Structure of the ATP binding domain from the Archaeoglobus fulgidus Cu+-ATPase.
ABSTRACT The P-type ATPases translocate cations across membranes using the energy provided by ATP hydrolysis. CopA from Archaeoglobus fulgidus is a hyperthermophilic ATPase responsible for the cellular export of Cu+ and is a member of the heavy metal P1B-type ATPase subfamily, which includes the related Wilson and Menkes diseases proteins. The Cu+-ATPases are distinct from their P-type counter-parts in ion binding sequences, membrane topology, and the presence of cytoplasmic metal binding domains, suggesting that they employ alternate forms of regulation and novel mechanisms of ion transport. To gain insight into Cu+-ATPase function, the structure of the CopA ATP binding domain (ATPBD) was determined to 2.3 A resolution. Similar to other P-type ATPases, the ATPBD includes nucleotide binding (N-domain) and phosphorylation (P-domain) domains. The ATPBD adopts a closed conformation similar to the nucleotide-bound forms of the Ca2+-ATPase. The CopA ATPBD is much smaller and more compact, however, revealing the minimal elements required for ATP binding, hydrolysis, and enzyme phosphorylation. Structural comparisons to the AMP-PMP-bound form of the Escherichia coli K+-transporting Kdp-ATPase and to the Wilson disease protein N-domain indicate that the five conserved N-domain residues found in P1B-type ATPases, but not in the other families, most likely participate in ATP binding. By contrast, the P-domain includes several residues conserved among all P-type ATPases. Finally, the CopA ATPBD structure provides a basis for understanding the likely structural and functional effects of various mutations that lead to Wilson and Menkes diseases.
Article: Reaction cycle of Thermotoga maritima copper ATPase and conformational characterization of catalytically deficient mutants.[show abstract] [hide abstract]
ABSTRACT: Copper transport ATPases sustain important roles in homeostasis of heavy metals and delivery of copper to metalloenzymes. The copper transport ATPase from Thermotoga maritima (CopA) provides a useful system for mechanistic studies, due to its heterologous expression and stability. Its sequence comprises 726 amino acids, including the N-terminal metal binding domain (NMBD), three catalytic domains (A, N, and P), and a copper transport domain formed by eight helices, including the transmembrane metal binding site (TMBS). We performed functional characterization and conformational analysis by proteolytic digestion of WT and mutated (NMBD deletion or mutation) T. maritima CopA, comparing it with Archaeoglobus fulgidus CopA and Ca(2+) ATPase. A specific feature of T. maritima CopA is ATP utilization in the absence of copper, to form a low-turnover phosphoenzyme intermediate, with a conformation similar to that obtained by phosphorylation with P(i) or phosphate analogues. On the other hand, formation of an activated state requires copper binding to both NMBD and TMBS, with consequent conformational changes involving the NMBD and A domain. Proteolytic digestion analysis demonstrates A domain movements similar to those of other P-type ATPases to place the conserved TGES motif in the optimal position for catalytic assistance. We also studied an H479Q mutation (analogous to one of human copper ATPase ATP7B in Wilson disease) that inhibits ATPase activity. We found that, in spite of the H479Q mutation within the nucleotide binding domain, the mutant still binds ATP, yielding a phosphorylation transition state conformation. However, covalent phosphoryl transfer is not completed, and no catalytic turnover is observed.Biochemistry 05/2009; 48(22):4871-80. · 3.42 Impact Factor
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ABSTRACT: Copper is an essential but potentially harmful trace element required in many enzymatic processes involving redox chemistry. Cellular copper homeostasis in mammals is predominantly maintained by regulating copper transport through the copper import CTR proteins and the copper exporters ATP7A and ATP7B. Once copper is imported into the cell, several pathways involving a number of copper proteins are responsible for trafficking it specifically where it is required for cellular life, thus avoiding the release of harmful free copper ions. In this study we review recent progress made in understanding the molecular mechanisms of copper transport in cells by analyzing structural features of copper proteins, their mode of interaction, and their thermodynamic and kinetic parameters, thus contributing to systems biology of copper within the cell.Cellular and Molecular Life Sciences CMLS 03/2010; 67(15):2563-89. · 6.57 Impact Factor
Article: Elucidation of the ATP7B N-domain Mg2+-ATP coordination site and its allosteric regulation.[show abstract] [hide abstract]
ABSTRACT: The diagnostic of orphan genetic disease is often a puzzling task as less attention is paid to the elucidation of the pathophysiology of these rare disorders at the molecular level. We present here a multidisciplinary approach using molecular modeling tools and surface plasmonic resonance to study the function of the ATP7B protein, which is impaired in the Wilson disease. Experimentally validated in silico models allow the elucidation in the Nucleotide binding domain (N-domain) of the Mg(2+)-ATP coordination site and answer to the controversial role of the Mg(2+) ion in the nucleotide binding process. The analysis of protein motions revealed a substantial effect on a long flexible loop branched to the N-domain protein core. We demonstrated the capacity of the loop to disrupt the interaction between Mg(2+)-ATP complex and the N-domain and propose a role for this loop in the allosteric regulation of the nucleotide binding process.PLoS ONE 01/2011; 6(10):e26245. · 4.09 Impact Factor