Abstract
Zinc transporter 8 (ZnT8) is mainly expressed in pancreatic islet β cells and is responsible for H+- coupled uptake (antiport) of Zn2+ into insulin secretory granules. Structures of human ZnT8 and its prokaryotic homolog YiiP have provided structural basis for constructing a plausible transport cycle for Zn2+. However, the mechanistic role that H+ plays in the transport process remains elusive. Here we present two cryo-EM structures of ZnT8 from Xenopus tropicalis (xtZnT8) captured in the presence of either abundant Zn2+ or abundant H+. Combined with a microscale thermophoresis analysis, our data suggest that binding of Zn2+ to the transmembrane Zn2+-binding site drives xtZnT8 to the outward-facing state. Surprisingly, binding of H+ to xtZnT8 is not sufficient to drive the transporter to an inward-facing state, suggesting that protonation alone is not a determining factor to establish an inward-facing conformation during Zn2+ transport. Instead, the role of protonation appears to unbind and release Zn2+ from the transmembrane site in the outward-facing state of xtZnT8, thus allowing an inward-facing isomerization to occur for the next cycle.
Introduction
Zinc is the second most abundant trace metal in cells and plays critical roles in many biological processes, including cell growth and development, functioning of the central nervous system and the immune system1-3. A variety of enzymes also require Zn2+ for their biological functions1-3. Meanwhile, excessive cytoplasmic free Zn2+ is highly toxic. Therefore, the intracellular homeostasis of Zn2+ is tightly controlled, mainly by two classes of solute carrier (SLC) transporters: the ZRT/IRT-like proteins (ZIPs, or SLC39), and the cation-diffusion facilitators (CDFs, or SLC30), also known as zinc transporters (ZnTs)3-5. These two transporter families mediate influx and efflux of Zn2+ into and out of the cytoplasm of the cell, respectively. Among ZnTs, ZnT8 is mainly expressed in pancreatic islet β cells and is responsible for H+-coupled uptake/antiport of Zn2+ into insulin secretory granules, in which Zn2+ is complexed with insulin in a crystalline form and is co-secreted with insulin6. ZnT8 is often targeted by autoantibodies in type 1 diabetes7,8, whereas mutations and single nucleotide polymorphisms of ZnT8 are associated with type 2 diabetes9-13.
High-resolution structures of the ZnT family were first obtained with its prokaryotic homolog, YiiP from Escherichia coli (ecYiiP)14,15 or Shewanella oneidensis (soYiiP)16-18, revealing a homodimeric configuration of YiiP. The former was captured in an outward-facing conformation while the latter in an inward-facing conformation, based on which an alternating-access model was proposed for Zn2+ transport in YiiP17,19-21. Recently, structures of human ZnT8 (hZnT8) were determined by single-particle electron cryomicroscopy (cryo-EM)22, also as a homodimer (Fig. 1A). Four Zn2+-binding sites were identified in each hZnT8 protomer: one STMD site at the center of the transmembrane domain (TMD), two adjacent SCTD sites in the carboxy-terminal domain (CTD), and one Sinterface site at the interface between TMD and CTD (Fig. 1A). STMD is the primary site for Zn2+ binding and transport, whereas the SCTD sites are formed by residues from both protomers and contribute to hZnT8 dimerization and stability. These three Zn2+-binding sites are highly conserved in the ZnT family (Supplementary Fig. 1), including the prokaryotic YiiP. On the other hand, the Sinterface site is much less conserved. In hZnT8, it is formed by residues from both CTD and a loop region between TM2 and TM322, while in YiiP it is formed solely by residues from the TM2-TM3 loop14,17. In some species like mouse and rat, no Zn2+-coordinating residue (Cys, His, Glu or Asp) is present within the predicted TM2-TM3 loop (Supplementary Fig. 1), further indicating that Sinterface may not be conserved. Nonetheless, it has been hypothesized that Sinterface may increase the local concentration of Zn2+ to facilitate its binding to the STMD site in ZnT822.
The hZnT8 structures were captured in both outward-facing and inward-facing states, allowing construction of a Zn2+ transport cycle for eukaryotic ZnT8 transporters22. In both conformations, the two SCTD sites of each hZnT8 protomer are occupied by two Zn2+ with little conformational change, indicating that the SCTD Zn2+ do not participate directly in the transport cycle. Therefore, a simplified alternating-access model proposed previously could well explain the transport function for ZnT8 in the following steps22 (Fig. 1B). (1) Due to the slightly basic cytosolic pH23,24, a counter-transported H+ is released to the cytosol from ZnT8 in the inward-open state, allowing a cytosolic Zn2+ to bind to the STMD site. (2) Zn2+ binding drives ZnT8 to the outward-facing state (i.e. the inward-to-outward-facing transition). (3) The STMD-bound Zn2+ is released to the granule lumen while the acidic luminal pH25 protonates the STMD site of ZnT8. (4) ZnT8 transits back to the inward-facing state (i.e. the outward-to-inward-facing transition), ready for the next cycle. The model is consistent with the structural and functional data reported for ZnTs regarding H+-coupled Zn2+ binding and transport22,26,27. However, mechanistic details of the H+ function in this process remain elusive.
In this study, we report two cryo-EM structures of ZnT8 from Xenopus tropicalis (xtZnT8) captured in the presence of abundant Zn2+ or abundant H+. Together with a microscale thermophoresis analysis that monitors conformational changes of xtZnT8, our findings help to elucidate the role of protonation during Zn2+ transport in ZnT8.
Results
Cryo-EM structure of xtZnT8 with Zn2+ in an outward-facing state
Based on this model, Zn2+ and H+ appear to be the two driving factors for directional conformational changes in the transport cycle of ZnT8 (Fig. 1B, step 2 and step 4, respectively). Herein, we determined two structures of xtZnT8 in the presence of either abundant Zn2+ (1 mM Zn2+, 3.85 Å, termed xtZnT8-Zn2+) (Supplementary Fig. 2) or abundant H+ (pH 5.5, 3.72 Å, termed xtZnT8-H+) (Supplementary Fig. 3) by cryo-EM, to analyze the Zn2+/H+-driven conformations. XtZnT8 shares 58% sequence identity and 83% sequence similarity to hZnT8 (Supplementary Fig. 1), and it is also organized as a homodimer similarly to hZnT8 and YiiP.
In the presence of Zn2+, xtZnT8-Zn2+ resembles closely the hZnT8-Zn2+ structure (PDB: 6XPE) and adopts an outward-facing conformation in both protomers (Fig. 2A). Structural alignment between individual protomers from xtZnT8-Zn2+ and hZnT8-Zn2+ yielded an RMSD of 1.18 Å with all Cα atoms aligned, while the major deviation came from relatively flexible loop regions of the two proteins. Similar to hZnT8-Zn2+, xtZnT8-Zn2+ has one Zn2+ bound at the STMD site with a tetrahedral coordination, which is formed by His100 and Asp104 from TM2, and His225 and Asp229 from TM5 (Fig. 2B and Supplementary Fig. 1). The STMD residues are highly conserved in eukaryotic species with the 2-His-2-Asp configuration, whereas prokaryotic YiiP proteins have a slightly different 1-His-3-Asp configuration21,28. Like the hZnT8-Zn2+ structure, the STMD-bound Zn2+ in xtZnT8-Zn2+ is accessible by solvent from the luminal side (Fig. 2C), indicating that the transporter is in an outward-open state. This result is consistent with the notion that Zn2+ binding at STMD drives ZnT8 to the outward-facing conformation (Fig. 1B, step 2), which is also observed in YiiP19. Interestingly, this STMD-bound Zn2+ was not released automatically from the STMD site in the outward-open state, suggesting that a Zn2+-releasing mechanism is required for eukaryotic ZnT8. Similar to hZnT8-Zn2+, each protomer of xtZnT8-Zn2+ has two adjacent SCTD sites (SCTD1 and SCTD2), which are formed by the HCH (His45-Cys46-His47) motif from one protomer and multiple Zn2+-coordinating residues from the other protomer, occupied by two Zn2+ (Supplementary Fig. 1 and 4A).
Meanwhile, two major differences were observed between the xtZnT8-Zn2+ and hZnT8-Zn2+ structures. First, the region between the HCH motif and TM1 (termed HCH-TM1-linker) assumed an α helical structure as a continuation of the TM1 helix in xtZnT8-Zn2+. In hZnT8-Zn2+, HCH-TM1-linker was not resolved (Supplementary Fig. 4B), suggesting that this region may adopt a flexible/unordered conformation, which may be involved in the outward-to-inward-facing transition (see the Discussion section). Second, even in the presence of 1 mM Zn2+, no Zn2+ density was observed for the Sinterface site in xtZnT8-Zn2+ (His131/His350) (Supplementary Fig. 4C), which corresponds to His137/His345 in hZnT8 (Supplementary Fig. 1). This result suggests that the function of Sinterface is less conserved.
Cryo-EM structure of xtZnT8 at a low pH still in an outward-facing state
Then, we analyzed the structure of xtZnT8-H+ at pH 5.5, which is close to the internal pH of insulin secretory granules (5∼6)25. It is tempting to postulate that binding of H+ to STMD of ZnT8 would drive the transporter to the inward-facing state (Fig. 1B, step 4), which serves as a H+-coupling mechanism for Zn2+ antiport. However, intriguingly, the xtZnT8-H+ structure also adopts an outward-facing conformation, similar to xtZnT8-Zn2+ (Fig. 2D). This data suggests that protonation alone of xtZnT8 is not sufficient to drive the transporter to the inward-facing conformation.
Superposition of the two xtZnT8 structures yielded an all-atom RMSD of 0.58 Å, indicating that xtZnT8-H+ and xtZnT8-Zn2+ are very alike (Fig. 2D). The most prominent difference between the two structures is that the side chain of His100 in xtZnT8-H+ is pointing toward the luminal side, thus disrupting the tetrahedral coordination for Zn2+ at the STMD site (Fig. 2E). Two histidines of STMD (His100 and His225) are likely protonated at pH 5.5, which would cause disruption of Zn2+ coordination. Therefore, protonation of xtZnT8 in the outward-facing state likely causes unbinding and release of Zn2+ from STMD, as proposed previously for hZnT822 and YiiP17,19. Consistently, no Zn2+ density was observed at STMD in xtZnT8-H+ (Fig. 2F). Another difference between the two xtZnT8 structures is that a portion of TM2 (Ala93-Ala98), which is close to the luminal side, appears more flexible in xtZnT8-Zn2+ than in xtZnT8-H+, and therefore is less visible in the former map (Supplementary Fig. 4D). This is reminiscent of hZnT8-Zn2+ (PDB: 6XPE) and the structure of its STMD double-mutant (hZnT8D110N/D224N, PDB: 6XPD), which mimics a protonated state at the positions 110 and 224, and doesn’t bind Zn2+ at the mutated STMD site22. Compared to hZnT8D110N/D224N, the hZnT8-Zn2+ structure also shows a less defined TM2 portion near the luminal side (Supplementary Fig. 4E). These results suggest that Zn2+ binding at STMD increases the flexibility of this near-lumen TM2 region, whereas H+ binding at STMD stabilizes this region. Different conformations of the near-lumen TM2 region may play a role in regulating accessibility and binding of Zn2+/H+ to the STMD site in ZnT8.
Conformational changes of xtZnT8 during Zn2+/H+ binding
We then used the microscale thermophoresis (MST) technique to probe conformational changes of xtZnT8 during Zn2+/H+ binding. MST measures the thermophoretic mobility of a protein in solution, which is mainly affected by its shape and size29, and so the conformational information of the protein may be inferred. First, we tested the effect of Zn2+ binding to a wild-type xtZnT8 sample, which has been purified in the absence of Zn2+ at a physiological pH of 7.5. MST analysis showed that the plot of normalized fluorescent signals against Zn2+ concentrations readily fit an inverse S-shaped curve (Fig. 2G), with a higher level of fluorescent signals at lower Zn2+ concentrations and a lower level of fluorescence at higher Zn2+ concentrations. The equilibrium dissociation constant (Kd) for Zn2+ is 4.91 ± 0.39 µM, slightly larger than previously reported affinity of STMD for Zn2+ in YiiP14,28. This data indicates that xtZnT8 transits from a lower thermophoretic mobility state (termed “low-mobility” state) to a higher thermophoretic mobility state (termed “high-mobility” state) upon Zn2+ binding (Fig. 2G), which likely corresponds to the xtZnT8-Zn2+ structure (outward-facing state) (Fig. 2A). Therefore, it is reasonable to postulate that the “low-mobility” state may correspond to an inward-facing-like state.
Then we monitored conformational changes of purified xtZnT8 during H+ binding in the absence of Zn2+. Surprisingly, titrating xtZnT8 with H+ also yielded an inverse S-shaped MST curve similar to the Zn2+ titration (Fig. 2H), suggesting that protonation of xtZnT8 also transits the transporter from the “low-mobility” state (inward-facing-like) to a “high-mobility” state (outward-facing), which is consistent with the xtZnT8-H+ structure (Fig. 2D). The equilibrium dissociation constant (Kd) for H+ is 0.28 ± 0.02 µM, which corresponds to ∼pH 6.55, close to the pKa of histidine side chain (∼6.0). This data further indicates that H+ binding to xtZnT8 is less likely a driving factor for the inward-facing conformation.
Discussion
In this study, we provide insight into the driving force of ZnT8 to its inward-facing state (Fig. 1B, step 4). From a thermodynamic perspective, the outward-to-inward-facing transition of ZnT8 is achieved either by an external driving factor with energy input, or by an internal thermodynamic equilibrium of conformational isomerization. Apparently, counter-transport of H+ along its electrochemical gradient would be a tempting driving factor to couple to the antiport of Zn2+ in ZnT8. However, our cryo-EM structure of xtZnT8-H+ (Fig. 2D) and its thermophoretic mobility analysis (Fig. 2H) refute protonation as the determining factor that drives the outward-to-inward-facing transition in ZnT8. Therefore, our data would favor the second possibility that the inward-facing conformation could be achieved by thermodynamic conformational equilibrium of ZnT8. Meanwhile, our data also show that Zn2+ binding to STMD drives xtZnT8 to the outward-facing state (Fig. 2A and 2G). So, protonation of the STMD site to disrupt Zn2+ coordination and to release Zn2+ would be a prerequisite for the inward isomerization of ZnT8. This hypothesis is consistent with a heterogeneous conformation observed for hZnT8 captured in the absence of Zn2+ (PDB: 6XPF), which contains one protomer in the outward-facing conformation while the other in the inward-facing conformation with no Zn2+ in the STMD site22.
Furthermore, a particularly intriguing region of eukaryotic ZnT8 proteins is the HCH motif and HCH-TM1-linker. The HCH motif participates directly in the formation of the SCTD sites and remains mostly unchanged during the Zn2+ transport cycle22. However, TM1 moves substantially during the outward-to-inward-facing transition of ZnT8. For example, Glu66 near the amino-end of TM1 in hZnT8 moves > 10 Å away from the dimer center during the outward-to-inward-facing transition (PDB: 6XPF, protomer B to A) (Supplementary Fig. 4F). The nearly fixed HCH motif and the highly mobile TM1 indicate that HCH-TM1-linker has to unwind from an α helical structure (outward-facing state) to an unordered loop structure (inward-facing state), to accommodate the increased distance between the HCH motif and the TM1 helix (Supplementary Fig. 4F). Consistently, HCH-TM1-linker was not resolved in the hZnT8 structures (Supplementary Fig. 4B), suggesting that this region may adopt a flexible/unordered conformation. It is noteworthy that prokaryotic YiiP proteins have no HCH motif, and their SCTD sites do not involve any residues before the TM1 helix14,17. Therefore, compared to prokaryotic YiiP proteins, eukaryotic ZnT8 proteins have one more structural coupling (i.e. HCH-to-TM1) between the CTD and TMD regions, which may provide additional mechanism for regulating conformational changes as previously suggested22.
Data Availability
The cryo-EM density maps for xtZnT8-Zn2+ and xtZnT8-H+ have been deposited in the Electron Microscopy Data Bank under the accession IDs EMD-33619 and EMD-33620, respectively. The atomic coordinates of the xtZnT8-Zn2+ and xtZnT8-H+ structures have been deposited in the Protein Data Bank under the accession codes 7Y5G and 7Y5H, respectively. Three previously published hZnT8 structures and one soYiiP structure used in this study are available in the Protein Data Bank under accession codes 6XPD, 6XPE, 6XPF and 5VRF. Source data of Kd values are provided with this paper.
Author Contributions
Senfeng Zhang, Functional study, Cryo-EM sample preparation, Data analysis; Chunting Fu, Functional study, Cryo-EM data processing, Data analysis; Yongbo Luo, Cryo-EM data collection, Data analysis; Qingrong Xie, Functional study, Data analysis; Tong Xu, Functional study; Ziyi Sun, Conceptualization, Supervision, Data analysis; Zhaoming Su, Supervision, Data analysis; Xiaoming Zhou, Conceptualization, Supervision, Cryo-EM model building and validation, Data analysis. Ziyi Sun and Xiaoming Zhou wrote the manuscript with input from all authors.
Competing Interests
The authors declare no competing financial interests.
Acknowledgements and Funding
Cryo-EM data were collected at SKLB West China Cryo-EM Center and were processed at the Duyu High Performance Computing Center of Sichuan University. This work was supported in part by the National Natural Science Foundation of China (NSFC) grant 31770783 to Xiaoming Zhou and 32070049 to Zhaoming Su, the 1.3.5 Project for Disciplines of Excellence grant by West China Hospital of Sichuan University to Xiaoming Zhou and Zhaoming Su, and Ministry of Science and Technology of China grant 2021YFA1301900 to Zhaoming Su.