Energy Coupling and Stoichiometry of Zn2+/H+ Antiport by the Cation Diffusion Facilitator YiiP

YiiP is a prokaryotic Zn2+/H+ antiporter that serves as a model for the Cation Diffusion Facilitator (CDF) superfamily, members of which are generally responsible for homeostasis of transition metal ions. Previous studies of YiiP as well as related CDF transporters have established a homodimeric architecture and the presence of three distinct Zn2+ binding sites named A, B, and C. In this study, we use cryo-EM, microscale thermophoresis and molecular dynamics simulations to address the structural and functional roles of individual sites as well as the interplay between Zn2+ binding and protonation. Structural studies indicate that site C in the cytoplasmic domain is primarily responsible for stabilizing the dimer and that site B at the cytoplasmic membrane surface controls the structural transition from an inward facing conformation to an occluded conformation. Binding data show that intramembrane site A, which is directly responsible for transport, has a dramatic pH dependence consistent with coupling to the proton motive force. A comprehensive thermodynamic model encompassing Zn2+ binding and protonation states of individual residues indicates a transport stoichiometry of 1 Zn2+ to 2–3 H+ depending on the external pH. This stoichiometry would be favorable in a physiological context, allowing the cell to use the proton gradient as well as the membrane potential to drive the export of Zn2+.

Suppl. Figure 4. Determination of D287A and D287A/H263A structures by cryo-EM (A,B) SDS-PAGE and elution profiles from SEC purifications.Images come from a single gel with the molecular weight markers (116,66,45,35,25,18.4,14.4 kDa).Position and presence of the double peak is consistent with the higher order oligomerization seen during image analysis.(C,D) Corrections for motion and CTF were followed by two rounds of 2D classification and ab initio structure determination.(E,F) Hetero-refinement was used to select a homogeneous set of particles for non-uniform refinement.(G,H) Final structures are characterized by FSC and by local resolution, illustrated by the plots and by surface coloring, respectively.Suppl. Fig. 5. Interactions between the TM2/TM3 loop and the CTD.(A) In the WT structure, the TM2/TM3 loop (blue) contacts the TM6/CTD linker from the opposing protomer with proximity of D72 and R210.(B) In the D70A_asym structure, the Zn 2+free TM2/TM3 loop extends to interact with the H1 helix in the CTD of the opposing protomer.(C) In the D287A/H263A structure, the TM2/TM3 loop extends towards CTD's of two different protomers (e.g., TM2/TM3 loop from chain B inserts between CTD's of chains A and D).(D) In the other monomer of D287A/H263A, the TM2/TM3 loop interacts with the TM6/CTD linker of an opposing chain (e.g., the loop from chain A interacts with the linker from chain B).In these chains, the TM6/CTD linker has refolded into one, long continuous helix.
Suppl.Fig. 6.Measuring Zn 2+ affinity by MST (A-D) SEC elution profiles for YiiP constructs.WT protein is included for comparison but was not analyzed by MST.Elution volumes of the peak are indicated on each plot, which are almost identical for all constructs.(E-G) Raw data from MST for the titrations at pH 7. The blue rectangles correspond to the time window used for initial fluorescence levels and the red rectangles correspond to the time window used for the thermophoresis rates.Each plot includes 48 traces that represent data from 16 Zn 2+ concentrations used for the titration, each of which were measured in triplicate.(H) SEC profiles of the D287A mutant in the presence and absence of Fab.The shift in position of the main peak in the presence of Fab is consistent with a complex between the homodimer and two Fab's and the secondary peak at 9.9 ml is consistent with the dimer of dimers (4 YiiP + 4 Fab) seen during cryo-EM process (Suppl.Fig. 4).(I) SEC profiles of the D287A/H263A mutant in the presence and absence of Fab.Although the position of the peak in the absence of Fab is consistent with the YiiP homodimer seen with other mutants, the position of the main peak in the presence of Fab is consistent with a dimer of dimers and the secondary peak with even higher order oligomers.
Suppl.Fig. 7. Walk of replica simulations in CpHMD pH-replica exchange (REX) through pH space.
Each panel shows how one replica simulation (Rep:0 to Rep:29) changes over the course of the simulation (Frame number) its current pH state, as indicated by the "pH replica" on the ordinate.The pH replicas range from pH 1.5 (pH replica 0) to 11.5 (pH replica 29).Multiple replicas sample most of the pH range and many move across the especially relevant range between pH replica 9 (pH 4) and pH replica 22 (pH 9), indicating that the REX procedure samples pH space well.
Suppl.Fig. 8. Convergence of the deprotonated fraction for titratable residues in CpHMD simulations.
Residues in site A (D47, D51, H155, D159) and site B (D70, H73, H77) from both protomers (A and B) are shown.The instantaneous deprotonated fraction S is plotted as a function of simulation time and pH value, sampled across all replicas in the replica exchange simulation.The deprotonated fractions generally converge to a stable value after about 8 ns with similar behavior in protomers A and B, thus indicating sufficient sampling during the CpHMD simulations.
Suppl.Fig. 9. Titration curves for titratable residues in CpHMD simulations.Residues in site A (D47, D51, H155, D159) and site B (D70, H73, H77) are shown from both protomers (A and B).The unprotonated fraction S from the end of the CpHMD simulations is shown as a function of pH (black points).The Hill equation (generalized Henderson-Hasselbalch equation) is fitted to the data (black line).The estimated pKa is indicated as a red line at S=0.5.As described in the text, H73/H77 were considered to be coupled and were analyzed in aggregate; the two pKas for the combined system (H73AH77A and H73BH77B) are represented by the pair of red lines in the last two panels.

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Fig. 10.Site A Protonation and Zn 2+ -binding states by CpHMD and MST inference.(A).Populations of protonated states obtained directly from CpHMD simulations for site A, in the absence of Zn 2+ .Data for protomer A and protomer B were combined.Color coded definitions of individual states are shown below with a "1" indicating the protonated state and absence of a number the unprotonated state of each corresponding residue.(B) Populations of protonated states from an inverse Multibind model based on inferring microscopic pKa values directly from the CpHMD populations in (A).(C) Population of protonated states resulting from MST inference, which relies on the MC method to refine microscopic model parameters based on experimental MST data.Populations represent the Zn 2+ -free states although the model incorporates the whole range of Zn 2+ bound states [see (F)].Only states S0, S3, S7, S11 have appreciable occupancy (>0.0001 maximum probability) although all states contribute to aggregated state probabilities in D and E. (D) MST inference results represented as populations of aggregated states defined by the total number of protons bound in the absence of Zn 2+ .(E) Deprotonated fraction of each site A residue as a function of pH, averaged over all possible states of the MST inference model in the absence of Zn 2+ .Data points (symbols) were generated from the model; then the Hill-Langmuir equation was fit to the generated data to obtain an effective per-residue pKa (listed in Table 4).(F) Population of states derived from the MST inference model as a function of Zn 2+ concentration at various pH values.S0Zn (dotted line) denotes the fully deprotonated, Zn 2+ -bound state, which is the only populated Zn 2+ -bound state predicted by the MST inference.Suppl.Fig. 11.Site B Protonation and Zn 2+ -binding states by CpHMD and MST inference.(A).Populations of protonated states obtained directly from CpHMD simulations for site B, in the absence of Zn 2+ .Data for protomer A and protomer B were combined.Color coded definitions of individual states are shown below with a "1" indicating the protonated state and absence of a number the unprotonated state of each corresponding residue.(B) Populations of protonated states from an inverse Multibind model based on inferring microscopic pKa values directly from the CpHMD populations in (A).(C) Population of protonated states resulting from MST inference.For this analysis, a symmetry restraint was imposed on H73 and H77 as described in the text.Populations are shown in the absence of Zn 2+ although the model incorporates the whole range of Zn 2+ concentrations [see (G)].(D) MST inference results represented as populations of aggregated states defined by the total number of protons bound in the absence of Zn 2+ .(E) Deprotonated fraction of each site B residue as a function of pH, averaged over all possible states of the MST inference model in the absence of Zn 2+ .Data points were generated from the model and the Hill-Langmuir equation was fit to D70 data (solid black line) to obtain an effective per-residue pKa; data for H73 and H77 are not correctly modelled by a Hill fit (not plotted).(F) H73 and H77 were treated as a coupled system and the total deprotonated fraction as a function of pH was fit with the "coupled titration model" (solid black line), resulting in two effective pKa values.(G) Population of states from the MST inference mode as a function of Zn 2+ concentration at various pH values.Unlike site A, multiple Zn 2+ -bound states (S0Zn, S1Zn, S2Zn, S3Zn) are populated at lower pH values.Suppl.Fig.12.Summary of structures from CDF transporters.Cartoon representations of most structures so far determined.The structures have been grouped according to conformational state: symmetric IF, symmetric OF and asymmetric states.PDB accession codes are indicated for previously published structures together with concentrations of Zn 2+ present in the solution and the number of ions observed in the structures.Accession codes for current work are listed in Table1.Although the sites been named differently for Znt7 and Znt8, the A,B,C nomenclature has been used to simplify the comparison.The helices in the TMD are shown in rainbow colors starting with blue for TM1 and red for TM6.CTD's are represented as a triangle.Zn 2+ ions as magenta spheres.Hydrophobic residues forming the so-called hydrophobic gate (Leu154 and Leu199 in SoYiiP) are shown as sticks.Disordered in TM2/TM3 loops is indicated by a blurred line.Suppl.Fig.13.Water accessibility of site A. Caver Analyst(Jurcik et al., 2018)  was used to map cavities starting from Site A and leading towards the cytoplasm.(A) Model of WT YiiP with the cavity depicted a series of spheres, color coded according to the radius.(B) Model of D70A_asym shows an IF conformation on the left and a much constricted cavity in the occluded protomer on the right.(C) Radii are plotted along the contour of the respective cavities shown in panels A and B. This plot shows that the occluded protomer in D70A_asym falls well below the radius of 1.4 Å, typically taken as the limit for water accessibility.