Engineering ATPase activity in the isolated ABC cassette of human TAP1
Engineering ATPase activity in the isolated ABC cassette of human TAP1
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DOI：
10.1074/jbc.m601131200
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发表时间：
2006-09-15
影响因子：
4.8
通讯作者：
Schmitt, Lutz
中科院分区：
文献类型：
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作者：
Ernst, Robert;Koch, Joachim;Schmitt, Lutz
与抗原处理相关的人类转运蛋白(TAP)将抗原肽从胞浆转运到内质网腔。TAP的功能单位是由TAP1和TAP2亚基组成的异源二聚体，这两个亚基都是ABC转运蛋白家族的成员。ABC转运蛋白是依赖于ATP的泵、通道或受体，由四个模块组成：两个核苷酸结合域(NBD)和两个跨膜域(TMD)。尽管TMD在序列上相当不同，但NBD在结构和功能上是保守的。有趣的是，TAP1的NBD在氨基酸位置上包含突变，这些突变被认为是催化活性所必需的。TAP1不是谷氨酸，而是天冬氨酸和谷氨酰胺，而不是保守的组氨酸，后者被认为是关键。我们利用这种退化来评估这两种氨基酸对工程TAP1-NBD突变体ATPase活性的单独贡献。根据我们的结果，推测了这两种基本氨基酸在突变的TAP1运动区的ATP水解中的催化等级。
The human transporter associated with antigen processing ( TAP) translocates antigenic peptides from the cytosol into the endoplasmic reticulum lumen. The functional unit of TAP is a heterodimer composed of the TAP1 and TAP2 subunits, both of which are members of the ABC-transporter family. ABC-transporters are ATP-dependent pumps, channels, or receptors that are composed of four modules: two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Although the TMDs are rather divergent in sequence, the NBDs are conserved with respect to structure and function. Interestingly, the NBD of TAP1 contains mutations at amino acid positions that have been proposed to be essential for catalytic activity. Instead of a glutamate, proposed to act as a general base, TAP1 contains an aspartate and a glutamine instead of the conserved histidine, which has been suggested to act as the linchpin. We used this degeneration to evaluate the individual contribution of these two amino acids to the ATPase activity of the engineered TAP1-NBD mutants. Based on our results a catalytic hierarchy of these two fundamental amino acids in ATP hydrolysis of the mutated TAP1 motor domain was deduced.