The Holo-form of the Nucleotide Binding Domain of the KdpFABC Complex from Escherichia coli Reveals a New Binding Mode

Abstract

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...

Cellular copper distribution: a mechanistic systems biology approach

“…,K ? -ATPase (PDB code 1MO8 [78]) and the bacterial Kdp-ATPase (PDB code 2A00 [79]), in agreement with the fact that their sequences are overall the most conserved within all P-type ATPases [80]. In particular also the conserved phosphorylation loop in P-domain (DKTGT), which contains the invariant Asp residue, adopts similar conformation in the copper-transporting ATPases and in several SERCA structures [58,76,77].…”

“…,K ? -ATPase [78,79]. Moreover, at the rim of the ATP-binding cavity, there are positively charged amino acids (Arg489 and Arg506 in Ca 2?…”

The Binding Mode of ATP Revealed by the Solution Structure of the N-domain of Human ATP7A

“…However, these interactions with the N-domain appear to be relatively loose. Indeed the N-domain of Kdp-ATPase, similarly to the N-WLN protein as already mentioned (36), does not discriminate between AMP, ADP, and ATP, indicating that the energetics of the phosphate-protein interactions is modest (41). In the 1VFP structure of the full-length SERCA protein, the ␥-phosphate group of the bound ATP analogue interacts with the P-domain (38).…”

“…However, there are notable variations in the way each of these domains binds ATP. In the bacterial Kdp-ATPase, the crucial protein-ATP interactions involve hydrogen bonding of the adenine NH 2 group to the side chain of Asp 344 , astacking interaction between the adenine ring and Phe 377 and a -cation interaction between the adenine ring and the side chain of conserved Lys 395 (41). In the N-domain of rat Na ϩ ,K ϩ -ATPase, the amino protons of the base form a hydrogen bond to the O⑀1 atom of Gln 482 , whereas the adenine ring is again involved in a -stacking interaction with Phe 475 (40).…”

Sulfate Acts as Phosphate Analog on the Monomeric Catalytic Fragment of the CPx-ATPase CopB from Sulfolobus solfataricus

“…Structural models of the nucleotide-binding fragments of several ATPases, such as the renal Na + /K + -ATPase and the bacterial K + -ATPase have been determined by X-ray crystallography 16 or NMR spectroscopy. 17,18 Recently the structures of the catalytic fragment of the copper-translocating CPx-ATPase of Archaeoglobus fulgidus and of the nucleotide-binding domain of the Wilson disease protein have been published. 19,20 Here, we present our first step in the strategy to establish a model of the Sulfolobus solfataricus CPx-ATPase.…”