Cryoelectron Microscopy of an ATP-dependent Cu pump from Archaeoglobus fulgidis

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CopA is an ATP-dependent Cu pump that belongs to the P1 subfamily of P-type ATPases. This subfamily shares core catalytic domains with other members of the family, such as Ca-ATPase and Na,K-ATPase, for which the X-ray crystal structures have been determined. Members of the P1 family are characterized by extended N-termini, which contain tandem repeats of metal binding domains (MBD). Compared to other P-type ATPases, P1 pumps have two extra transmembrane helices near the N-terminus and truncation of four C-terminal transmembrane helices. CopA from Archaeoglobus fulgidis is an unusual P1 pump because, in addition to the N-terminal MBD, it also has a C-terminal MBD. Although there is some uncertainty about their precise function, MBDs have been proposed to participate in regulation, in targeting, and in transfer of Cu to the transport sites. To study the structural disposition of the MBDs, we have expressed constructs of CopA with truncation of the N-terminus and the C-terminus either individually or together. We have used cryoelectron microscopy and helical reconstruction to determine structures of these constructs. Comparison of the double truncation with the C-terminal truncation revealed the location of the N-terminal domain. We constructed an atomic model by fitting X-ray crystal structures of relevant fragments into our map, which suggests a regulatory role for the N-terminal domain. By imaging somewhat wider tubular crystals with better order, we have been able to determine the structure of the C-terminal truncation at higher resolution. This new structure reveals the architecture of the transmembrane domain and allows us to place the extra two transmembrane helices with greater precision. Additionally, we are working on a structure of the N-terminal truncation, which should reveal the location of the C-terminal domain and help us determine its role in Cu transport.