The Architecture of CopA from Archeaoglobus fulgidus Studied by Cryo-Electron Microscopy and Computational Docking

Abstract

Summary CopA uses ATP to pump Cu across cell membranes. X-ray crystallography has defined atomic structures of several related P-type ATPases. We have determined a structure of CopA at 10 Å resolution by cryo-electron microscopy of a new crystal form and used computational molecular docking to study the interactions between the N-terminal metal binding domain (NMBD) and other elements of the molecule. We found that the shorter chain lipids used to produce these crystals are associated with movements of the cytoplasmic domains, with a novel dimer interface and with disordering of the NMBD, thus offering evidence for the transience of its interaction with the other cytoplasmic domains. Docking identified a binding site that matched the location of the NMBD in our previous structure by cryo-electron microscopy, allowing a more detailed view of its binding configuration and further support for its role in autoinhibition.

The Confluence of Heavy Metal Biooxidation and Heavy Metal Resistance: Implications for Bioleaching by Extreme Thermoacidophiles

“…In the hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidus, two copper-transporting ATPases, CopA and CopB, were isolated, characterized and found to activated on Cu(I) and Cu(II), respectively [194,195]. The CopA of A. fulgidus has been extensively studied with regards to its metal binding, actuator, ATP binding domains, and interaction with chaperones [196][197][198][199][200][201][202].…”

Characterization of the P IB -Type ATPases Present in Thermus thermophilus

“…The P IB -ATPases include the two human Cu ϩ -ATPases in which mutations are responsible for Menkes and Wilson diseases (17). Structures have been reported for the Cu ϩ pumps from Legionella pneumophila by X ray (28) and from Archaeoglobus fulgidus by cryoelectron microscopy and computer docking (2).…”

Membrane protein structure determination by electron crystallography

“…Like AQP0, crystals of the Cu transporter CopA were produced by reconstitution into exogenous lipids. Unlike AQP0, a radically different crystal form resulted from changing the lipid from DOPC to a mixture of DMPC and DOPE (4:1 weight ratio), even though the crystallization conditions otherwise remained the same [11]. Although the resolution was too low to evaluate the lipid interactions at an atomic scale, it was clear that the membrane domain tilted by 30° in DMPC, which is consistent with the 25% decrease in thickness of the hydrophobic core of DMPC membranes relative to DOPC [12].…”

“…The first report showed changes in the cytoplasmic domains consistent with addition of a phosphate analogue [26]. Unpublished comparison of the more recent structure from electron crystallography [11] with the even more recent structure from X-ray crystallography [27] not only confirms the influence of phosphate analogues on the juxtaposition of cytoplasmic domains, but also illustrates how bilayer-induced shear of the membrane helices pulls on one of the cytoplasmic domains and pushes the pump towards the conformation that binds Cu + (Fig. 2a–d).…”