Metal binding properties of Escherichia coli YjiA, a member of the metal homeostasis-associated COG0523 family of GTPases

Abstract

GTPases are critical molecular switches involved in a wide range of biological functions. Recent phylogenetic and genomic analyses of the large, mostly uncharacterized COG0523 subfamily of GTPases revealed a link between some COG0523 proteins and metal homeostasis pathways. In this report, we detail the bioinorganic characterization of YjiA, a representative member of COG0523 subgroup 9 and the only COG0523 protein to date with high-resolution structural information. We find that YjiA is capable of binding several types of transition metals with dissociation constants in the low micromolar range and that metal binding affects both the oligomeric structure and GTPase activity of the enzyme. Using a combination of X-ray crystallography and site-directed mutagenesis, we identify, among others, a metal-binding site adjacent to the nucleotide-binding site in the GTPase domain that involves a conserved cysteine and several glutamate residues. Mutations of the coordinating residues decrease the impact of metal, suggesting that metal binding to this site is responsible for modulating the GTPase activity of the protein. These findings point toward a regulatory function for these COG0523 GTPases that is responsive to their metal-bound state.

Figure 1

Cobalt and nickel binding to WT YjiA. (A) The difference spectra of 20 μM YjiA incubated with 20 μM NiCl₂ (—) or 20 μM CoSO₄ (---) display ligand-to-metal charge-transfer (LMCT) signals in the region of 250–400 nm. The extinction coefficient was calculated on the basis of the protein concentration. (B) To determine the affinity of apo-WT YjiA for Ni(II), the metal was titrated into a sample of 250 nM YjiA and the increase in absorption at 340 nm was used to calculate the fractional saturation of the protein (●). Data like those shown were fit to the Hill equation to yield an apparent K_d of (3.7 ± 0.3) × 10^–6 M [n = 1.2 ± 0.3 (—)] (see the text). Likewise, binding of Co(II) to 5 μM YjiA can be monitored at 350 nm (■) and fit to yield an apparent K_d of (2.0 ± 0.1) × 10^–5 M [n = 1.7 ± 0.2 (---)].

Figure 2

Zinc binding to YjiA. Once Zn(II) binds, the maximal absorption of zincon shifts from 488 to 620 nm. It takes 140 μM ZnSO₄ to saturate 140 μM zincon (●), consistent with a 1:1 stoichiometry. In a competition between 140 μM zincon and 10 μM apo-YjiA (■), the spectrum of zincon does not change until after the addition of 20 μM ZnSO₄, suggesting that there are two Zn(II) sites on YjiA that can outcompete zincon. It takes an additional 160 μM ZnSO₄ to saturate the 620 nm signal, suggesting that there are two additional sites in YjiA capable of competing with zincon for Zn(II). In the case of the E37A/C66A/C67A mutant (▼), the initial plateau region is not observed, but 180 μM ZnSO₄ is still required to saturate zincon, suggesting that the mutant YjiA can still bind four Zn(II) ions but with affinities weaker than that of the WT protein.

Figure 3

Structure of WT YjiA and location of the metal-binding site in the primary structure of the GTPase domain. (A) Sequence alignment of the GTPase domain regions between the Walker A and Walker B motifs of representative G3E GTPases generated by the COBALT sequence alignment program (available online at http://www.ncbi.nlm.nih.gov/tools/cobalt/). Located between the Walker A and B motifs is a putative metal-binding motif, the location of which is common among the G3E GTPases. The two glutamates mutated in this study are highlighted by orange boxes. The species from which the protein sequences were derived and the starting sequence positions (in brackets) are as follows: YjiA, E. coli (11); YeiR, E. coli (9); CobW, P. denitrificans (18); YciC, B. subtilis (11); Nha3, Rhodococcus sp. N-771 (13); UreG, Klebsiella aerogenes (14); EcHypB, E. coli (111); HpHypB, Helicobacter pylori (53). (B) The structure of apo-YjiA (PDB entry 1NIJ), previously published, features two domains. The N-terminal GTPase domain possesses a typical G3E GTPase fold with a central β-sheet core surrounded by α-helices. Located on one of the central β-strands is the conserved C₆₄XCC₆₇ motif. Glu37 and Glu42 are near this motif (inset).

Figure 4

Effect of metal on the quaternary structure of YjiA. In the absence of metal, 60 μM YjiA elutes at a volume corresponding to a monomer (solid black line). The addition of 2 equiv of NiCl₂ (solid red line), CoSO₄ (dashed black line), or ZnSO₄ (solid blue line) results in the formation of dimeric and oligomeric species. Incubation of YjiA with 2 equiv of GDP (dashed red line) or GTP (dashed blue line) followed by chromatography with 400 μM nucleotide in the running buffer resulted in only a small portion of dimeric protein. The chromatographic traces (monitored at 280 nm) are representative data sets from experiments with a Superdex 200 10/300 analytical column equilibrated with 25 mM HEPES (pH 7.6), 200 mM NaCl, and 5 mM MgCl₂. The ticks at the top of the graph denote the elution volumes of the protein standards. From left to right, the identities of the standards (and their molecular masses) are thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B₁₂ (1.4 kDa), respectively.

Figure 5

Crystal structure of Zn(II)-bound WT YjiA. (A) Overall structure of the two YjiA protomers in the asymmetric unit (yellow and green ribbons). Symmetry-related molecules involved in Zn(II) binding are shown as gray ribbons. Four types of Zn(II)-binding sites are observed in the structure: a bridging site (labeled B), an internal site (labeled C), and two types of surface sites (labeled D and E). Bound Zn(II) ions are shown as purple spheres and coordinating residues as sticks. A Zn anomalous difference Fourier map is contoured around bound Zn(II) ions at 5σ. (B) Close-up view of the bridging Zn(II)-binding site, located on a 2-fold axis between the two protomers in the asymmetric unit. Zn(II) is coordinated by E74 and H114 from both protomers. (C) Close-up view of the internal Zn(II)-binding site. The side chains of E37, E42, and C66 (green carbons) coordinate the Zn(II), with an open coordination sphere probably occupied by a water molecule. The structure of apo-WT YjiA (PDB entry 2NIJ(17)) is superimposed and shown with magenta carbons. The region around the Zn(II)-binding site undergoes conformational changes upon Zn(II) binding, as indicated by the arrows. The Zn(II)-binding site is also located in the proximity of the nucleotide binding site, as demonstrated by modeling studies with a GTP analogue-bound HypB structure [PDB entry 2HF8, GTPγS shown with cyan carbons and Mg(II) shown as an orange sphere]. (D and E) Close-up views of the two types of surface sites, located at crystal contacts between YjiA protomers in the asymmetric unit and crystallographically related molecules. Zn(II) is coordinated by the side chains of E167 and/or H170 from one molecule and H187 from the other molecule.

Figure 6

E37 or E39 of YjiA could coordinate nucleotide-associated Mg(II). GTP analogue-bound HypB (cyan carbons, PDB entry 2HF8(18)) is superimposed onto the structure of Zn(II)-bound WT YjiA (green carbons). Bound GTPγS is shown with cyan carbons, and GTPγS-associated Mg(II) is shown as an orange sphere. D75 of HypB is involved in Mg(II) coordination. Both E37 and E39 of YjiA are in the proximity of the modeled Mg(II), as indicated by the purple dashed lines, raising the possibility that these residues are involved in Mg(II) coordination in YjiA. Notably, E37 is also involved in binding Zn(II) (purple sphere). E39 was truncated to the C_β atom because of a lack of electron density for the side chain.