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.