Structural basis of ion uptake in copper-transporting P1B-type ATPases

Abstract

Copper is essential for living cells, yet toxic at elevated concentrations. Class 1B P-type (P1B-) ATPases are present in all kingdoms of life, facilitating cellular export of transition metals including copper. P-type ATPases follow an alternating access mechanism, with inward-facing E1 and outward-facing E2 conformations. Nevertheless, no structural information on E1 states is available for P1B-ATPases, hampering mechanistic understanding. Here, we present structures that reach 2.7 Å resolution of a copper-specific P1B-ATPase in an E1 conformation, with complementing data and analyses. Our efforts reveal a domain arrangement that generates space for interaction with ion donating chaperones, and suggest a direct Cu+ transfer to the transmembrane core. A methionine serves a key role by assisting the release of the chaperone-bound ion and forming a cargo entry site together with the cysteines of the CPC signature motif. Collectively, the findings provide insights into P1B-mediated transport, likely applicable also to human P1B-members.

Diverse roles of the metal binding domains and transport mechanism of copper transporting P-type ATPases

“…Separately, we instead prepared the sample with a combination of copper and reducing agent, with the ambition to recover an early E1 conformation. We also studied the effect of no such supplementation to HMA4, which has previously yielded an early E1 state of AfCopA 26 . These various conditions yielded four structures, determined at overall resolutions of 3.3 (HMA4 BeF ), 3.2 (HMA4 AlF ), 3.6 (HMA4 apo ), and 3.7 Å (HMA4 Cu ) (Fig.…”

“…14 ). In comparison to available structural information on P 1B-1 -ATPases, HMA4 apo/Cu are most alike the early inward-open E1 structure of AfCopA (RMSD 4.1 and 3.8 Å) 26 , while HMA4 AlF /HMA4 BeF are most reminiscent of the outward-facing structure of LpCopA captured using AlF 4 - (RMSD 1.8 and 1.7 Å, Supplementary Table 2 ) 24 . Equivalently, the position of the omnipresent TGE- and DKTGT-motifs in the soluble domains shift from distant in HMA4 apo /HMA4 Cu to adjacent to each other in HMA4 BeF /HMA4 AlF , the latter representing a requirement to permit dephosphorylation (Fig.…”

“…It has previously been proposed that ATP turn-over of the soluble domains in P 1B -ATPases is different from other P-type ATPase classes, but the validity of these studies could be questioned considering the spread in origin of the compared members and because different experimental techniques have been used to obtain the structures 23 – 26 , 33 . Our HMA4 structures clearly support that the E1, but not E2P, conformation of P 1B-1 -ATPases is unique.…”

“…Hitherto, structural information on Cu + -ATPases is limited to the E2P and a subsequent E2.P i transition state of dephosphorylation of CopA from Legionella pneumophila (LpCopA) 23 , 25 , the E2P state of ATP7B from Xenopus tropicalis (XtATP7B) 24 , and an E1 state of CopA from Archaeoglobus fulgidus (AfCopA) and hATP7B 26 , 33 . These structures generally lack the MBDs, but the structural information for XtATP7B revealed the presence of the two MBDs closest to the core, MBD −2 and MBD −1 (previously MBD5 and MBD6, respectively), while the hATP7B structure displayed presence of one MBD (see more below).…”

Susceptibility of Cu(I) transport ATPases to sodium dodecyl sulfate. Relevance of the composition of the micellar phase

“…Cu(I) transport ATPases constitute a group of integral membrane proteins involved in active transport of Cu(I) across cell membranes coupled to the hydrolysis of ATP [1]. They belong to the P-type ATPases family, all sharing some common structural features [2] including a transmembrane region -composed by 6-8 α-helices arranged in a helix bundle- and three cytoplasmic domains: the catalytic one, composed by subdomains P (phosphorylation) and N (nucleotide binding), the so-called actuator (A) and a metal binding domain at the N terminal extreme (N-MBD) (Fig. 1).…”