Engineering ATPase Activity in the Isolated ABC Cassette of Human TAP1

Abstract

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. ATP-binding cassette (ABC)3 -transporters form a large superfamily of primary transmembrane proteins (1), which ultimately use the energy of ATP hydrolysis to translocate their substrate or allocrite (2), the term we prefer to distinguish the transported substrate from ATP. Found in all three kingdoms of life, ABC-transporters contain certain sequence motifs such as the Walker A and B motifs, the C-loop (ABC signature motif), and the D-loop (3, 4). These sequences serve as diagnostic tools to identify ABC-transporters. Furthermore, all members of this family show a modular architecture composed of two nucleotide-binding domains (NBDs), which contain all the conserved sequence motifs, and two transmembrane domains (TMDs). In archea and eubacteria, these four modules are generally encoded on separate genes, whereas they are fused on a single polypeptide chain ("full-size" transporter) in eukarya. Additionally, fusions of one NBD and one TMD, which are called "half-size" transporters, are found in all organisms. The functional unit of these systems would be a homo-or heterodimer of two half-size transporters. Examples of the latter are hemolysin B (HlyB) from Escherichia coli (5) and the human transporter associated with antigen processing (TAP) (6).Human TAP, located in the membrane of the endoplasmic reticulum (ER), is a key component of major histocompatibility complex class I-mediated adaptive immunity (7). The functional TAP complex is a heterodimer composed of TAP1 and TAP2, both of which comprise a TMD-NBD fusion. It is now firmly established that the allocrites of TAP, antigenic peptides, which are generated in the cytosol by proteasomal degradation (8, 9), are transported in an ATP-dependent manner across the ER membrane for subsequent loading of major histocompatibility complex class I molecules (10). After passing the ER quali...

Direct Sensing of Intracellular pH by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl− Channel

“…This suggests that H-bond formation at site 2 enhances channel activity by accelerating NBD dimerization and stabilizing the ATP bound conformation of the NBD1: NBD2 dimer. Consistent with this idea, acidic pH enhanced greatly the formation of an engineered TAP1 homodimer (11).…”

“…Mechanism of ATP Hydrolysis at Site 2-A common feature of the ATP binding sites of the ABC transporters CFTR and TAP is their asymmetric ATPase activity (11,29). In an engineered human TAP1 homodimer, the ATP-binding site with ATPase activity contains a catalytic dyad composed of a histidine residue in the H-loop and a glutamic acid residue distal to the Walker B motif (11,31).…”

“…In an engineered human TAP1 homodimer, the ATP-binding site with ATPase activity contains a catalytic dyad composed of a histidine residue in the H-loop and a glutamic acid residue distal to the Walker B motif (11,31). However, instead of hydrolyzing ATP by general base catalysis, this dyad employs substrate-assisted catalysis (SAC) for this reaction (11,31). In SAC, the interaction of the substrate, ATP, with catalytic water via its ␥-phosphate is stabilized by an H-bond between the dyad histidine and the ␥-phosphate of ATP (31).…”

“…However, the ATPase activity of some ABC transporters (e.g. the transporter associated with antigen processing (TAP)) is pH i -dependent (11), while the ATPase activity of an NBD1-RD-CFTR fusion protein is optimal at neutral pH (12). These data raise the possibility that pH i might regulate directly CFTR activity.…”

The DrrAB Efflux System of Streptomyces peucetius Is a Multidrug Transporter of Broad Substrate Specificity

“…Most commonly, a glutamine replaces the histidine in the Switch motif, and an aspartate replaces the glutamate in the Walker B region. When present (for example, in TAP1 and LmrC), these deviations are seen to result in asymmetrical ATP binding pockets with one site being catalytically non-functional (33,34). Interestingly, however, despite the presence of the non-canonical glutamine residue (Gln 197 followed by Tyr 198 , resulting in the QY sequence) in the Switch motif of DrrA (and its close prokaryotic homologs, Fig.…”