Critical hydrophobic interactions between phosphorylation and actuator domains of Ca2+-ATPase for hydrolysis of phosphorylated intermediate.
Functional roles of seven hydrophobic residues on the interface between the actuator (A) and phosphorylation (P) domains of sarcoplasmic reticulum Ca2+-ATPase were explored by alanine and serine substitutions. The residues examined were Ile179/Leu180/Ile232 on the A domain, Val705/Val726 on the P domain, and Leu119/Tyr122 on the loop linking the A domain and M2 (the second transmembrane helix). These residues gather to form a hydrophobic cluster around Tyr122 in the crystal structures of Ca2+-ATPase in Ca2+-unbound E2 (unphosphorylated) and E2P (phosphorylated) states but are far apart in those of Ca2+-bound E1 (unphosphorylated) and E1P (phosphorylated) states. The substitution-effects were also compared with those of Ile235 on the A domain/M3 linker and those of T181GE of the A domain, since they are in the immediate vicinity of the Tyr122-cluster. All these substitutions almost completely inhibited ATPase activity without inhibiting Ca2+-activated E1P formation from ATP. Substitutions of Ile235 and T181GE blocked the E1P to E2P transition, whereas those in the Tyr122-cluster blocked the subsequent E2P hydrolysis. Substitutions of Ile235 and Glu183 also blocked EP hydrolysis. Results indicate that the Tyr122-cluster is formed during the E1P to E2P transition to configure the catalytic site and position Glu183 properly for hydrolyzing the acylphosphate. Ile235 on the A domain/M3 linker likely forms hydrophobic interactions with the A domain and thereby allowing the strain of this linker to be utilized for large motions of the A domain during these processes. The Tyr122-cluster, Ile235, and T181GE thus seem to have different roles and are critical in the successive events in processing phosphorylated intermediates to transport Ca2+.
Wang
Guoli
G
Department of Biochemistry, Asahikawa Medical College, Asahikawa 078-8510, Japan.
Yamasaki
Kazuo
K
Daiho
Takashi
T
Suzuki
Hiroshi
H
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000409:Alanine / Q000737:chemistry; D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006868:Hydrolysis; D007532:Isoleucine / Q000737:chemistry; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000737:chemistry; D010766:Phosphorylation; D011188:Potassium / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D012694:Serine / Q000737:chemistry; D013696:Temperature; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry
D010710:Phosphates; D007532:Isoleucine; D014443:Tyrosine; D012694:Serine; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D007930:Leucine; D000409:Alanine; D011188:Potassium; D002118:Calcium