Abstract
The copper-transporting ATPase ATP7A has an essential role in human physiology. ATP7A transfers the copper cofactor to metalloenzymes within the secretory pathway; inactivation of ATP7A results in an untreatable neurodegenerative disorder, Menkes disease. Presently, the mechanism of ATP7A-mediated copper release into the secretory pathway is not understood. We demonstrate that the characteristic His/Met-rich segment Met(672)-Pro(707) (HM-loop) that connects the first two transmembrane segments of ATP7A is important for copper release. Mutations within this loop do not prevent the ability of ATP7A to form a phosphorylated intermediate during ATP hydrolysis but inhibit subsequent dephosphorylation, a step associated with copper release. The HM-loop inserted into a scaffold protein forms two structurally distinct binding sites and coordinates copper in a mixed His-Met environment with an ∼2:1 stoichiometry. Binding of either copper or silver, a Cu(I) analog, induces structural changes in the loop. Mutations of 4 Met residues to Ile or two His-His pairs to Ala-Gly decrease affinity for copper. Altogether, the data suggest a two-step process, where copper released from the transport sites binds to the first His(Met)(2) site, triggering a structural change and binding to a second 2-coordinate His-His or His-Met site. We also show that copper binding within the HM-loop stabilizes Cu(I) and protects it from oxidation, which may further aid the transfer of copper from ATP7A to acceptor proteins. The mechanism of copper entry into the secretory pathway is discussed.
Highlights
Copper is required for normal cell homeostasis and serves as an essential cofactor for a variety of metalloenzymes involved in neurotransmitter biosynthesis, amidation of neuroendocrine peptides, vasculature and connective tissue formation, and many other processes
Our results suggest that a sequence insert in the lumenal portion of ATP7A, which is absent in ATP7B, binds copper and influences the rate of copper release from ATP7A
The First Lumenal Loop of ATP7A Contains a Unique Sequence Insert—To identify structural element(s) that may contribute to the faster rate of copper release by ATP7A, we carried out sequence alignments of human Cu-ATPases and their orthologues
Summary
Copper is required for normal cell homeostasis and serves as an essential cofactor for a variety of metalloenzymes involved in neurotransmitter biosynthesis, amidation of neuroendocrine peptides, vasculature and connective tissue formation, and many other processes. The transport of copper by Cu-ATPases requires the hydrolysis of ATP and involves the formation of a transient phosphorylated intermediate (catalytic phosphorylation, see Fig. 1A) This step depends on Cu(I) binding to the intramembrane transport sites [4, 5]. Severe disorders due to ATP7A or ATP7B inactivation indicate that these Cu-ATPases do not fully complement each other’s activity This is in part due to different levels of expression of ATP7A and ATP7B in tissues and their distinct trafficking behavior in polarized epithelia [8]. Kinetic studies of catalytic phosphorylation and dephosphorylation show that ATP7A performs each of these steps faster than ATP7B [2] These functional differences may become critical in Menkes and Wilson diseases, when the function of one of the Cu-ATPases is lost and cells have to rely solely on activity of the remaining Cu-ATPase. Human Cu-ATPases are large membrane proteins, consisting of an JOURNAL OF BIOLOGICAL CHEMISTRY 26585
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