Identification of the Transmembrane Metal Binding Site in Cu+-transporting PIB-type ATPases

Mandal, A.; Yang, Ying; Kertesz, Tzipporah M.; Argüello, José M.

doi:10.1074/jbc.m410854200

Cited by 70 publications

(91 citation statements)

References 59 publications

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“…Our model of CopA also provides insight into the architecture of the transmembrane high-affinity copper binding sites, whose liganding amino acid residues on TM6-TM8 have been determined recently (Mandal et al 2004). Figure 5 displays the copper sites I and II, modeled by using the topology of the membrane helices of Model-2.…”

Section: Discussionmentioning

confidence: 99%

Structural model of the CopA copper ATPase of Enterococcus hirae based on chemical cross-linking

et al. 2008

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The CopA copper ATPase of Enterococcus hirae belongs to the family of heavy metal pumping CPx-type ATPases and shares 43% sequence similarity with the human Menkes and Wilson copper ATPases. Due to a lack of suitable protein crystals, only partial three-dimensional structures have so far been obtained for this family of ion pumps. We present a structural model of CopA derived by combining topological information obtained by intramolecular cross-linking with molecular modeling. Purified CopA was crosslinked with different bivalent reagents, followed by tryptic digestion and identification of cross-linked peptides by mass spectrometry. The structural proximity of tryptic fragments provided information about the structural arrangement of the hydrophilic protein domains, which was integrated into a three-dimensional model of CopA. Comparative modeling of CopA was guided by the sequence similarity to the calcium ATPase of the sarcoplasmic reticulum, Serca1, for which detailed structures are available. In addition, known partial structures of CPx-ATPase homologous to CopA were used as modeling templates. A docking approach was used to predict the orientation of the heavy metal binding domain of CopA relative to the core structure, which was verified by distance constraints derived from cross-links. The overall structural model of CopA resembles the Serca1 structure, but reveals distinctive features of CPx-type ATPases. A prominent feature is the positioning of the heavy metal binding domain. It features an orientation of the Cu binding ligands which is appropriate for the interaction with Cu-loaded metallochaperones in solution. Moreover, a novel model of the architecture of the intramembranous Cu binding sites could be derived.

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Section: Discussionmentioning

confidence: 99%

Structural model of the CopA copper ATPase of Enterococcus hirae based on chemical cross-linking

et al. 2008

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“…Due to the chemical similarities between Cu ϩ and Ag ϩ and between Zn 2ϩ and Cd 2ϩ , it is not surprising that P-type exporters specialize in detoxifying one of these two subsets (7,13,14,21,23,24). We have determined the metal specificities for 5 previously uncharacterized P-type transition-metal exporters, along with tentative assignments for 3 additional pumps exhibiting low levels of activity.…”

Section: Discussionmentioning

confidence: 99%

“…The CPC motif of TM6 of HmtA is followed by the conserved Ser of Zn 2ϩ /Cd 2ϩ exporters, whereas TM8 lacks the conserved Asp residue that has been shown to be functionally essential to this subgroup (28). The lack of conservation of this catalytically crucial Asp is highlighted by the conservation of a Ser residue that has been shown to participate in Cu ϩ /Ag ϩ recognition (23).…”

Section: Putative Amino Acid Motifs Contributing To Substrate Specifimentioning

confidence: 99%

“…Another is Asp of TM8 of Zn 2ϩ /Cd 2ϩ pumps, which is paralleled by Pro in Cu ϩ /Ag ϩ exporters. Importantly, mutational analyses of these residues (23,28) have demonstrated their importance for metal recognition. The newly identified WIYR/K motif (TM6) and Lys of TM7 (both specific to Zn 2ϩ /Cd 2ϩ pumps) present attractive targets for future studies.…”

Section: Putative Amino Acid Motifs Contributing To Substrate Specifimentioning

confidence: 99%

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A P-type ATPase importer that discriminates between essential and toxic transition metals

Lewinson

Lee

Rees

2009

Proc. Natl. Acad. Sci. U.S.A.

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Transition metals, although being essential cofactors in many physiological processes, are toxic at elevated concentrations. Among the membrane-embedded transport proteins that maintain appropriate intracellular levels of transition metals are ATP-driven pumps belonging to the P-type ATPase superfamily. These metal transporters may be differentiated according to their substrate specificities, where the majority of pumps can extrude either silver and copper or zinc, cadmium, and lead. In the present report, we have established the substrate specificities of nine previously uncharacterized prokaryotic transition-metal P-type ATPases. We find that all of the newly identified exporters indeed fall into one of the two above-mentioned categories. In addition to these exporters, one importer, Pseudomonas aeruginosa Q9I147, was also identified. This protein, designated HmtA (heavy metal transporter A), exhibited a different substrate recognition profile from the exporters. In vivo metal susceptibility assays, intracellular metal measurements, and transport experiments all suggest that HmtA mediates the uptake of copper and zinc but not of silver, mercury, or cadmium. The substrate selectivity of this importer ensures the high-affinity uptake of essential metals, while avoiding intracellular contamination by their toxic counterparts.membrane proteins ͉ transition-metal homeostasis ͉ heavy metal transporters ͉ P 1B ATPases M aintaining transition-metal homeostasis presents a unique challenge to all living organisms. Transition metals such as zinc, copper, and iron are essential to many physiological processes but are also toxic at elevated concentrations. Other metals, such as cadmium, silver, and mercury, exhibit acute toxicity by binding to macromolecules and perturbing their physiological interactions (1, 2). A diversity of membrane-embedded transporters translocate metals across the cell membrane, underlining the importance and the delicacy of this process. In prokaryotes, the concerted action of members of the RND, ABC, CDF, and P-type ATPase superfamilies functions to maintain appropriate intracellular concentrations of essential and toxic transition metals (3). In humans, perturbations in metal homeostasis lead to degenerative syndromes, cancer, and Wilson's and Menkes' diseases (4, 5).P-type ATPases constitute a superfamily of transporters characterized by unique signature motifs. The hallmark of this family of pumps is the formation of a phosphoenzyme intermediate (hence the name P-type ATPase), by the transfer of the ␥-phosphate from ATP to the highly conserved DKTGT motif (6). A family of P-type ATPases catalyzing the translocation of transition metals (also referred to as heavy-metal or type P 1B ATPases) has been identified (7,8), with members present in all kingdoms of life. Such P-type ATPases harbor a Cys-Pro-Xaa (or Xaa-Pro-Cys) motif, with Xaa ϭ Cys, Ser, or His, in their sixth transmembrane helix (TM6) that is essential for transport activity (7, 9, 10). Sequence homology of transition-metal P-type pump...

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“…Copper coordination in the M-domain is essential for ATPasedependent copper transport, and therefore three transmembrane helices contain invariant residues that coordinate copper in the M-domain (Fig. 1); the Cys-Pro-Cys (CPC) motif in helix 6 [24], the Asp-Tyr (YN) motif in helix 7, and the Met-Xaa-Xaa-Ser (MxxS) motif in helix 8 coordinate copper in the M-domain [26][27][28]. Copper export by ATP7A and ATP7B is essentially dependent on the cyclic ATP hydrolysis activity [25].…”

Section: Introductionmentioning

confidence: 99%

Posttranslational regulation of copper transporters

Berghe

Klomp

2009

J Biol Inorg Chem

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Identification of the Transmembrane Metal Binding Site in Cu+-transporting PIB-type ATPases

Cited by 70 publications

References 59 publications

Structural model of the CopA copper ATPase of Enterococcus hirae based on chemical cross-linking

Structural model of the CopA copper ATPase of Enterococcus hirae based on chemical cross-linking

A P-type ATPase importer that discriminates between essential and toxic transition metals

Posttranslational regulation of copper transporters

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