Perria Christopher L, Rajamanickam Vijayalakshmi, Lapinski Philip E, Raghavan Malini
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0620, USA.
J Biol Chem. 2006 Dec 29;281(52):39839-51. doi: 10.1074/jbc.M605492200. Epub 2006 Oct 26.
The transporter associated with antigen processing (TAP), a member of the ATP binding cassette (ABC) family of transmembrane transporters, transports peptides across the endoplasmic reticulum membrane for assembly of major histocompatibility complex class I molecules. Two subunits, TAP1 and TAP2, are required for peptide transport, and ATP hydrolysis by TAP1.TAP2 complexes is important for transport activity. Two nucleotide binding sites are present in TAP1.TAP2 complexes. Compared with other ABC transporters, the first nucleotide binding site contains non-consensus catalytic site residues, including Asp(668) in the Walker B region of TAP1 (in place of a highly conserved glutamic acid), and Gln(701) in the switch region of TAP1 (in place of a highly conserved histidine). At the second nucleotide binding site, a glutamic acid (TAP2 Glu(632)) follows the Walker B motif, and the switch region contains a histidine (TAP2 His(661)). We found that alterations at Glu(632) and His(661) of TAP2 significantly reduced peptide translocation and/or TAP-induced major histocompatibility complex class I surface expression. Alterations of TAP1 Asp(668) alone or in combination with TAP1 Gln(701) had only small effects on TAP activity. Thus, the naturally occurring Asp(668) and Gln(701) alterations of TAP1 are likely to contribute to attenuated catalytic activity at the first nucleotide binding site (the TAP1 site) of TAP complexes. Due to its enhanced catalytic activity, the second nucleotide binding site (the TAP2 site) appears to be the main site driving peptide transport. A mechanistic model involving one main active site is likely to apply to other ABC transporters that have an asymmetric distribution of catalytic site residues within the two nucleotide binding sites.
与抗原加工相关的转运体（TAP）是跨膜转运体ATP结合盒（ABC）家族的成员，它将肽转运过内质网膜，用于组装主要组织相容性复合体I类分子。肽转运需要两个亚基，即TAP1和TAP2，并且TAP1.TAP2复合体的ATP水解对于转运活性很重要。TAP1.TAP2复合体中有两个核苷酸结合位点。与其他ABC转运体相比，第一个核苷酸结合位点包含非保守的催化位点残基，包括TAP1的沃克B区域中的Asp（668）（取代高度保守的谷氨酸），以及TAP1的开关区域中的Gln（701）（取代高度保守的组氨酸）。在第二个核苷酸结合位点，一个谷氨酸（TAP2 Glu（632））紧跟在沃克B基序之后，并且开关区域包含一个组氨酸（TAP2 His（661））。我们发现，TAP2的Glu（632）和His（661）发生改变会显著降低肽转运和/或TAP诱导的主要组织相容性复合体I类分子的表面表达。单独改变TAP1的Asp（668）或与TAP1的Gln（701）联合改变对TAP活性只有很小的影响。因此，TAP1天然存在的Asp（668）和Gln（701）改变可能导致TAP复合体第一个核苷酸结合位点（TAP1位点）的催化活性减弱。由于其增强的催化活性，第二个核苷酸结合位点（TAP2位点）似乎是驱动肽转运的主要位点。一个涉及一个主要活性位点的机制模型可能适用于其他在两个核苷酸结合位点内催化位点残基呈不对称分布的ABC转运体。
