Zrt/Irt-like protein (ZIP) plays a basic role in metal metabolism/homeostasis, and is widely involved in many physiological and pathological processes. The lack of high-resolution structure of ZIP hinders the understanding of metal transport mechanisms. We report the crystal structure of two prokaryotic ZIPs (Cd2 + at 2.7 Å and Zn2 + at 2.4 Å) with a metal substrate in the lipid cubic phase. The structure reveals a novel 3 + 2 + 3TM structure, and a closed inward open conformation on the outside of the cell. The two metal ions were trapped in the middle of the membrane, accidentally forming a binuclear metal center. The structure of Zn2 + substitution indicates that the function of the two metal binding sites is asymmetric, and also reveals the way of zinc release. Pathogenic mutations, structure-directed mutagenesis, and cell-based zinc transport analysis patterns indicate that binuclear metal centers are essential for human ZIP4. A metal transport mechanism for the ZIP of Bordetella bronchis is proposed, which may be applicable to other ZIPs.
Zinc is an essential trace element in life. It is estimated that 10% of the proteins (about 3000 proteins) encoded by the human genome are zinc-binding proteins (1). Zinc is widely involved in various biological processes and plays a particularly important role in enzyme catalysis, gene regulation and macromolecular stability. Zinc is also an emerging signal molecule that regulates physiological and pathological events (2). Therefore, strict and precise control of intracellular zinc levels is required. In mammalian cells, the Zrt/Irt-like protein (ZIP) family [solute carrier family 39A (SLC39A)] and the ZnT family are responsible for the inflow and outflow of zinc, respectively (3-5). In humans, a total of 14 ZIPs have been identified, with different tissue/cell distributions and unique physiological functions (6). In particular, ZIP4 is essential for embryonic development and is specifically responsible for the absorption of zinc in the diet. Abnormal mutations in ZIP4 can lead to a fatal genetic disease, enteropathic enteropathitis (AE) (7, 8). Overexpression of ZIP4 is associated with pancreatic cancer (9). In order to understand the structural basis of ZIP4 dysfunction caused by mutations that cause AEs, and to facilitate the drug discovery of ZIP4 inhibitors, we previously solved the crystal structure of the extracellular domain (ECD) of mammalian ZIP4 (10), proving that ZIP4-ECD is Key regulatory areas for optimal zinc transportation. Here, we solved the crystal structure of pronuclear ZIP, which provided the first structural framework for understanding the metal transport mechanism of ZIP.
The ZIP family consists of thousands of integral membrane proteins in the three kingdoms of life, and all of its members share a conserved transmembrane domain (TMD) with eight transmembrane helices (TM). In a thorough screening of candidates for crystallographic studies, the ZIP of Bordetella bronchis (BdZIP) exhibited ideal solution behavior in detergents. BbZIP is purified in the presence of cadmium ions (Cd2 +), which is reported to be a substrate of BbZIP (11). However, purified proteins in the form of apolipoproteins tend to form aggregates, indicating that substrate binding is necessary for protein stability in detergents. Since Cd2 + binding protein seems to be more stable than Zn2 + binding protein, we chose Cd2 + in the following structural studies. Although detergent-solubilized proteins crystallize under various conditions, the crystals are difficult to diffract. Then, we switched to crystallization in the lipid cubic phase (LCP), which provides a nearly natural membrane environment for integral membrane proteins. The crystal diffraction effect obtained in 100 mM CdCl2 LCP with neutral pH is better than 3.1Å, and the gentle dehydration treatment further improves the resolution. We finally solved the structure by using selenomethionine (SeMet) substituted crystals at 2.7Å through single wavelength abnormal dispersion (SAD) (Table S1 and Figure S1).
A BbZIP molecule is observed in an asymmetric unit. The crystal packing analysis does not support strong interaction with neighboring molecules, and neighboring molecules are in the opposite direction (Figure S2). BbZIP is unlikely to form antiparallel dimers in the cell membrane because it has more TMs than the rarely reported antiparallel (or dual) transporters, which are usually very small, with only four TMs (12). Note that freshly purified BbZIP is usually run in two forms on size exclusion chromatography columns, with apparent molecular weights of ~160,000 and ~110,000 (Figure S3). The larger species (160 kDa) matches the reported BbZIP dimer in n-dodecyl-β-d-maltopyranoside (DDM) (11), and the smaller species (110 kDa) is most likely Corresponds to the monomer form, because theoretically the weight of the molecule BbZIP is 31,000. It is worth noting that both substances can be crystallized under the same conditions, and the current structure represents the conformation of the monomer state.
The structure of BbZIP shows that eight TMs form closely related helical bundles (Figure 1A), where TM2, TM4, TM5 and TM7 create an inner bundle surrounded by the other four TMs (Figure 1B), representing a novel overall fold Search for membrane proteins based on the similarity of three-dimensional protein structures on the Dali server (13). TM2 is unusually long (36 amino acid residues) with a knot in which the helical structure is partially unwound due to the conserved proline residue (P110) (Figure S4). TM4 and TM5 are also similar to the invariant proline residues (h means hydrophobic residues) in the metal-binding motifs “177HNhPEG182″ and “207QD/NhPEG212″, respectively. Further structural inspection revealed the symmetrical relationship in the structure. The first three TMs (TM1 to TM3) are symmetrically related to the last three TMs (TM6 to TM8) through a pseudo double axis, which is roughly parallel to the assumed membrane plane (Figure S5). TM4 and TM5, which are also symmetrically related through the same axis, are sandwiched by two 3-TM repeats. Therefore, the BbZIP structure shows an unusual 3 + 2 + 3TM architecture, which is unprecedented for transporters.
(A) Side view of BbZIP in the presumptive film with marked TM (α1 to α8). The protein is displayed in cartoon mode and colored in rainbow colors, with blue at the N end and red at the C end. The bound Cd2+ ions are shown as yellow-brown spheres. The dashed lines indicate disordered interhelical loops (the loop between TM3 and TM4 and the loop between TM7 and TM8). (B) The top view of BbZIP in the direction indicated by the black arrow in (A). The two Cd2 + binding sites in the transport pathway are labeled M1 and M2, respectively. (C) A cross-sectional view of the electrostatic potential diagram of BbZIP. Two highly negatively charged cavities are indicated by arrows. Note that the path from the entrance cavity to the metal binding site is blocked. (D) The electrostatic potential diagram of the outlet cavity.
In order to avoid exposure of hydrophobic residues to an aqueous environment, when the entire spiral bundle of BbZIP is embedded in the membrane, it must be tilted significantly (Figure 1, A and C and Figure S6), which is also supported by crystals. Monoolein in the lipid bilayer (Figure S2). According to the established topology, where both the N- and C-termini of ZIP are exposed to the extracellular space (6), the putative transport pathway outside the cell is blocked by highly conserved hydrophobic residues from the inner helix bundle TM (from TM2 M99 and A102, L200 and I204 from TM5, and M269 from TM7) (Figure 1, B and C, and Figure S4). The invariant S106 on TM2 is located at the bottom of the shallow and negatively charged inlet cavity. On the cytoplasmic side, the opening of the internal spiral bundle leads to an opening of the outlet cavity with a high negative electrostatic potential (Figure 1D). Therefore, the structure of BbZIP represents an inward conformation.
In the BbZIP structure crystallized in the presence of CdCl2, four heavy metal binding sites (M1 to M4) were identified (Figure 2, A and B). The abnormal signal derived from the data set collected at 1.81Å is much stronger than the abnormal signal collected at 0.98Å, excluding the presence of zinc, copper, cobalt, iron and nickel, but consistent with Cd2+. In order to further determine the identity of the bound metal, we performed an inductively coupled plasma mass spectrometry (ICP-MS) experiment on the high-purity BbZIP eluted from two consecutive desalting columns to remove free metal ions in the sample. The result showed that Cd2 + Is the most abundant transition metal (Figure S7). Compared with the hydrated Cd2 + ions at M3 and M4 at the membrane-water interface, the dehydrated Cd2 + ions at M1 and M2 are trapped by highly conserved residues, passing through about half of the membrane, and these residues are mainly derived from TM4. 177HNhPEG182 motif and “207QD / NhPEG212″ motif on TM5 (Figure 2, A and B, and Figure S4). The two Cd2 + ions at M1 and M2 are separated by 4.4 Å and are bridged by E181 on TM4 to form a binuclear metal center. Cd2 + at M1 is coordinated with the selenium atoms of H177, E181, Q207, E211 and M99 at TM2 and M2 Cd2+, with N178, E181, D208, E211, E239 (from TM6) and a water molecule. In the Cd2 +-bound natural crystal, the sulfur atom of M99 is also coordinated with Cd2 + at M1 (Figure S8). The kinks of TM4 and TM5 produced by P180 and P210 appear to promote metal binding by allowing chelating residues to approach and bind the metal in a synergistic manner.
(A) The combination of Cd2 + and BbZIP. The protein is in cartoon mode and has rainbow colors. Four Cd2+ binding sites are labeled (M1 to M4). The bound Cd2+ ions are shown as yellow-brown spheres. The area in the dashed frame is shown in (B). (B) An enlarged view of the center of the binuclear metal in the direction indicated by the arrow in (A). The blue grid shows the abnormal difference graph of Cd2 + ions at Md and M2 (σ=5). The data set was collected at 1.7969Å, and the abnormal signal generated by Cd was much stronger than at 0.9792Å. The metal chelating residues are marked with the same color as in (A) and shown as a bar. The dashed line indicates that the bonding range of the Cd 2+ ion to the coordination residue is 2.6 to 2.9. Ordered water molecules (W) are shown as small red spheres. (C) Metal combination with Zn2 + substituted BbZIP. Seven metal binding sites (M1 to M7) are marked. The bound Zn2+ and Cd2+ ions are shown as gray and yellow-brown spheres, respectively. The area in the dashed frame is shown in (D). (D) An enlarged view of the center of the binuclear metal in the direction indicated by the arrow in (C). The green grid shows the Fo-Fc omission of the metal at M1 and M2 (σ=5). The blue grid (σ=5) and the pink grid (σ=3) show the anomalous differences calculated from the data sets collected at 1.2782 and 1.3190Å, respectively. The K edge of zinc is 1.2835 Å. Mark the metal chelate residue and display it in a bar shape with the same color as in (C). The dotted line indicates that the bonding range of Zn2+ and Cd2+ ions to the coordination residues is 2.0 to 2.2 Å (for Zn2 +) and 2.6 to 2.8 Å (for Cd2 +). Ordered water molecules are shown as small red balls.
In order to study the combination of zinc and BbZIP, we immersed the Cd2 +-loaded crystal in 100°M ZnCl2 at 21°C for 40 hours, and collected two wavelengths of λ1 (1.2812Å, 9.68 keV) and λ2 ( 1.3225Å, 9.37 keV), slightly shorter than the K edge of zinc (1.2835Å, 9.66 keV), respectively. As expected, the completely exposed Cd2+ ions at M3 and M4 were completely replaced by high-concentration Zn2+ ions, which showed a strong abnormal signal at λ1, but no detectable abnormal signal at λ2 (Figure S9). Similarly, Cd2 + at M1 is replaced by Zn2 +, and the coordination environment has changed significantly: the side chains of M99 and H177 move away from the previously occupied coordination domain of Cd2 +, and Zn2 + is pentacoordinated by E181, Q207, E211 and two water molecules (Figure 2, C and D). On the contrary, although M2 is still mainly occupied by heavy atoms, which has been confirmed by the strong signal in the omission of Fo-Fc, the negligible abnormal signal at λ1 indicates that zinc is basically zero at M2 compared with Zn2+ at M1 (Figure 2, C and D). Consistently, the coordination environment of M2 did not change significantly, except that E211 wiggled slightly and became another bridge residue. This is an unexpected result, because the coordinating atoms at M2 are all oxygen atoms, and it prefers the “harder” Zn2 + ions compared to Cd2 + ions. The inability of Zn2+ to replace Cd2+ at M2 strongly suggests that, from the cytoplasmic point of view, the accessibility of M2 is much lower than that of M1. M2 is located slightly farther from the cytoplasm, and the path of metal release from M2 seems to be partially blocked by M1 (Figure 1, C and D). The structure of the Zn2+ substitution also revealed three other bound Zn2+ ions (M5 to M7) on the cytoplasmic side. It is worth noting that M5 is directly below M1 and coordinates with E276 (absolutely conserved residue in bacterial ZIP) and H177 (Figure 2D), which was previously chelated with Cd2+ on M1 (Figure 2B) , Which means that the side chain flip of H177 may be related to the metal release of M1. It is worth noting that multiple conserved zinc binding sites (M1, M5, M6, and M3; Figure S4) seem to constitute a pathway for zinc release into the cytoplasm.
Paired sequence alignments showed that there is considerable homology between BbZIP and human ZIP4 (hZIP4) in the entire TMD, especially in TM2 and the last five TMs (TM4 to TM8), these two proteins have 21% Identity and 61% similarity (Figure S10). . Therefore, the main structural features of BbZIP should be retained in hZIP4. Then, we used the zinc binding structure of BbZIP as a template to construct a TMD structure model of hZIP4 in homology modeling (Figure 3A). The overall model structure of hZIP4-TMD is very similar to the computational model based on predicted co-evolution of contact residues (Figure S11) (14), especially for the four closely related TMs (TM1, TM4, TM5 and TM6).
(A) TMD model structure of hZIP4. Homology modeling is performed using SWISS-MODEL server. The protein is displayed in cartoon mode. The mutated residues in AE are marked and highlighted in red. The area in the dashed frame is shown in (C). (B) Sequence alignment of all 14 human ZIPs in the BbZIP and TM4 and TM5 sections. Potential metal chelating residues contributing to M1 or M2 are highlighted in red. The bridging residues involve two metal binding sites. Highly conserved residues are drawn in white on a black background, while normally conserved residues are drawn on a gray background. (C) A magnified view of the dual-core metal center modeled in hZIP4. The metal chelator residues are compared as shown in Figure 2. The modeled Zn2+ ions are shown as gray spheres. The dashed line indicates the bonding of Zn 2+ to coordination residues in the range of 2.3 to 3.1. (D and E) Alanine scan (D) and arginine scan (E) of metal chelating residues in the putative binuclear metal center of hZIP4. The H379A/D375A double mutant is also included in (E). The activity of the mutant is expressed as the relative activity compared to the wild-type (WT) protein. Each data point represents the average of a total of nine calibration relative activities from three independent experiments. Error bar represents 1±SD; *P<0.01. Figure 3 shows data from three independent experiments, including the zinc absorption, total expression and surface expression of each mutant in human embryonic kidney (HEK) 293T cells. S15. Please note that since this assay has a significantly reduced affinity for the H540R variant for zinc, its relative activity in this experiment was underestimated by about 30%.
In order to understand the structural basis of ZIP4 dysfunction caused by AE mutations, 8 residues usually conserved between hZIP4 and BbZIP (Figure S10) were mapped to the hZIP4 model structure. As shown in Figure 3A, in addition to L372, the other seven residues are at or immediately adjacent to the TM-TM interface, implying a role in mediating TM packaging. For G330D, G374R and G630R, corresponding mutations in mouse ZIP4 (mZIP4) lead to a significant reduction in zinc transport activity, as well as reduced surface expression and glycosylation defects, strongly suggesting that these mutations severely affect protein folding and transport (15). Although the G526R counterpart in mZIP4 appears to be normal in terms of cell surface expression and glycosylation (15), the significant reduction in activity may be due to the interaction between G526 on TM5 and the highly conserved F519 on TM4. Caused by destruction. It is worth noting that in AE patients, L372 is topologically equivalent to M99 facing the binuclear metal center of BbZIP, replaced by proline (16) or arginine (17). Proline substitution of the same residue in mZIP4 resulted in protein folding defects (15), possibly due to TM2 kink. Differently, arginine mutations are most likely to affect the metal binding of the binuclear metal center and/or block the zinc transport pathway.
Because the residues that coordinate the metal ions at M1 and M2 are conserved (Figure 3B), we modeled the two Zn2+ ions in the hZIP4 structure and optimized them by manually adjusting the χ angle of the relevant residues The geometry of the coordination ball (Figure 3C). The modeled Zn2+ ions can be comfortably accommodated in two positions without significantly affecting the main chain structure. The Zn2+ ion at M1 is five-coordinated with H507, D511, H536 and H540 (corresponding to H177, E181, Q207 and E211 in BbZIP respectively), and the Zn2+ ion of M2 is coordinated with N508, D511 and E537 tetrahedrons (respectively Corresponds to N178, E181 and D208 in BbZIP). D511, which is topologically equivalent to bridging residue E181 in BbZIP, connects two Zn2+ ions that are 4.2Å apart.
To examine the importance of metal chelating residues in the putative binuclear metal center of hZIP4, we performed alanine scanning by substituting alanine residues for participating residues. As shown in Figure 3D, the zinc transport activity of the D511A variant was significantly reduced (approximately 20% of wild-type hZIP4), while the other variants (H507A, N508A, H536A, and E537A) only showed a moderately reduced activity, which indicates The integrity of the binuclear metal center is essential for metal transportation. Unexpectedly, the H540A mutation significantly increased the transport activity by more than two times, but it also reduced the apparent affinity for zinc (Figure S12). Because H540 is the closest to the outlet cavity among the metal chelating residues in the binuclear metal center, we hypothesized that H540 may play a role in controlling the metal release rate and/or the conformational conversion rate of the transporter during transportation.
In order to further explore the binding effect of the metal in the binuclear metal center, we performed an arginine scan by replacing a single metal chelating residue with an arginine residue (Figure 3E). D511 is not included because the alanine mutation has abolished this activity. The large and positively charged side chains of Arg are expected to eliminate metal bonding and prevent transportation routes. Facts have proved that H507R, H536R and E537R mutations lead to complete loss of zinc transport activity, indicating that disrupting the metal binding at M1 and M2 will severely disrupt the function of hZIP4. The N508R mutation reduced the activity by about 60%, and the residual activity indicated that the large side chain of the introduced Arg did not completely block the transport pathway. Similarly, the H540R variant showed activity comparable to that of the wild-type protein (or even slightly higher), but its activity and apparent affinity for zinc were greatly reduced compared to the H540A variant (Figure S12). Given that both N508 and H540 are close to the widely open outlet cavity, it is not surprising that the Arg residues at these two positions may be oriented in such a way that the transport pathway is still at least partially open.
The proposed metal transport pathway for BbZIP is shown in Figure 4. The inward open conformation of BbZIP is stabilized by the binding of the substrate at the center of the conserved binuclear metal in the middle of the transport pathway. The bound metal will be released into the cytoplasm through the histidine-rich ring connecting TM3 and TM4 through a string of metal Chelating residues (H177, E276, H275 and D144). The multiple weak zinc binding sites at the exit cavity constitute a “metal sink” to promote the release of metal from the binuclear metal center. Metal release may be driven by the repulsive electrostatic force between metal chelating residues and/or eliminate geometric constraints. Metal release may cause TM to rearrange, thereby creating an open channel outside the cell that is blocked by conserved hydrophobic residues. (M99 and A102 TM2, L200 and I204 on TM5, and M269 on TM7) are in an inwardly open configuration. In the oral cavity, two unchanging metal chelation residues (D113 and D305; Figure S4) may be crucial for the recruitment of metal substrates. The absolutely conservative S106 is located at the bottom of the entrance cavity, which means the function of guiding the metal substrate into the transmission path. The alanine mutation of H379 in hZIP4 is equivalent to S106 in BbZIP (Figure S10), which severely reduces zinc transport (14). The H379A/D375A double mutation further confirmed this result, which resulted in a 75% loss of activity (Figure 3E). According to the proposed transportation pathway (Figure 4), D375 on TM2 of hZIP4 is topologically equivalent to A102 of BbZIP (Figure S10), and is likely to be a pore lining residue on the outside of the cell.
The side view of the electrostatic potential surface of BbZIP is shown in the presumed film (solid line). The solid arrow indicates the proposed metal transport pathway connected through a series of metal binding sites, while the dashed arrow indicates the hypothetical pathway outside the cell, which is currently blocked by the hydrophobic residues of TM2 (M99 and A102) and TM5 (L200) I204) and TM7 (M269; not shown for clarity). The metal base is depicted as a light blue sphere. The residues involved in the marking mode are marked. Note that H177 may adopt two conformations, which implies that the metal is released from M1.
Structural inspection also revealed that TM1, TM4, TM5 and TM6 are closely connected to form a tight 4-TM bundle, which seems to be weakly correlated with the other four TMs (Figure S13). Since the metal captured in the binuclear metal center is mainly retained in this 4-TM bundle (TM2, through M99, only associates with Cd2 +, but not Zn2 +), the rigid rocking motion of the spiral bundle is coordinated The movement of the other four TMs (especially the long and curved TM2) will make the metal matrix alternately exposed to the extracellular space and cytoplasm (Figure S13). Although the structural features of an open conformation need to be established to establish a complete transport cycle, the lack of an open entrance on the outside of the cell in the current structure has shown that a significant conformational change is required for metal transport, which is compared with the typical ion channel, which passes through BbZIP. The flux is extremely slow (11).
Zinc homeostasis is vital to any organism. The ZIP family is a major player in the early stages of life evolution, which can regulate the import of zinc (and other transition metals such as iron and manganese) from the environment. From bacteria, archaea to humans, almost every species retains at least one zip gene in the genome. This fact reflects the evolutionary success of this ancient protein family. In order to understand the working mechanism of this conservative zinc transport machinery, whose efficiency has been proven for billions of years of development, we crystallized the prokaryotic ZIP in the LCP, which led us to discover a new transport folding structure and determine the transport Unexpected dual-core metal center path in the medium, and proposed metal transport mechanisms that may be applicable to other ZIPs.
Previous bioinformatics analysis showed that the ZIP family belongs to the drug/metabolite transporter (DMT) superfamily (18) with a predicted 3 + 5TM structure. Recent studies on the structure of DMT member SnYddG showed that the inverted 5-TM repeat The sequence is derived from the original 4-TM DMT member, such as EmrE (19). Unexpectedly, the BbZIP structure revealed an unusual 3 + 2 + 3TM arrangement, which raised the question of the origin of the ZIP series. Because it does not include the symmetrically related substrate binding TM (TM4 and TM5), but is sandwiched between the primary sequence and tertiary structure by two 3-TM repeats, the BbZIP structure represents a different structure from any known vector Unprecedented folding. As an ancient transporter family that exists in almost every species, ZIP may have diverged in the early stages of evolution and evolved from the original 3-TM fragments observed in many transporters (20). ZIPs may have completed the 3 + 2 + 3TM structure through gene replication and the acquisition of two other TMs with metal binding motifs, and developed into metal transporters. Therefore, unlike other families, the ZIP family represents a fairly distant branch of the DMT superfamily.
Unexpectedly, a binuclear metal center was found in the transportation route of BbZIP. Binuclear metal centers are known for their catalytic effects in enzymes (16, 17), including membrane enzymes (21, 22), but no known carrier has been found in the transport pathway. The unique coordination environment and significantly different interchangeability indicate that the roles of M1 and M2 are asymmetric. Because the Cd2+ ions at M1 can be easily replaced by Zn2+, and M1 is closer to the putative entrance on the outside of the cell (Figure S14), we assume that M1 is located exactly in the transport pathway to allow the metal to quickly bind and in the release process freed. transport. Since a calculation study shows that the second metal in the binuclear metal cluster weakens the binding affinity, but increases the metal selectivity of the first metal binding site (23), we assume that the metal bound to M2 may be Play a regulatory role in terms of regulatory nature. (Affinity, selectivity, kinetics, etc.) By affecting the bridging residues, the charge and geometry of the metal chelating residues at M1 are affected. In addition, both M1 and M2 are within the metal transmission path, and the different exchangeability at M1 and M2 may reflect the sequence of metal release from the binuclear metal center. That is, the metal bound at M2 must slowly pass through M1 and then be released into the cytoplasm. However, the activity of D511A in hZIP4 was significantly reduced, indicating that the two metal ions bridging the binuclear metal center are essential for zinc transport.
Together, the first atomic structure framework of ZIP proposed in this work, coupled with the unexpected binuclear metal center in the transport path, laid the foundation for further research on the unique metal transport mechanism and substrate specificity of ZIP . The clarification of the structure-function relationship of ZIP will not only help to discover drugs for ZIP inhibitors against cancer (9, 24) and inflammatory diseases (such as osteoarthritis) (25), but also contribute to the protein engineering of plant ZIP. This is the main transition metal transporter used in the roots of many plants for nutritional fortification (26, 27) and phytoremediation (28).
The gene of BbZIP (B. Bronchiseptica; National Biotechnology Center Information Reference Code: WP_010926504) with the best E. coli codon was synthesized and subcloned into the pLW01 vector (a gift from L. Waskell of the University of Michigan). A thrombin cleavage site is inserted between the His6-tag and BbZIP. The complementary DNA of the full-length hZIP4 from the mammalian gene collection was obtained from GE Healthcare (GenBank code: BC062625). The hZIP4 gene was subcloned into a modified pEGFP-N1 vector (Clontech), in which the downstream EGFP (enhanced green fluorescent protein) gene was replaced by a hemagglutinin (HA) tag. All mutations were performed using QuikChange Mutagenesis kit (Agilent). The genes, plasmids and primers are listed in Table S2. 1-oleoyl-rac-glycerin (monoolein) was purchased from Sigma-Aldrich. l(+)-selenomethionine was purchased from Acros Organics. Reagents and tools for protein crystallization are available from Hampton Research. Thrombin was purchased from Novagen.
HEK293T cells (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (Invitrogen) and 1x Gibco antibiotic-antifungal drug (Invitrogen). Plasmids were prepared by Qiagen Plasmid Maxi Kit (Qiagen) and used for transient transfection of HEK293T cells. The cells were seeded on a 24-well plate with poly-d-lysine (Corning BioCoat) at a density of 1×104 cells per well, and transfected with Lipofectamine 2000 (Invitrogen) after overnight culture. After transfection, the cells were cultured at 37°C and 5% CO 2 for 24 hours before activity determination and Western blotting.
The expression of hZIP4-HA (and other constructs and mutants) was detected by Western blot using an anti-HA antibody (catalog number 26183, Thermo Scientific Pierce), as described previously (10). The expression level of hZIP4-HA protein (and other constructs and mutants) on the cell surface is measured by surface-binding anti-HA antibodies that recognize the C-terminal HA tag (15, 29). Cells from multiple sub-wells of the same 24-well plate are used for zinc uptake determination to determine the surface expression level of the corresponding protein. After washing twice with Dulbecco’s phosphate buffered saline (DPBS; Sigma-Aldrich), the cells were fixed in 4% formaldehyde for 10 minutes at room temperature. Then the cells were washed 3 times with DPBS and incubated with anti-HA antibody (3μgml-1) for 1 hour at room temperature. The cells were washed five times in DPBS to remove unbound antibodies, and then lysed by ultrasound in SDS-polyacrylamide gel electrophoresis (PAGE) sample loading buffer. A 10% SDS-PAGE was used to separate the cell lysate containing the soluble anti-HA antibody bound to the surface ZIP4, and transfer it to a polyvinylidene fluoride membrane. As a load control, an anti-β-actin antibody was used to detect β-actin levels at a dilution of 1:2500 (catalog number 4970, Cell Signaling Technology). Goat anti-mouse immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP) (1:2000 dilution; catalog number 32230, Thermo Scientific Pierce) or goat anti-rabbit IgG (1:5000 dilution; catalog No. #) Detected bound primary antibody 7074, cell signaling technology) HRP through chemiluminescence. The heavy chain band of the anti-HA antibody was used to quantify the surface expression level of ZIP4. The surface expression level quantified using the Image Lab (Bio-Rad) program is expressed as a ratio to the surface expression level of wild-type hZIP4, and then used to calibrate the apparent zinc uptake activity (see below).
The zinc absorption assay is slightly different from the previously reported protocol (10). Twenty-four hours after transfection (cell confluence is about 90%), wash the cells with 300 μl zinc uptake buffer containing 10 mM Hepes, 142 mM NaCl, 5 mM KCl, and 10 mM glucose (pH 7.3). Under the conditions, in the same buffer, use the specified concentration of ZnCl2 (containing 20% 65ZnCl2) to represent the concentration of 300μl of zinc absorption buffer, for 20 minutes, and gently shake. In the 30-minute incubation time, HEK293T cells overexpressing wild-type hZIP4 showed linear uptake of zinc (Figure S16A). Add 300μl stop buffer (ice-cold zinc uptake buffer containing 1 mM EDTA) to stop the uptake. Transfer the resuspended cells in each well (24-well plate) to a 1.5 ml centrifuge tube and collect by centrifugation at 100 g for 5 minutes at 4°C. The precipitated cells were gently washed twice with 300 μl zinc stop buffer, lysed in 400 μl uptake buffer containing 0.5% Triton X-100, and then transferred to a polystyrene test tube. The radioactivity associated with the cells was measured with a Packard Cobra Auto-Gamma gamma counter, and the zinc content was calculated using a standard curve drawn by plotting the amount of 65Zn radioactivity versus 65ZnCl2. The cells transfected with the empty vector were treated in the same way and used as a control in the assay. By subtracting the zinc uptake in the control cells from the zinc uptake in the cells transfected with the ZIP4 gene, the amount of zinc transported into the cells by ZIP4 was calculated.
In the alanine and arginine scanning experiments, 10 μM ZnCl2 was used in the transport analysis. In each individual experiment, at least three replicates of at least three independent experiments were performed on each alanine (or arginine) mutant. The amount of zinc accumulated in the cells is adjusted by the protein concentration, and the protein concentration is determined by the Bradford method (Bio-Rad). Due to the differences in transient expression between different batches of cells, we did not directly merge the values from independent experiments. In contrast, after subtracting the background from the control sample, the activity was normalized by setting the reading of wild-type hZIP4 to 100%, and the activity of the mutant was expressed as a percentage of the wild-type hZIP4 activity. Then, the relative activity was calibrated using the quantitative surface expression level, and finally it was used for statistical analysis using the Student’s t test method. Each data point shown in Figure 3 (D and E) represents the average value of a total of nine calibrated relative zinc absorption activities from three independent experiments. The data of each experiment is shown in Figure 1. S15.
The absorption of zinc by the variants at different zinc concentrations was measured, and the kinetic parameters of H540A were determined (Figure S12A) (10). Use the series mode and the tool weighting method in Origin to curve fit the Hill model. Due to very low activity or complete cancellation, we did not perform curve fitting on D511A, H507R, H536R, E537R and H379A/D375A, and determined the Kms of other alanine and arginine variants (Figure S12B). We noticed that the linear range of zinc absorption by the H540A variant was reduced to about 20 minutes (or less than 20 minutes) (Figure S16B), which may be because its zinc transport activity is much higher than that of the wild-type protein. Therefore, the zinc absorption determination of H540A also requires 15 minutes of incubation time, and the measured Km is slightly greater than the Km obtained at 20 minutes (Figure S12B).
The expression of BbZIP was induced in the C41 (DE3) pLysS (Lucigen) strain in the LBE-5052 medium (30) and grown at room temperature for 24 hours before harvesting. To produce SeMet-substituted BbZIP, cells were cultured in M9 minimal medium supplemented with SeMet at 37°C, and then 0.2mM isopropyl-β-d-sulfur was used at OD600 (optical density at 600nm) = 0.6 Galactosylpyranoside induction. Before harvest, the cells were cultured for another 16 hours at room temperature. Prepare spheroplasts (31) and suspend them in a buffer containing 20 mM Hepes (pH 7.3), 300 mM NaCl, 0.25 mM CdCl2, and cOmplete protease inhibitor (Sigma-Aldrich). DDM (Anatrace) is added to partially solubilize the membrane, and the final concentration is 1.5% (w/v). Use HisPur cobalt resin (Thermo Fisher Scientific) in 20 mM Hepes (pH 7.3), 300 mM NaCl, 5% glycerol, 0.25 mM CdCl2 and 0.1% DDM to purify proteins with His6 tag. After removing the imidazole using an Amicon centrifugal filter, the protein sample was treated with thrombin overnight at 4°C, and then loaded onto the Superdex with 10 mM Hepes (pH 7.3), 300 mM NaCl, 5 equilibrated with a 200 column (GE Healthcare)% glycerol, 0.25 mM CdCl 2 and 0.05% DDM. The purification and crystallization of SeMet substituted proteins are the same as natural proteins, except that 1 mM tris(2-carboxyethyl)phosphine is added to all solutions.
The purified BbZIP protein was concentrated to 15 mg/ml using an Amicon centrifugal filter with a molecular weight cutoff of 30,000. Then use two connected syringes to mix the protein solution and the molten monoolein at a ratio of 2:3 (protein/monoolein; v/v). All crystallization experiments were performed using Gryphon crystallization robot (Art Robbins Instruments). The LCP sandwich assembly (Hampton Research) was used to sandwich 50 nanoliters of BbZIP-monoolein mixture covered by 800 nl of well fluid. The rod-shaped crystals appear approximately at 21°C and 25°C containing 33% (w/v) polyethylene glycol 400 (PEG 400), 100 mM NaCl, 100 mM CdCl2 and 100 mM tris-HCl (pH 7.5). Two weeks. It grows to full size in 4 weeks. The crystals were dehydrated with slightly higher concentrations of PEG 400, 100 mM NaCl, 100 mM CdCl2, and 100 mM tris-HCl (pH 7.5) for 24 hours, and then collected with MiTeGen micro-mesh and quickly frozen in liquid nitrogen. After removing the Cd2+-containing solution from the crystal drops, the natural BbZIP crystals were soaked in 35% PEG 400, 100 mM NaCl, 100 mM ZnCl2, and 100 mM tris-HCl (pH 7.5) for 48 hours, and then harvested.
X-ray diffraction data is collected in the Life Sciences Collaborative Visiting Team (LS-CAT) (21-ID-D) and the General Medicine and Cancer Institute Collaborative Visiting Team (GM/CA-CAT) (23-ID-B and 23) -ID-D) Use a 20 or 10μm beam on the Advanced Photon Source (APS) on the Eiger 9M or Pilatus 6M detector, and use the Charge Coupled Device detector on the beamline 5.0 at the Berkeley Center for Structural Biology (BCSB). 2 is carried out in Advanced Light Source (ALS) facility. LCP crystals are positioned and centered using a gridding strategy. The diffraction data set of SeMet crystal is 0.9792Å, which is used for experimental phasing. SeMet crystal in the 1.8066Å data set is used to detect Cd2 + abnormal signals. In order to clearly determine the identity of the bound metal in the crystal impregnated with Zn2+, two data sets were collected at 1.2812 and 1.3225 Å on the same natural crystal.
The diffraction data of SeMet BbZIP crystals are indexed, integrated and scaled by HKL2000 (32). In Phenix, AutoSol used a SAD of 2.7Å to determine the experimental stage (33) and identified all 12 selenium atoms in a BbZIP. The experimental electron density map is very clear, enough to identify eight TMs and trace the backbone of the polypeptide chain. The iterative model was established and improved in COOT (34) and CCP4 suite (35) or refmac5 in phenix.refine. Using the structure of Cd2 + as a search template, the structure of Zn2 + substitution was resolved to 2.4 Å by molecular replacement. Except for the SeMet dataset collected at 0.9792Å, all other datasets were indexed and integrated by xdsapp (36), and scaled in CCP4 by Aimless. The space group name was changed from C2 to I2 (all shared Space group 5). This will not affect structure determination, model construction, optimization and crystallographic statistics. All maps of protein structure are generated by PyMOL v1.3 (Schrödinger LLC).
Thermo Scientific iCAP Q ICP-MS was used to measure the metal content in BbZIP samples. The free Cd2 + in the high-purity BbZIP is removed by using two consecutive PD-10 desalting columns (GE Healthcare). Treat the control sample in the same way (gel filtration buffer plus 1% DDM). The protein sample, control sample and buffer solution were heated in 2 M HNO3 at 65°C for 20 minutes, left overnight at room temperature, and centrifuged at 14,000 g for 20 minutes. The common transition metals (Cr, Mn, Fe, Co, Ni, Cu, Zn, Ag, Cd, Sn, and Pb) in the supernatant were analyzed by ICP-MS for metal concentrations (parts per billion). The protein concentration was determined by using the Bradford method.
Perform homology modeling on the SWISS-MODEL server (https://swissmodel.expasy.org/interactive) (37). The TMD structure of hZIP4 (residues 327 to 647) was modeled using a Zn2+ substituted structure (2.4Å) as a structural template. For better modeling, the residues in the predicted unstructured loops (between TM3 and TM4 and between TM7 and TM8) are deleted from the hZIP4 sequence to obtain the GMQE (Global Model Quality Estimate) score of the model Is 0.65. 3A. The local quality assessment based on the QMEAN scoring function (38) showed that the residues in TM2 and TM4 to TM7 scored the highest (approximately or higher than 0.7) than other regions of the model structure, indicating that the reliability of these regions is higher, including putative Transportation routes and binuclear metal centers. First, add Zn2 + ions to the same position as the Zn2 + substitution structure in the model. In order to meet the geometric constraints of Zn2+ coordination, the χ angles of the side chains of potential metal chelating residues were manually adjusted in COOT, and the metal positions were slightly adjusted. In the final model, due to side chain adjustment and Zn2+ modeling, there is no obvious spatial conflict.
For supplementary materials for this article, please see http://advances.sciencemag.org/cgi/content/full/3/8/e1700344/DC1
Figure. S13. The release of metal from the binuclear metal center contributes to the conformational change.
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The structure of the ZIP zinc transporter shows an inward configuration with a binuclear metal center in the transport pathway.
The structure of the ZIP zinc transporter shows an inward configuration with a binuclear metal center in the transport pathway.
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Post time: Oct-09-2020