Abstract
We report the solution NMR structures of the N-domain of the Menkes protein (ATP7A) in the ATP-free and ATP-bound forms. The structures consist of a twisted antiparallel six-stranded beta-sheet flanked by two pairs of alpha-helices. A protein loop of 50 amino acids located between beta 3 and beta 4 is disordered and mobile on the subnanosecond time scale. ATP binds with an affinity constant of (1.2 +/- 0.1) x 10(4) m(-1) and exchanges with a rate of the order of 1 x 10(3) s(-1). The ATP-binding cavity is considerably affected by the presence of the ligand, resulting in a more compact conformation in the ATP-bound than in the ATP-free form. This structural variation is due to the movement of the alpha1-alpha2 and beta2-beta 3 loops, both of which are highly conserved in copper(I)-transporting P(IB)-type ATPases. The present structure reveals a characteristic binding mode of ATP within the protein scaffold of the copper(I)-transporting P(IB)-type ATPases with respect to the other P-type ATPases. In particular, the binding cavity contains mainly hydrophobic aliphatic residues, which are involved in van der Waal's interactions with the adenine ring of ATP, and a Glu side chain, which forms a crucial hydrogen bond to the amino group of ATP.
Highlights
Localize with copper(I)-transporting ATPases in the transGolgi network and require the ATPase-mediated copper transport for the formation of the holo-enzymes [5]
All P-type ATPases are multi-domain membrane proteins, sharing a basic core architecture [10]. This comprises a hydrophilic region protruding into the cytosol, which contains the Actuator domain (A-domain) and the ATP-binding domain, which in turn can be further separated into two smaller domains named the phosphorylation domain (P-domain) and the nucleotide-binding domain (N-domain)
The chemical shift changes increased the human MNK protein (N-MNK) and determined the solu- upon increasing ATP concentration, indicating that ATP binds tion structures of its ATP-free and ATP-bound forms using to the protein
Summary
Localize with copper(I)-transporting ATPases in the transGolgi network and require the ATPase-mediated copper transport for the formation of the holo-enzymes [5]. All P-type ATPases are multi-domain membrane proteins, sharing a basic core architecture [10] This comprises a hydrophilic region protruding into the cytosol, which contains the Actuator domain (A-domain) and the ATP-binding domain, which in turn can be further separated into two smaller domains named the phosphorylation domain (P-domain) and the nucleotide-binding domain (N-domain). In the present work we report the solution structure of both the ATP-free and ATP-bound forms of the N-domain of the human MNK protein Their comparison provides a detailed analysis of the conformational changes that the N-domain undergoes upon nucleotide binding, which is a crucial requisite for the thorough understanding of the enzymatic cycle of PIB-ATPases
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