Wang Guoli, Yamasaki Kazuo, Daiho Takashi, Suzuki Hiroshi
Department of Biochemistry, Asahikawa Medical College, Asahikawa 078-8510, Japan.
J Biol Chem. 2005 Jul 15;280(28):26508-16. doi: 10.1074/jbc.M503789200. Epub 2005 May 17.
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+.
通过丙氨酸和丝氨酸替换，研究了肌浆网Ca2 + -ATP酶的驱动域（A）和磷酸化域（P）之间界面上七个疏水残基的功能作用。所检测的残基包括A域上的Ile179 / Leu180 / Ile232，P域上的Val705 / Val726，以及连接A域和M2（第二个跨膜螺旋）的环上的Leu119 / Tyr122。在未结合Ca2 +的E2（未磷酸化）和E2P（磷酸化）状态的Ca2 + -ATP酶晶体结构中，这些残基聚集在Tyr122周围形成疏水簇，但在结合Ca2 +的E1（未磷酸化）和E1P（磷酸化）状态的晶体结构中相距较远。还将这些替换效应与A域/M3连接子上的Ile235以及A域的T181GE的替换效应进行了比较，因为它们紧邻Tyr122簇。所有这些替换几乎完全抑制了ATP酶活性，但不抑制由ATP形成Ca2 +激活的E1P。Ile235和T181GE的替换阻断了E1P向E2P的转变，而Tyr122簇中的替换阻断了随后的E2P水解。Ile235和Glu183的替换也阻断了EP水解。结果表明，Tyr122簇在E1P向E2P转变过程中形成，以配置催化位点并使Glu183正确定位以水解酰基磷酸。A域/M3连接子上的Ile235可能与A域形成疏水相互作用，从而使该连接子的应变在这些过程中用于A域的大运动。因此，Tyr122簇、Ile235和T181GE似乎具有不同的作用，并且在处理磷酸化中间体以转运Ca2 +的连续事件中至关重要。
