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

Melina Haupt,Marc Bramkamp,Markus Heller,Murray Coles,Gabriele Deckers-Hebestreit,Brigitte Herkenhoff-Hesselmann,Karlheinz Altendorf,Horst Kessler

doi:10.1074/jbc.m508290200

Melina Haupt, Marc Bramkamp + Show 6 more

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https://doi.org/10.1074/jbc.m508290200

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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 pi-pi stacking and a cation-pi 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.

Highlights

Family of P-type ATPases, which play a fundamental role in the transport of heavy metal, alkali, and earth-alkali ions
Three-dimensional Structure of Nucleotide-bound KdpB nucleotide binding domains (KdpBN)—We have previously published the NMR solution structure of the apo-form of the nucleotide binding domains of KdpB termed KdpBN, together with a model of the holo-form based on data for the apo-form, which suggested that no significant conformational changes occur within the enzyme upon nucleotide binding [10]
In the very well studied case of P-loop ATPases, ATP is bound via a hydrophobic ␲-␲ stacking to a conserved phenylalanine or tyrosine residue on the surface of the enzyme, and the triphosphate group of ATP caps the N terminus of an ␣-helix, leaving it embedded in a close network of hydrogen bonds

Summary

Introduction

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 Ca2ϩATPase and the Naϩ,Kϩ-ATPase, have been studied intensively in recent years. In Escherichia coli, the KdpFABC complex serves as a highly specific potassium transport system, which is only synthesized when the cells’ need for Kϩ can no longer be satisfied by the constitutive Kϩ transport systems [5] This P-type ATPase differs from most others by the fact that catalytic activity (KdpB) and ion transport (KdpA) are associated with two different subunits (6 – 8). Nucleotide Bound State of KdpBN formulated notion that KdpB belongs to type I P-type ATPases [12, 13], this study corroborates that KdpB is more closely related to alkali- and earth-alkali-transporting P-type ATPases, sharing most similarities with the proton pump (type III)

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