Figures and data
Overview of the cryo-EM structures.
Density maps and corresponding atomic models are shown for each mutant. Fab molecules are colored orange (light chain) and red (heavy chain) with YiiP colored in cyan, blue, purple and green, depending on the mutant. The homo-dimers adopt C2 symmetry for WT (A), D51A (B) and D287A (D) mutants, but a bend between TMD and CTD break this symmetry for D70A (C). The D287A/H263A mutant (E) forms a dimer of dimers in which the Fab molecules are rather disordered. Two conformations were observed for D70A, both of which are shown in panel C: D70A_sym on the left and D70A_asym on the right. Location Zn2+ binding sites, membrane boundaries as well as the topology of the YiiP protomer (inset) are shown in panel A; rectangles and arrows represent α-helices and β-sheets, respectively. Insets in panels B-D show the coordination geometry at the individual sites. Although the resolution was not always sufficient to uniquely define the side chain orientations, the maps are fully consistent with coordination geometry originally defined in the higher resolution X-ray structure (Lu et al., 2009).
Structure determination of YiiP mutants
Comparisons of atomic models.
(A) Overlay of the TMD for WT (cyan) and D51A (blue) structures. This view is from the cytoplasmic side of the membrane, tilted slightly to show site A in protomer A. Core helices (M2, M3, M6) are well aligned, but there are substantial displacements of the peripheral helices (M1, M4, M5), presumably due to lack of Zn2+ binding at site A serving to bridge M2 and M5. (B) Overlay of the TMD for WT (cyan) and D70A_sym (purple) shows a very similar configuration of helices and an intact dimeric interface. This view is directly along the two-fold axis from the cytoplasmic side of the membrane. (C) Structure of D70A_asym viewed along the membrane plane showing the asymmetry between the two TMD’s. The protomer on the left (chain B) adopts an IF conformation, whereas the protomer on the right (chain A) adopts a novel occluded conformation that includes a reconfigured TM2/TM3 loop (blue) making a novel interaction with the CTD. (D) Overlay of the TMD for WT (cyan) and D70A_asym (plum) showing the occluded conformation adopted by protomer A (top) and the IF conformation adopted by protomer B (bottom). L199 and L154 (spheres) make van der Waals interactions that appear to stabilize the occluded conformation. These views are from the cytoplasmic side of the membrane, slightly tilted to show the respective protomers. (E-F) Overlays of D70A_asym (plum) and D70A_sym (purple) structures after alignment of core helices TM3 and TM6. Structures are viewed parallel to the membrane plane in E and along the two-fold axis from the cytoplasmic side of the membrane in F. A significant shift in the position of the CTD is apparent in E and bottom panel in F. Despite this shift and substantial conformational changes in the occluded protomer A, the dimer interface in the TMD (TM3 and TM6) is well aligned (F, top panel). (G) Domain swapped dimer of dimers adopted by the D287A/H263A construct. Dimerization of TMD’s involves interaction between one dark-green and one light-green molecule (e.g., TMDa and TMDb, where “a” and “b” refer to chain ID), whereas dimerization of CTD’s involves interaction between either two dark-green or two light-green molecules (e.g., CTDa and CTDd). The linker between M6 and the CTD adopts a long straight helix in chains b and d, but remains an unstructured loop in chains a and c. This view is from the cytoplasm looking toward the membrane surface. (H) Overlay of the TMD for WT (cyan) and D287A/H263A structures viewed along the dimer axis from the cytoplasmic side of the membrane shows a good match, indicating that disruption of site C affects mainly the configuration of the CTD.
Zn2+ removal from site B in the TM2/TM3 loop.
(A) Per-residue RMSF of the WT, holo structure with Zn2+ present (cyan) and absent (purple) at site B demonstrates a notable increase in fluctuations in the TM2/TM3 loop. The dashed line indicates the boundary between TMD and CTD. (B) Per-residue RMSF for simulations with empty site B using three different alignment schemes: the entire molecule (“All”), transmembrane domain (“TMD”), or C-terminal domain (“CTD”). Analogous data for the WT, holo structure have previously been published (Fig. 3i in Lopez-Redondo et al., 2021). (C) Distributions of distance between Cα atoms from Asp72 in one chain and Arg210 in the opposite chain during simulations of the WT, holo structure, WT apo structure with Zn2+ absent from all sites, WT structure with site B empty, and the D72A mutant with Zn2+ present at all three sites. The sharp peak at ∼5 Å from the holo structure suggests a salt bridge that is less stable in the D72A mutant and disrupted when Zn2+ is absent from site B. (D) Angle between the TMD and CTD in simulations in the presence (cyan) and absence (purple) of Zn2+ at site B; the D72A mutant in the holo state is also shown on the right. The distribution of angles, on the right, highlight greater mobility either with site B is empty or with the D72A mutation. (E,F) Structure of the WT, holo YiiP dimer showing global C2 symmetry about a vertical axis and juxtaposition of the TM2/3 loop (blue) with the TM6/CTD linker from the opposing protomer (orange-to-red). Rainbow colors progress from blue to red moving from the N-terminus of one protomer to the C- terminus of the other protomer. (G,H) D70A_sym structure shows a kink between TMD and CTD and disordering of the TM2/3 loop. Both protomers are in the IF state. (I,J) D70A_asym structure showing further twisting of the CTD and asymmetry of the TMD’s. The TM2/3 loop is disordered in the protomer on the left (chain B), but adopts a novel interaction with the CTD in the protomer on the right (chain A). (K,L). Electrostatic surface of D70A_asym showing a negatively charged cavity leading to site A on the left, but an occluded cavity with positive charge on the right. Note that L is at an oblique angle looking down on the M2-M3 loop.
pH dependence of Zn2+ binding.
MST was used to experimentally measure Zn2+ binding affinity at each site (panels A, B and C). These data were combined with a thermodynamic model, represented in panel H, with parameters derived either from CpHMD (panels D and E) or MST inference (panels F and G). (A) For MST studies of site A, the D70A/D287A/H263A construct was used with Zn2+ buffered either with NTA (pH 7.0 and 7.4) or with citrate (pH 5.6, 6.0 and 6.5). Curves represent the law of mass action with Kd values listed in Table 2. The relatively poor fit at pH 7.4 may reflect the fact that the Kd (1 nM) is lower than the minimum protein concentration (8 nM) supported by the assay, thus moving the system from the binding to the titration regime (Jarmoskaite et al., 2020). (B). For MST studies of site B, the D51A/D287A/H263A construct was used and Zn2+ was buffered with citrate. (C) For MST studies of site C, the D51A/D70A construct was used and Zn2+ was buffered with NTA at pH 7 or with citrate at the other pH’s. (D,E) Curves represent predictions of a thermodynamic model with pKa values for either site A residues (Asp47, Asp51, His153, Asp157) or site B residues (D70, H73, H77) taken from CpHMD simulations. These pKa values are listed in Table 4 and the distributions of protonation states are shown in Suppl. Figs. 10 and 11. Symbols represent experimental MST data as in A and illustrate a poor fit using these parameters. Thermodynamic modeling was not possible for site C because of instability of the CTD in CpHMD simulations in the absence of Zn2+. (F,G). Curves represent predictions of the MST inference algorithm with corresponding pKa values listed in Table 4. Again, symbols represent experimental MST data and show the excellent fit using these refined parameters. (H) Schematic representation of the microscopic thermodynamic model for site A. Titratable residues (D47, D51, D159, H155) are represented either as black squares for the protonated state, or white squares for the deprotonated state. Binding of one Zn2+ ion to the site is indicated by a filled magenta circle. Transitions are only possible between states connected by lines (protonation/deprotonation) or corresponding states connected by double-sided arrows (Zn2+ binding/release). An analogous model for site B comprised D70, H73 and H77 (not depicted).
Binding affinity for individual Zn2+ sites measured by MST* or deduced by the MST inference algorithm§
Transport cycle for YiiP.
According to the alternating access paradigm, YiiP toggles between the OF and IF states via an intermediate occluded state. We assume that these changes occur independently in each protomer based on asymmetry seen in structures of soYiiP and Znt8 (Xue et al., 2020). Thus, our model depicts changes in the right-hand protomer while the left-hand protomer remains in a resting IF state. Although these intermediate states are informed by current and past structural work, they do not precisely conform to solved structures but represent hypothetical states that we believe to exist under physiological conditions. (A) Zn2+ is released to the periplasm. The TM2/TM3 loop is depicted interacting with the CTD in a Zn2+-free, extended conformation, as seen in our D70A_asym structure. (B) The release of Zn2+ is promoted by the low pH of the periplasm and results in protonation of two residues in site A, or potentially three residues at lower pH. (C-D) The protonated form transitions to the IF state via an occluded state. In the IF state, the Zn2+-free TM2/TM3 loop is released by the CTD and becomes disordered. (E) Zn2+ is recruited to site B, inducing an ordered conformation of the TM2/TM3 loop that folds onto the CTD enabling interaction of Asp72 and Arg210. (F) Zn2+ is transferred from a relatively low affinity site B to the much higher affinity site A via a negatively charged access channel, thus displacing two protons. (G) This transfer induces a Zn2+-bound, occluded conformation in which the CTD tilts toward the occluded protomer and interacts with the TM2/TM3 loop in its Zn2+-free, extended conformation. Features of the model are illustrated in the middle, boxed panel with desaturated colors. Zn2+ ions are depicted as magenta spheres and protons with a “+”. The CTD is pink with two Zn2+ ions constitutively bound at site C. The scaffolding membrane helices (TM3 and TM6) are blue and the transport domain (TM1,2,4,5) is yellow. The TM2/TM3 loop is blue and depicted with dashed lines in the disordered state. Created with biorender.com.
© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.
Binding affinity and Hill coefficients derived from MST data*
pKa values of residues determined by CpHMD simulations (Hill-Langmuir) and MST inference
MD simulations
