken together, the local pocket architecture is crucial for anion binding and therefore important for pendrin’s anion transport and exchange function. We performed the electrophysiological recording and compared mutants P142A, Y105A, S408A and N457A with wild-type pendrin. Consistent with the structural findings, all four mutations significantly influenced the transport of Cl−( Fig. 2e and Extended Data Fig. 1). Particularly, P142A and N457A showed severe reduction of Cl− current, and both sites have corresponding pathogenetic mutations 26,27. Both residues are supposed to be essential to the function of pendrin. Coincidently, the allelic residue of pendrin P142 is Alanine in prestin and SLC26A9. Therefore, P142 is assumed to have a specific role in pendrin to regulate Cl− transport and exchange.
HCO3− binds the same pocket as Cl−
In order to obtain HCO3−-bound Pendrin (Pendrin-HCO3−), pendrin in Cl− buffer was exchanged to a buffer containing HCO3− using gel-filtration and frozen for cryo-EM data collection. The cryo-EM structure was determined to 3.5 Å resolution with C2 symmetry. Similar as pendrin-Cl−, pendrin-HCO3− also forms symmetric homodimer with two inward-open protomers. Anion HCO3− was clearly observed as well in the binding pocket, which is about 2 Å away from Cl− binding position (Fig. 2h). Though Y105 is too far to interact with, HCO3− forms extra hydrogen bonds with the backbone nitrogen of S408 and L407.
Pendrin forms asymmetric homodimer in the presence of two different anions
As abovementioned, in the presence of a single anion, Cl− or HCO3−, only inward-open pendrin was observed in our cryo-EM structures. The inward-open state may represent the most thermodynamically stable conformation but not dynamically favorite for anion transport and exchange. However, in physiological circumstance, pendrin is exposed a mixture of different anions. As an exchanger, pendrin could bind either anion (of the exchange pair) at both inward- and outward-open states. Therefore, we investigated pendrin in a buffer containing two different anions, such as Cl−/HCO3−, Cl−/I−, or HCO3− /I− anion pairs.
Pendrin purified in 100 mM NaCl was mixed with the same volume of 300 mM NaHCO3, resulting a buffer containing 50 mM NaCl and 150 mM NaHCO3. The sample was then concentrated and used for cryo-EM data collection. The data was processed without symmetry. Surprisingly, the cryo-EM structural determination of pendrin in the presence of Cl−/HCO3− (pendrin-Cl−/HCO3−) mixture revealed three distinct states. Only ~ 15% percent particles remained symmetric inward-open state. Another 15% percent particles represent the symmetric outward-open state in which the cavity of the binding pocket is connected to the outside. This outward-open conformation of TMD is similar to that of SLC4A1 (anion exchanger 1, AE1) TMD crystal structure which is stabilized in outward state by antibody 25. Interestingly, the majority of particles (~ 70%) form asymmetric homodimers composed of one inward-open protomer and the other outward-open protomer (Fig. 3a). In this asymmetric dimer, a clear anion density was observed in the inward-open protomer, which is assigned to the identical Cl− occupying position as that of pendrin-Cl symmetric structure. While in the outward-open protomer, there were two individual densities identified in the cavity above the binding pocket (Fig. 3b). And the open cavity forms a positive charged docker, in which the side chains of K237 and Q230 are close enough to interact with two anion densities, respectively (Fig. 3c). This docker would stabilize and bind anions, in which way it is believed to help to recruit anions from the outside.
Similarly, the pendrin sample in the anion pair of Cl−/I− (1:3) (pendrin-Cl−/ I−) was prepared and used for structure determination. Symmetric inward-open and asymmetric homodimers were observed with approximate percentage of 75% and 25%, respectively. Finally, the structures of pendrin in HCO3−/I− (1:3) (pendrin-HCO3−/I−) was determined, which also revealed two distinct states: 75% symmetric inward-open and 25% asymmetric states (Extended Data Fig. 3). The combination of Cl− and HCO3− provides the most varieties of conformations, hereafter pendrin-Cl−/HCO3− structures are used for illustration.
Remarkable changes among the TMD of the inward-open and outward-open pendrin protomers were observed. The major difference is the relative position of the gate and the core regions (Fig. 3d). When the gate regions are superimposed, the core region in the outward-open state (versus the inward-open state) rotates roughly by 15° and translates 9 Å towards the extracellular side (Fig. 3e-g). This movement is characteristic in SLC26 family members and the relative value is the biggest among published data 20,21,23. While in this movement, the binding pocket carried the anion towards outside, and release it to the outward cavity (Fig. 3e). And these two individual densities we captured at the same time reveals an exchanging moment that one anion reaches the outward cavity from inside and another anion is recruited by local charge to reverse the process simultaneously.
In addition, the density of relatively stable cholesterol can be seen in all conformations, despite the completeness differences. Since cholesterol is believed to influence the localization and diffusion of prestin in plasma membrane 28, this may be characteristic for the interactions between SLC26 family members and plasma membranes.
The STAS domain modulates pendrin transport function
The STAS domain takes 4 β-strands as the core which is surrounded by 4 α-helixes, extending the lateral helix Cα1b to link IVS (Fig. 3h). The starting residues 515–545 of STAS domain form a long loop region, however, it is well resolved due to the interactions with Cβ3, Cα5 and NTD. The pathogenetic mutations we investigated with electrophysiology, Y530H, T721M, and D724N, which severely affected Cl− transport, are located around this loop region 29 (Fig. 4b,c). This indicate that the structural stability of the STAS domain has dramatic influence on the transport function. Similar to prestin and SLC26A9, pendrin’s dimerization interface is mainly formed by STAS domains. The STAS domains of two protomers contact closely face to face on a relatively flat surface, and two NTD’s Nβ1s parallel inversely below the STAS domains. In the dimerization interface, S552 forms a hydrogen bond with S666 from the other protomer, which would be destroyed by pathogenetic mutation S552I 29 (Fig. 4e,f). Referring to the complete loss of Cl− permeability in electrophysiological experiment, we hypothesized that the instability of dimerization would disable the transport function of Pendrin (Fig. 4d).
The STAS of one protomer not only interacts with the STAS of another protomer, but also contacts the TMD of another protomer, as the platform formed by Cα1, Cα1b, and Cα2 is directly below the anion transport pathway (Fig. 4b). Moreover, an anion pre-binding site between loop Cβ3-Cα1 and loop Cβ4-Cα2, according to the rat prestin X-Ray structure 30, forms an interface with TM12, TM13 and TM14. Pathogenetic mutations Y556C, F667C, and G672E of this region, would significantly affect Cl− and I− transport 27. When the TMDs were superimposed, the helix Cα1b rotated about 6° from inward state to outward state (Fig. 3i,j). Interestingly, Cα1b provides a completely positively charged platform, therefore the movement may significantly contribute to the initiation of anion transport within STAS domain, subsequently facilitating the anion transport or exchange (Fig. 4a).
Cl−/HCO3− and Cl−/I− exchange function of pendrin
To verify the Cl−/HCO3− exchange function of pendrin, we used pH sensitive fluorescent probe BCECF to reflect HEK293T intracellular concentration of HCO3− in different bath 31. And for Cl−/I− exchange function, halides-sensitive EYFP with different sensitivity to Cl− and I− was co-transfected with pendrin to detect the intracellular changes of Cl− and I− concentration 32. As a positive control, the wild type pendrin showed remarkable exchange functions in both experiments (Fig. 5H and 5M). Thereafter, pathogenetic mutations reported to affect the exchanger function (E303Q, F335L, G209V and G672E) were tested and distinct responses were detected (Fig. 5a).
E303 is located at the core-gate interface, although it is far from the anion pathway, mutation E303Q still lose anion exchange capability (Fig. 5e). According to both exchange experiments, E303Q is no longer permeable to Cl−, resulting in the blocked exchange, but still retains the permeability to I− (Fig. 5f,g,k,n). Glutamine would reverse the nearby surface charge (from negative to positive), thus affecting the thermodynamics of conformational change. F335 is located at the protein-lipid interface and appears to have strong interactions, as excess lipid density is seen next to F335 in cryo-EM map (Fig. 5d). In fluorescence experiments, F335L was substantially weakened on both exchanges (Fig. 5f,g,l,o). Structurally, the side chain of leucine may disrupt protein-lipid interactions, thereby impairing the allosteria of TMD. It may indicate that relatively immobilized protein-lipids interactions are essential for the normal function of pendrin. G209 is located on the interface between TMD and STAS domain, and pendrin G209V was reported to located on plasma membrane (PM) and show severe reduction of I− transport 26 (Fig. 5b). However, our fluorescence assays not only demonstrated the functional impairment but also displayed intracellular localization of pendrin G209V (Fig. 5f,i). According to the cryo-EM structure, side chain of valine would increase steric hindrance of core-gate interface and contribute the positive surface charge. These changes might influence the allosteria of TMD and pendrin localization on plasma membrane. G672, conserved with Rat Prestin, is located at the hypothetic pre-binding site in the STAS domain (Fig. 5c). In Cl−/I− exchange assays, G672E lost I− transport capacity, but maintains Cl− permeability (Fig. 5f,j). The long side chain of glutamic acid would extend into the cleft, perhaps altering pendrin’s anion selectivity. In summary, the pendrin structures provide a plausible guideline for us to understand pathogenetic mutations at a molecular level.
Mapping of critical pathogenetic mutations
Pendrin is the best-studied family member due to the enormous mutations that have been implicated in Pendred syndrome. These mutations showed a very dense distribution present in almost every region of pendrin 29. Notably, a single missense would lead to dysfunction or loss of function. Our cryo-EM pendrin structures make it possible to rationalize previous mutational studies. Of all the identified mutations, we analyzed those with a well-defined cellular localization verified by experiments (Table S3). Therefore, we expect to explain how PM localization dysfunction is derived, and why membrane-localized pendrin mutants cannot transport anions properly.
There are six missense variants, which still secure pendrin PM localization, vary in function impairment. R409H located in the binding pocket changes the hydrogen bond that guarantees stability, causing reduction of Cl− and I− transport 26. Anion access pathway related mutation E303Q would reverse local charge to cause function impairment. Two mutation variants apart from anion exchange pathway, F335L would influence the protein-lipid interaction, and G209V could affect core-gate interface stability, therefore they could affect exchange function as well. The rest two S28R and C565Y variants remain unclear on function impairment interpretation.
Mostly, 14 out of the 22 missense variants would mis-localize in the endoplasmic reticulum (ER) or cytoplasm. Among these 7 variants, in TMD (G102R, P123S, V239D, E384G, N392Y, L445W, and G497S), introduce new steric hindrances between different TM helixes, which would alter the position and orientation of the helixes, thus eventually disordering the overall arrangement of core and gate regions. Besides, V138 is closely connected to the binding pocket, and mutation V138F will affect organization of the loop next to TM3. Similarly, T410M providing a large side chain will also affect the loop next to TM3. For R185T at the core-gate interface and L236P at the protein-lipids interface, the charge change may lead to local misfolding. It is noteworthy that mutations in intracellular STAS domain also affect pendrin’s PM localization. Y530H/S, T721M and H723R are concentrated at the bottom of pendrin, participating in the stabilization of STAS domain and NTD, and also determining the orientation of Cα4. Additionally, Y556C and G672E in TMD-STAS interface would only partially affect PM localization. This indicates that these mutations on the interface modify local stability which subsequently impair PM localization.
Structural comparison of pendrin, prestin and SLC26A9
The overall fold of pendrin is similar to prestin 20,23 and SLC26A9 21,22, however, the atomic cryo-EM structures of pendrin reveal its intrinsic features of asymmetric homodimer as an exchanger, given the symmetric homodimers resolved for all other SLC26 family members 20–23. The structures of mouse SLC26A9 have two states: inward-open (PDBID: 6RTC) and intermediate (PDBID: 6RTF) states 21. The dolphin prestin structures include several states, including an inward-like prestin- SO42− (PDBID: 7S9C), intermediate-like prestin-Cl− (PDBID: 7S8X) and several other states in between these two states 23.
When superimposing the inward-state protomers, the TMD regions of pendrin, prestin and SLC26A9 are relatively similar (RMSD < 1.3Å, Table S2). The major difference is that the STAS domain of SLC26A9 has a distinct angular offset from pendrin and prestin, while the latter two basically overlap with each other.
Comparing outward-open pendrin with prestin and SLC26A9 in various intermediate states, we could observe more differences. Among all SLC26 structures, outward-open pendrin’s core region has the largest rotation, and its anion binding pocket is also the uppermost and close to the outward-open state of AE1 (PDBID: 4YZF, Fig. 6a). When focused on anion binding pocket, within a conserved framework, residue differences were found at key sites between pendrin, prestin and SLC26A9. Sequence alignment showed that pendrin residues Q101, F141, L407 and S408 are invariant, but not Y105, P140, P142 and R409 (Extended Data Fig. 6). Different from the allelic residue of prestin and SLC26A9’s phenylalanine, pendrin Y105 has the phenolic hydroxyl group, which increases the charge of binding pocket and enhances the electrostatic attraction to anions. Pendrin’s P140-F141-P142 hydrophobic fragment has slightly different allelic residues in prestin and SLC26A9, PFA and TFA, respectively. The diversity of these three consecutively amino acids tunes the surface shape and charge of the pocket, and may cause differences in interaction intensity between the pocket and anions. Finally, pendrin R409 is conserved in most SLC26A members except SLC26A1, SLC26A2 and SLC26A9. In SLC26A1 and SLC26A2 it is replaced by Lysine, which might be related to the specific function in SO42− transport; while in SLC26A9, this residue was replaced by Valine. Within the binding pocket, pendrin R409 provides the only positive charge and forms multiple hydrogen bonds with neighboring residues for stability, as does in Prestin. However, Valine in SLC26A9 only has a hydrophobic short side chain, which weakens the overall positive charge of pocket. In summary, surface charge of the binding pocket may directly define the anion selectivity, which may be the reason why these three members are so different in terms of anion selectivity and function.
Inverted alternate-access exchange mechanism
The competitive binding of anion pair in binding pocket, which pivot points the exchange cleft’s access direction, determines the conformation of the TMD and probably modulates that of STAS as well. Here, we have resolved a wide spectrum of pendrin structures flash frozen under various conditions. On the basis of structural analysis together with physiological and biochemical assays, we hypothesized the working model of pendrin as an anion exchanger. The symmetric inward-open conformation most likely represents the energy favorite state, existing in the single binding-anion circumstance, Cl− for instance (Fig. 6e). When bicarbonate was added to a high concentration, HCO3− replaces Cl− from the same binding site in protomer B. This causes local conformation changes, with the binding pocket of protomer B translating towards the extracellular side, while protomer A stays unchanged (Fig. 6f and Supplementary Video 1). To accommodate this change, the core region rotates about 15° against the gate region, indicating the elevator transport mechanism 21 (Fig. 6c). Eventually this conformational change would end up with the outward-open state (Fig. 6d), at which HCO3− diffuses out and Cl− binds to reverse the exchange process (Fig. 6f). The significant rotation of Cα1b of protomer B, occurring at the end stage of anion releasing prior to anion uptake, may facilitate the on-set of protomer A’s anion secretion process (Fig. 6h). Therefore, the coincidence of secretion and uptake in this asymmetric homodimer, shapes the molecular basis of electroneutral exchange of pendrin with the so-called inverted alternate-access mechanism.
To the best of our knowledge, the asymmetric structure was never observed before for any other anion exchanger. Moreover, some important pathogenetic mutations were mapped on the structure, functional studies were also performed to interpret the structure-function relationships. All above-mentioned would provide a framework for us to understand more pathogenetic mutations and could shed light on therapeutic discovery.