PDBsum entry 2a00

Hydrolase

PDB id: 2a00

Contents

Protein chain

136 a.a. *

Ligands

ANP

* Residue conservation analysis

PDB id:

2a00

Name:

Hydrolase

Title:

The solution structure of the amp-pnp bound nucleotide binding domain of kdpb

Structure:

Potassium-transporting atpase b chain. Chain: a. Fragment: kdpbn, nucleotide binding domain of kdpb. Synonym: potassium-transporting p-type atpase, kdpfabc, atp phosphohydrolase [potassium- transporting] b chain, potassium binding and translocating subunit b. Engineered: yes

Source:

Escherichia coli. Organism_taxid: 562. Gene: kdpb. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

NMR struc:

19 models

Authors:

M.Haupt,M.Bramkamp,M.Coles,K.Altendorf,H.Kessler

Key ref:

M.Haupt et al. (2006). The holo-form of the nucleotide binding domain of the KdpFABC complex from Escherichia coli reveals a new binding mode. J Biol Chem, 281, 9641-9649. PubMed id: 16354672 DOI: 10.1074/jbc.M508290200

Date:

15-Jun-05 Release date: 20-Dec-05

Protein chain

P03960  (KDPB_ECOLI) -  Potassium-transporting ATPase ATP-binding subunit from Escherichia coli (strain K12)

Seq: 682 a.a.

Struc: 136 a.a.

Enzyme reactions

• Enzyme class: E.C.7.2.2.6 - P-type K(+) transporter.
• Reaction: K⁺(out) + ATP + H2O = K⁺(in) + ADP + phosphate + H⁺

K(+)(out) + ATP + H2O = K(+)(in) (Bound ligand (Het Group name = ANP) matches with 81.25% similarity) + ADP + phosphate + H(+)

• Cofactor: Mg(2+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1074/jbc.M508290200

J Biol Chem 281:9641-9649 (2006)

PubMed id: 16354672

The holo-form of the nucleotide binding domain of the KdpFABC complex from Escherichia coli reveals a new binding mode.

M.Haupt, M.Bramkamp, M.Heller, M.Coles, G.Deckers-Hebestreit, B.Herkenhoff-Hesselmann, K.Altendorf, H.Kessler.

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.

Selected figure(s)

Figure 7.
FIGURE 7. Zoom into the nucleotide binding pocket. The stereo view of the binding pocket backs the critical role of Phe^377 and Lys^395 for nucleotide binding, depicted are all residues that are within a sphere of 10 Å around the nucleotide.

Figure 8.
FIGURE 8. Clip to fit. The schematic picture of the nucleotide binding pocket illustrates the simple and energy-saving nucleotide binding mode, enabling the rapid nucleotide exchange necessary for a functional reaction cycle. Highlighted are the residues Phe^377, Lys^395, Arg^317, and Arg^382. Asp^344 is represented as a negative charge at the bottom of the binding pocket by a red circle.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2006, 281, 9641-9649) copyright 2006.

Figures were selected by the author.