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P-type ATPases are ubiquitously abundant enzymes involved in active transport of charged residues across biological membranes. The KdpB subunit of the prokaryotic Kdp-ATPase (KdpFABC complex) shares characteristic regions of homology with class II-IV P-type ATPases and has been shown previously to be misgrouped as a class IA P-type ATPase. Here, we present the NMR structure of the AMP-PNP-bound nucleotide binding domain KdpBN of the Escherichia coli Kdp-ATPase at high resolution. The aromatic moiety of the nucleotide is clipped into the binding pocket by Phe 377 and Lys 395 via a -stacking and a cation-interaction, respectively. Charged residues at the outer rim of the binding pocket (Arg 317 , Arg 382 , Asp 399 , and Glu 348 ) stabilize and direct the triphosphate group via electrostatic attraction and repulsion toward the phosphorylation domain. The nucleotide binding mode was corroborated by the replacement of critical residues. The conservative mutation F377Y produced a high residual nucleotide binding capacity, whereas replacement by alanine resulted in low nucleotide binding capacities and a considerable loss of ATPase activity. Similarly, mutation K395A resulted in loss of ATPase activity and nucleotide binding affinity, even though the protein was properly folded. We present a schematic model of the nucleotide binding mode that allows for both high selectivity and a low nucleotide binding constant, necessary for the fast and effective turnover rate realized in the reaction cycle of the Kdp-ATPase.Ion transport is a vital prerequisite for cellular life, because ions play a crucial role in many biochemical processes, either as cofactors for enzyme function, as substrates for sym-and antiport with other substances, or maintenance of cellular pH or turgor. To maintain ion gradients across their membranes, cells employ highly sophisticated transport systems. Primary transport systems are driven by ATP to achieve transport against concentration gradients. A classic representative is the family of P-type ATPases, which play a fundamental role in the transport of heavy metal, alkali, and earth-alkali ions. Because of their enormous importance, eukaryotic P-type ATPases, such as the Ca 2ϩ -ATPase and the Na ϩ ,K ϩ -ATPase, have been studied intensively in recent years. A major breakthrough was achieved by Toyoshima et al.(1-3) with the elucidation of the Ca 2ϩ -ATPase structure in different reaction cycle intermediates. In addition, the structure of the nucleotide binding domain of the Na ϩ ,K ϩ -ATPase was solved by x-ray crystallography (4). However, distantly related members of the P-type ATPase group are less well examined, and in particular, the important question of the evolutionary relationship of the different P-type ATPases remains uncertain. An unusual prokaryotic P-type ATPase, the KdpFABC complex, serves as a good model to address several questions for both mechanistic and evolutionary aspects. In Escherichia coli, the KdpFABC complex serves as a highly specific potassium transport system, which is on...

Background:The peripheral stator stalk of Escherichia coli ATP synthase contains two b subunits. Results: Using disulfide bond formation, one b subunit was cross-linked to a, ␣, and ␦ and the other to ␤. Conclusion:The b subunits adopt distinct positions within the stator to generate stability. Significance: The different positions imply different roles in counteracting the torque generated by the rotor.

The Methanocaldococcus jannaschii (formerly Methanococcus jannaschii) protein Mj0968 has been reported to represent a soluble P-type ATPase [Ogawa et al., FEBS Lett. 471 (2000) 99^102]. In this study, we report that the heterologously expressed Mj0968-His 10 protein exhibits high rates of phosphatase activity, whereas only very low ATPase activity was measured. Replacement of the aspartate residue in the DSAGT motif (D7A), which becomes phosphorylated during the reaction cycle of P-type ATPases, does not a¡ect the V max , but only the K M of the reaction. Labeling studies with [Q Q-32 P]ATP and [K K-32 P]ATP revealed that the previously reported labeling experiments do not necessarily show phosphorylation of Mj0968, but rather point to ATP binding. Binding studies with trinitrophenyl adenosine nucleotides showed low apparent K d values for those molecules. These results provide evidence that the native function of Mj0968 seems to be that of a phosphatase, rather than that of an ATP-hydrolyzing enzyme. ß

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