Archaeoglobus fulgidus CopB is a thermophilic Cu2+-ATPase: functional role of its histidine-rich-N-terminal metal binding domain.
P1B-type ATPases transport heavy metal ions across cellular membranes. Archaeoglobus fulgidus CopB is a member of this subfamily. We have cloned, expressed in Escherichia coli, and functionally characterized this enzyme. CopB and its homologs are distinguished by a metal binding sequence Cys-Pro-His in their sixth transmembrane segment (H6) and a His-rich N-terminal metal binding domain (His-N-MBD). CopB is a thermophilic protein active at 75 degrees C and high ionic strength. It is activated by Cu2+ with high apparent affinity (K1/2 = 0.28 microm) and partially by Cu+ and Ag+ (22 and 55%, respectively). The higher turnover was associated with a faster phosphorylation rate in the presence of Cu2+. A truncated CopB lacking the first 54 amino acids was constructed to characterize the His-N-MBD. This enzyme showed reduced ATPase activity (50% of wild type) but no changes in metal selectivity, ATP dependence, or phosphorylation levels. However, a slower rate of dephosphorylation of the E2P(Cu2+) form was observed for truncated CopB. The data suggest that the presence of the His residue in the putative transmembrane metal binding site of CopB determines a selectivity for this enzyme that is different for that observed in Cu+/Ag+-ATPases carrying a Cys-Pro-Cys sequence. The His-NMBD appears to have a regulatory role affecting the metal transport rate by controlling the metal release/dephosphorylation rates.
Mana-Capelli
Sebasián
S
Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, Massachusetts 01609, USA.
Mandal
Atin K
AK
Argüello
José M
JM
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D019843:Archaeal Proteins; D019611:Archaeoglobus fulgidus / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003300:Copper / Q000737:chemistry; D000079922:Copper Transport Proteins; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D007477:Ions; D007700:Kinetics; D008670:Metals / Q000737:chemistry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010802:Phylogeny; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013997:Time Factors
D019843:Archaeal Proteins; D001425:Bacterial Outer Membrane Proteins; D027682:Cation Transport Proteins; C489503:CopB protein, Archaea; D000079922:Copper Transport Proteins; D007477:Ions; D008670:Metals; D006639:Histidine; D003300:Copper; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases