Fusca Tiziana, Bonza Maria Cristina, Luoni Laura, Meneghelli Silvia, Marrano Claudia Adriana, De Michelis Maria Ida
Dipartimento di Biologia L. Gorini, Università di Milano, Istituto di Biofisica del Consiglio Nazionale delle Ricerche, Sezione di Milano, via G. Celoria 26, Milano 20133, Italy.
J Biol Chem. 2009 Nov 6;284(45):30881-8. doi: 10.1074/jbc.M109.006148. Epub 2009 Sep 9.
ACA8 is a type 2B Ca(2+)-ATPase having a regulatory N terminus whose auto-inhibitory action can be suppressed by binding of calmodulin (CaM) or of acidic phospholipids. ACA8 N terminus is able to interact with a region of the small cytoplasmic loop connecting transmembrane domains 2 and 3. To determine the role of this interaction in auto-inhibition we analyzed single point mutants produced by mutagenesis of ACA8 Glu(252) to Asn(345) sequence. Mutation to Ala of any of six tested acidic residues (Glu(252), Asp(273), Asp(291), Asp(303), Glu(302), or Asp(332)) renders an enzyme that is less dependent on CaM for activity. These results highlight the relevance in ACA8 auto-inhibition of a negative charge of the surface area of the small cytoplasmic loop. The most deregulated of these mutants is D291A ACA8, which is less activated by controlled proteolysis or by acidic phospholipids; the D291A mutant has an apparent affinity for CaM higher than wild-type ACA8. Moreover, its phenotype is stronger than that of D291N ACA8, suggesting a more direct involvement of this residue in the mechanism of auto-inhibition. Among the other produced mutants (I284A, N286A, P289A, P322A, V344A, and N345A), only P322A ACA8 is less dependent on CaM for activity than the wild type. The results reported in this study provide the first evidence that the small cytoplasmic loop of a type 2B Ca(2+)-ATPase plays a role in the attainment of the auto-inhibited state.
ACA8是一种2B型Ca(2+) -ATP酶，其N端具有调节作用，钙调蛋白（CaM）或酸性磷脂的结合可抑制其自身抑制作用。ACA8的N端能够与连接跨膜结构域2和3的小细胞质环区域相互作用。为了确定这种相互作用在自身抑制中的作用，我们分析了通过对ACA8的Glu(252)至Asn(345)序列进行诱变产生的单点突变体。六个测试的酸性残基（Glu(252)、Asp(273)、Asp(291)、Asp(303)、Glu(302)或Asp(332)）中的任何一个突变为丙氨酸都会产生一种对CaM活性依赖性较低的酶。这些结果突出了小细胞质环表面积负电荷在ACA8自身抑制中的相关性。这些突变体中失调最严重的是D291A ACA8，其通过可控蛋白水解或酸性磷脂激活的程度较低；D291A突变体对CaM的表观亲和力高于野生型ACA8。此外，其表型比D291N ACA8更强，表明该残基在自身抑制机制中更直接地参与其中。在其他产生的突变体（I284A、N286A、P289A、P322A、V344A和N345A）中；只有P322A ACA8对CaM活性的依赖性比野生型低。本研究报告的结果提供了第一个证据表明2B型Ca(2+) -ATP酶的小细胞质环在达到自身抑制状态中起作用。
