Difference in Stability of the N-domain Underlies Distinct Intracellular Properties of the E1064A and H1069Q Mutants of Copper-transporting ATPase ATP7B

Abstract

Wilson disease (WD) is a disorder of copper metabolism caused by mutations in the Cu-transporting ATPase ATP7B. WD is characterized by significant phenotypic variability, the molecular basis of which is poorly understood. The E1064A mutation in the N-domain of ATP7B was previously shown to disrupt ATP binding. We have now determined, by NMR, the structure of the N-domain containing this mutation and compared properties of E1064A and H1069Q, another mutant with impaired ATP binding. The E1064A mutation does not change the overall fold of the N-domain. However, the position of the ␣1,␣2-helical hairpin (␣-HH) that houses Glu 1064 and His 1069 is altered. The ␣-HH movement produces a more open structure compared with the wild-type ATP-bound form and misaligns ATP coordinating residues, thus explaining complete loss of ATP binding. In the cell, neither the stability nor targeting of ATP7B-E1064A to the trans-Golgi network differs significantly from the wild type. This is in a contrast to the H1069Q mutation within the same ␣-HH, which greatly destabilizes protein both in vitro and in cells. The difference between two mutants can be linked to a lower stability of the ␣-HH in the H1069Q variant at the physiological temperature. We conclude that the structural stability of the N-domain rather than the loss of ATP binding plays a defining role in the ability of ATP7B to reach the trans-Golgi network, thus contributing to phenotypic variability in WD.The human copper-transporting ATPase ATP7B regulates copper levels in the cytosol and within the secretory pathway (1). Genetic mutations that disrupt the transport function of ATP7B lead to Wilson disease (WD), 4 a severe metabolic disorder associated with copper accumulation in the liver, brain, and other tissues (2). The WD phenotypes are rather diverse. The disease is characterized by significant variability in the age of onset, severity, and manifestations, which include hepatic, neurological, and psychiatric symptoms. More than 350 WDcausing mutations have been described. The majority of these mutations are single base pair substitutions that do not eliminate synthesis of the full-length ATP7B. Despite considerable effort, no strong correlations have been observed between specific mutations and the disease phenotype, necessitating better understanding of the effects that mutations have on ATP7B structure, function, and intracellular behavior.ATP7B transports copper from the cytosol across cellular membranes using the energy of ATP hydrolysis. ATP binds to the cytosolic ATP-hydrolyzing domain, which consists of the N-and P-domains. The bulk of the ATP molecule is bound to the N-domain (3, 4), whereas the ␥-phosphate extends to the P-domain to transiently phosphorylate the invariant aspartate residue Asp 1027 . The structure of the N-domain has been solved for human ATP7B (4) and several orthologs (5-8). These studies have identified invariant residues contributing to the ATP binding pocket. In ATP7B, these residues are Glu 1064 , Glu 1068 , His 1069 , Gly 1101 , Asn 115...

The cytosolic chaperone α-Crystallin B rescues appropriate folding and compartmentalization of misfolded multispan transmembrane proteins

“…This process may occur, since the third and fourth Cu-binding domains in the Nterminal portion of ATP7B have been shown to aggregate in vitro (Banci et al, 2008). Given that the H1069Q mutation might affect the strength of interaction between NBD and N-terminal domains of ATP7B (Dmitriev et al, 2011), the third and fourth Cu-binding domains in the N-terminal portion of the mutant ATP7B molecules could potentially acquire more freedom to oligomerise and generate ATP7B-H1069Q aggregates. CRYAB may counteract these aggregation events by holding the Nterminal tails of ATP7B-H1069Q molecules from oligomerization and in this way may help the mutant be exported from the ER.…”

Distinct phenotype of a Wilson disease mutation reveals a novel trafficking determinant in the copper transporter ATP7B

“…Two well-characterized trafficking signals encoded within the primary sequences of both copper-ATPases have been identified: (i) F 37 -E 45 in ATP7B, which is required for retention of ATP7B in the TGN (−Cu) and targeting to the apical region in polarized hepatic cells (+Cu, anterograde trafficking) (11); and (ii) di-and trileucines in ATP7A and ATP7B's C-termini, which are needed for retrograde trafficking to the TGN (−Cu) (12,41,42). Trafficking also requires MBD5 or MBD6 (10), the C terminus (12), nucleotide binding-domain stability (43), and ATPase-dependent coppertransport activity (31).…”

Linkage Disequilibrium and Haplotype Analysis of theATP7BGene in Alzheimer's Disease

“…This evidence is in agreement with some previous studies, which demonstrated that ATP7B mutation in transmembrane and ATP-binding domains (block 1) have a strong impact on protein function. [33][34][35] It is noteworthy that rs1061472 and rs732774, strongly associated with AD in our study population, are located in transduction and transmembrane domains.…”