The nucleotide binding sites in A-ATP synthases are located at the interfaces of subunit A and B, which is proposed to play a regulatory role. Differential binding of MgATP and -ADP to subunit B has been described, which does not exist in the related alpha and B subunits of F-ATP synthases and V-ATPases, respectively. The conserved phosphate loop residues, histidine and asparagine, of the A-ATP synthase subunit B have been proposed to be essential for gamma-phosphate interaction. To investigate the role of these conserved P-loop residues in nucleotide-binding, subunit B residues H156 and N157 of the Methanosarcina mazei Go1 A-ATP synthase were separately substituted with alanine. In addition, N157 was mutated to threonine, because it is the corresponding amino acid in the P-loop of F-ATP synthase subunit alpha. The structures of the subunit B mutants H156A, N157A/T were solved up to a resolution of 1.75 and 1.7 angstrom. The binding constants for MgATP and -ADP were determined, demonstrating that the H156A and N157A mutants have a preference to the nucleotide over the wild type and N157T proteins. Importantly, the ability to distinguish MgATP or -ADP was lost, demonstrating that the histidine and asparagine residues are crucial for nucleotide differentiation in subunit B. The structures reveal that the enhanced binding of the alanine mutants is attributed to the increased accessibility of the nucleotide binding cavity, explaining that the structural arrangement of the conserved H156 and N157 define the nucleotide-binding characteristics of the regulatory subunit B of A-ATP synthases. (C) 2012 Elsevier Inc. All rights reserved.