ATPase gene transfer and mutational analysis of the cation translocation mechanism.
The peptide segment interposed between cation binding and phosphorylation domains retains a high degree of homology in all cation transport ATPases. Mutational analysis and chimeric replacements of Ca2+ ATPase components with corresponding Na+,K(+)-ATPase components indicate that this segment is utilized by various cation ATPases as a common structural device for a long-range functional linkage of enzyme phosphorylation and cation transport. Vectorial displacement of bound cation is rendered possible by a transmembrane channel formed by four clustered helices (M4, M5, M6, and M8). Originating from the four helices, the oxygen functions of Glu309, Glu771, Thr799, Asp800, and Glu908 form a duplex Ca2+ binding site in the middle of the channel, while Lys297 seals the luminal end of the channel with its positively charged side chain. The perturbation triggered by enzyme phosphorylation is apparently transmitted through the linkage segment to produce rotational displacement of the M4 helix with minimal change of secondary structure. The cation binding site is thereby disrupted and the Lys297 side chain removed, permitting Ca2+ to dissociate in exchange for H+ and to flow through the luminal end of the channel.
Inesi
G
G
Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore 21201, USA.
Lewis
D
D
Sumbilla
C
C
Nandi
A
A
Kirtley
M
M
Ordahl
C P
CP
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004721:Endoplasmic Reticulum / Q000201:enzymology; D018014:Gene Transfer Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D011993:Recombinant Fusion Proteins; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium