De novo design of a transmembrane Zn²⁺-transporting four-helix bundle

Abstract

The design of functional membrane proteins from first principles represents a grand challenge in chemistry and structural biology. Here, we report the design of a membrane-spanning, four-helical bundle that transports first-row transition metal ions Zn(2+) and Co(2+), but not Ca(2+), across membranes. The conduction path was designed to contain two di-metal binding sites that bind with negative cooperativity. X-ray crystallography and solid-state and solution nuclear magnetic resonance indicate that the overall helical bundle is formed from two tightly interacting pairs of helices, which form individual domains that interact weakly along a more dynamic interface. Vesicle flux experiments show that as Zn(2+) ions diffuse down their concentration gradients, protons are antiported. These experiments illustrate the feasibility of designing membrane proteins with predefined structural and dynamic properties.

Fig. 1. Computational design and molecular dynamics simulations of Rocker

(A) Schematic of the goal of obtaining conformational exchange between two oppositely oriented symmetry-frustrated states without being trapped in a symmetric state with both sites simultaneously occupied. (B) Metal site consists of a set of ExxH motifs and a single Glu residue from each of the tight dimers. (C) Helical-wheel diagram of Rocker peptide. (D) The repacking algorithm placed Ala residues at the tight interface and Phe residues at the loose interface. Empty metal site on the left is omitted for clarity. (E) MD simulation of the design model with two Zn²⁺ ions placed at one metal site show stable interhelical distances for both tight and loose interfaces. Continuing the simulation after removing the Zn²⁺ ions maintained the tight interfaces, but resulted in an increased interhelical distance at the loose interface.

Fig. 2. Structure of Rocker

(A) Axial view of the x-ray crystal structures from three different packing environments (green, cyan, and magenta for space groups of increasing symmetries, P4₃2₁2, I4₁22, and I2₁3, respectively) superimposed on the tight dimeric subunit of the design model (white), with the Ala residues (spheres from the cyan structure with highest resolution of 2.7 Å) forming the tight interface as anticipated. (B) Metal-binding residues (sticks) from the crystal structures can chelate with a change in rotamers without encountering any unfavorable steric interactions within the dimer. (C) A close-up of the metal site with 2mF_obs –DF_calc map contoured at 1.0 σ for the 2.7 Å–resolution structure.

Fig. 3. Evidence for Zn²⁺ binding and tetramerization of Rocker in micelles and lipid bilayers

(A) ¹H-NMR of Rocker in D₂O and deuterated DPC (at 900 MHz ¹H field) shows significant changes to the side-chain chemical shifts as Zn²⁺ is titrated into the solution. The changes level off when Zn²⁺ reaches two equivalents per tetramer, which indicates the expected binding stoichiometry. (B) 1D ¹³C CP-MAS spectra of apo Rocker in DMPC bilayers from 233 K to 313 K show invariant peak positions across a wide temperature range, which indicates single species with conformational dynamics in bilayers. (C) 2D ¹³C-¹³C 2D correlation spectra of apo and Zn²⁺ bound Rocker with 500-ms mixing. Relevant 1D cross sections are plotted to compare cross-peak intensities. L19-I5 cross peaks (blue annotations) are observed, indicating antiparallel packing. Compared with the apo sample, the Zn²⁺-bound sample lacks the L19α-I5δ peak in the 56-ppm cross section and has much weaker A8/22-I5 and A8/22-L19 cross peaks, which indicate that Zn²⁺ binding loosens interhelical packing. The proximal L19 and I5 are shown on a structural model of Rocker. (D) ¹⁹F CODEX data of DMPC-bound Rocker with para-¹⁹F-Phe14 at 220 K and 8 kHz MAS (error bar, SD propagated from signal-to-noise). The CODEX intensity decays to 1/n of 0.34, where n is the oligomer number; this indicates that the peptide assembles into a species larger than dimers. The CODEX decay is well fit (solid lines) using ¹⁹F-¹⁹F distances found in inward-facing rotamers of Phe14 in an antiparallel tetramer, consistent with the crystal structures. The data rule out outward-facing orientations of Phe14 (dashed lines), which suggests that Glu4 and Glu18 face the pore.

Fig. 4. Antiporter-like function of Rocker

(A) Examples of liposome flux assays using fluorescent indicators for characterizing the cotransportation function are illustrated. (B) Ion selectivity in Rocker is shown via a Zn²⁺ efflux assay. Rocker can specifically transport Zn²⁺ (blue) but not Ca²⁺ (green), at a level higher than passive leakage (gray). Mutating the first-shell ligands via E4/E18-to-Q substitutions abrogates Zn²⁺ transport (gold). (C) Rocker-mediated, outward [Zn²⁺] gradient-driven proton-antiport against an outward pH gradient is shown by measuring net inward proton flux (red, right axis) and the outward Zn²⁺ flux (blue, left axis). (D) Rocker-mediated, H⁺-driven Zn²⁺ antiport under symmetrical Zn²⁺ concentration is shown using both the outward proton flux (red, right axis) and the inward Zn²⁺ flux (blue, left). (E) Representative traces show the dependence of initial rates of transport on the exterior Zn²⁺ concentration. (F) The initial rates of transport of Zn²⁺ and H⁺ from panel (E) increase with increasing exterior Zn²⁺ concentration, following Michaelis-Menten kinetics with a K_M of ~ 280 µM. (Error values in text are SD propagated from curve fitting.)