Planar substrate-binding site dictates the specificity of ECF-type nickel/cobalt transporters

Abstract

The energy-coupling factor (ECF) transporters are multi-subunit protein complexes that mediate uptake of transition-metal ions and vitamins in about 50% of the prokaryotes, including bacteria and archaea. Biological and structural studies have been focused on ECF transporters for vitamins, but the molecular mechanism by which ECF systems transport metal ions from the environment remains unknown. Here we report the first crystal structure of a NikM, TtNikM2, the substrate-binding component (S component) of an ECF-type nickel transporter from Thermoanaerobacter tengcongensis. In contrast to the structures of the vitamin-specific S proteins with six transmembrane segments (TSs), TtNikM2 possesses an additional TS at its N-terminal region, resulting in an extracellular N-terminus. The highly conserved N-terminal loop inserts into the center of TtNikM2 and occludes a region corresponding to the substrate-binding sites of the vitamin-specific S components. Nickel binds to NikM via its coordination to four nitrogen atoms, which are derived from Met1, His2 and His67 residues. These nitrogen atoms form an approximately square-planar geometry, similar to that of the metal ion-binding sites in the amino-terminal Cu(2+)- and Ni(2+)-binding (ATCUN) motif. Replacements of residues in NikM contributing to nickel coordination compromised the Ni-transport activity. Furthermore, systematic quantum chemical investigation indicated that this geometry enables NikM to also selectively recognize Co(2+). Indeed, the structure of TtNikM2 containing a bound Co(2+) ion has almost no conformational change compared to the structure that contains a nickel ion. Together, our data reveal an evolutionarily conserved mechanism underlying the metal selectivity of EcfS proteins, and provide insights into the ion-translocation process mediated by ECF transporters.

Figure 1

Overall structure of TtNikM2. (A) The X-ray fluorescence spectrum as fitted shows multiple elements, e.g., Cl, K, Ca, Fe, Ni and Zn ions. The nickel ion's Kα and Kβ emission lines are shown as solid-dot purple lines. (B) Overall structure of TtNikM2. The TtNikM2 is shown in cartoon model, colored by a rainbow fashion. The Ni²⁺ ion is shown as a sphere. The secondary structural elements, N- and C-termini are indicated. (C) TtNikM2 is represented as an electrostatic surface model and two views related by 180° rotation along the vertical axis are shown. The blue, white, and red shadings represent positively charged, neutral, and negatively charged surface regions, respectively.

Figure 2

TtNikM2 has an additional transmembrane helix and an extra N-terminal loop compared to vitamin-specific S components. For TtNikM2, the extra N-terminal helix and loop are shown in blue and the rest of the protein is colored in cyan. RibU, ThiT, BioY are colored in wheat, slate, and lime, respectively. The right panel shows substrate-binding sites with 90° rotation to the left panel. The rioflavin, thiamine and biotin are colored in yellow, magenta and orange in sticks, respectively. The Ni²⁺ is shown as a lightpink sphere.

Figure 3

The N-terminus of TtNikM2 is responsible for Ni²⁺ binding. (A) The N-terminal loop of TtNikM2 inserts into a deep pocket. The α1-helix and the N-terminal loop are shown in ribbon representation and the rest of the protein is shown in an electrostatic potential surface. The blue, white, and red shadings represent positively charged, neutral, and negatively charged surface regions, respectively. The Ni²⁺ ion is shown as a sphere. (B) A close view of the N-terminal substrate-binding pocket. The residues from N-terminal loop and from the rest of the protein are shown in yellow and magenta sticks, respectively. The water molecule is shown as a red sphere. The Ni²⁺-nitrogen coordinate bonds and hydrogen bonds are shown as green and red dashed lines, respectively. (C) The sequence alignment of the highly conserved N-terminus (amino acids 1-9) of CbiMs and NikMs from different species.

Figure 4

The approximately square-planar geometry of Ni²⁺-binding site. (A) The detailed binding mode of the Ni²⁺. The residues involved in Ni²⁺ coordination and stabilization of the binding pocket are shown as sticks. The Ni²⁺-nitrogen coordinate bonds and hydrogen bonds are shown as green and red dashed lines, respectively. (B) Comparison of the Ni K-edge XANES spectra of the NiO, Ni(dmg)₂ in solid and TtNikM2 in solution. The observed distinct shoulder feature at 8339.5 eV present in the TtNikM2 and Ni(dmg)₂ is attributed to the 1s-to-4p_z electronic transition in a square-planar metal complex. The structures of the Ni²⁺-binding sites of NiO, Ni(dmg)₂ and TtNikM2 are shown in sticks. (C) The electron distribution of the Ni²⁺-binding pocket. The residues are shown in stick model, with hydrogen atoms displayed. The distributions of electrons are indicated by δ⁻ and δ⁺. (D) Ni²⁺-uptake activity of E. coli cells containing the RcNik(MN)QO transporter from R. capsulatus with wild-type or mutant RcNikM. Each assay was performed in triplicate. Mean values ± standard deviations are shown. Numbers in parentheses correspond to residue positions in RcNikM, where the numbering differs between RcNikM and TtNikM.

Figure 5

The approximately square-planar geometry of the metal-binding site dictates substrate specificity of TtNikM2. (A) Structure of the TtNikM2 nickel coordinate cavity. The nitrogen atoms are defined as N1, N2, N3, and N4. (B) Binding energy of M²⁺ + L⁻→ ML⁺(M represents different divalent metal ions) as calculated with BHaH functional. (C) Splitting of the 3d orbital in NiL⁺ and 3D contours (isovalue = 0.03) of Kohn-Sham molecular orbitals. The calculated Kohn-Sham energy levels and the contour diagrams of the frontier molecular orbitals of NiL⁺ are shown, where Ni²⁺ has a d⁸ electron configuration. (D) Structural alignment of the TtNikM2-nickel and TtNikM2-cobalt structures. The structures with nickel and cobalt are shown in magenta and slate, respectively. The residues binding the metal ions are shown in sticks.