Mutagenesis of the residues forming an ion binding pocket of the NtpK subunit of Enterococcus hirae V-ATPase

Abstract

The crystal structures of the Na(+)- and Li(+)-bound NtpK rings of Enterococcus hirae V-ATPase have been obtained. The coupling ion (Na(+) or Li(+)) was surrounded by five oxygen atoms contributed by residues T64, Q65, Q110, E139, and L61, and the hydrogen bonds of the side chains of Q110, Y68, and T64 stabilized the position of the E139 γ carboxylate essential for ion occlusion (PDB accession numbers 2BL2 and 2CYD). We previously indicated that an NtpK mutant strain (E139D) lost tolerance to sodium but not to lithium at alkaline pHs and suggested that the E139 residue is indispensable for the enzymatic activity of E. hirae V-ATPase linked with the sodium tolerance of this bacterium. In this study, we examined the activities of V-ATPase in which these four residues, except for E139, were substituted. The V-ATPase activities of the Q65A and Y68A mutants were slightly retained, but those of the T64A and Q110A mutants were negligible. Among the residues, T64 and Q110 are indispensable for the ion coupling of E. hirae V-ATPase, in addition to the essential residue E139.

Fig 1

Na⁺ and Li⁺ dependence of purified V-ATPase containing an NtpK mutation. V-ATPases were purified (16) from 25DKD/pHEexK (wild type) or from 25DKD harboring the NtpK mutant plasmids. The effects of NaCl (A and C) and LiCl (B and D) on ATP hydrolytic activities of purified V-ATPases were examined at 2 mM ATP, as described in Materials and Methods (15). The results are shown with error bars and represent the means ± standard deviations calculated from three independent experiments. The insets (A and B) show ATP hydrolytic activities with lower concentrations of salts. Symbols: ●, wild type; ×, T64A mutant; △, Q65A mutant; ▲, Y68A mutant; □, Q110A mutant.

Fig 2

Li⁺ extrusion by whole cells expressing NtpK mutant enzymes. Strains NKD/pHEexK (A), NKD/pHEex (B), NKD/T64A (C), NKD/Q65A (D), NKD/Y68A (E), and NKD/Q110A (F) were cultured in NaTY medium and then loaded with Li⁺ as described in Materials and Methods. The extrusion of Li⁺ was initiated by the addition of 10 mM glucose at 0 min (filled symbols, with glucose; open symbols, without glucose). The results are representative of data from two independent experiments.

Fig 3

Side-view models of the ion binding sites of E. hirae NtpK and the I. tartaricus c subunits. The ion binding site in E. hirae NtpK (A) consists of three helices (h2, h3, and h4) from the single NtpK subunit, while that in the I. tartaricus c subunits (B) consists of two helices (h1′ and h2′) from one c subunit and one helix (h2) from the neighboring c subunit. Since the Li⁺-bound structure of the I. tartaricus c subunits has not been solved so far, Na⁺-bound structures are shown (11, 18). The residues involved in ion binding are shown in a stick representation. Dashed lines indicate Na⁺-O bonds and potential hydrogen bonds, with distances in angstroms. Sodium ions are shown as black spheres.