Nucleotide Interactions with Membrane-bound Transporter Associated with Antigen Processing Proteins

Abstract

The transporter associated with antigen processing (TAP) contains two nucleotide-binding domains (NBD) in the TAP1 and TAP2 subunits. When expressed as individual subunits or domains, TAP1 and TAP2 NBD differ markedly in their nucleotide binding properties. We investigated whether the two nucleotide-binding sites of TAP1/TAP2 complexes also differed in their nucleotide binding properties. To facilitate electrophoretic separation of the subunits when in complex, we used TAP complexes in which one of the subunits was expressed as a fluorescent protein fusion construct. In binding experiments at 4°C using the photo-cross-linkable nucleotide analogs 8-azido-[␥-32 P]ATP and 8-azido-[␣-32 P]ADP, TAP2 was found to have reduced affinity for nucleotides compared with TAP1, when the two proteins were separately expressed. Complex formation with TAP1 enhanced the binding affinity of the TAP2 nucleotide-binding site for both nucleotides. Binding analyses with mutant TAP complexes that are deficient in nucleotide binding at one or both sites provided evidence for the existence of two ATP-binding sites with relatively similar affinities in TAP1/TAP2 complexes. TAP1/TAP2 NBD interactions appear to contribute at least in part to enhanced nucleotide binding at the TAP2 site upon TAP1/TAP2 complex formation. Binding analyses with mutant TAP complexes also demonstrate that the extent of TAP1 labeling is dependent upon the presence of a functional TAP2 nucleotide-binding site.The transporter associated with antigen processing (TAP) 1 complex is an integral part of the major histocompatibility complex class I antigen presentation pathway (1). TAP is comprised of two related subunits, TAP1 and TAP2, which are necessary and sufficient for peptide translocation from the cytosol into the endoplasmic reticulum (2, 3). Both proteins contain an N-terminal membrane-spanning region (MSR) and a C-terminal nucleotide-binding domain (NBD). Peptide translocation by TAP complexes is preceded by peptide binding to the cytosolic face of TAP1/TAP2 complexes, a step that appears to be nucleotide binding-independent, at least at low temperatures (4, 5). However, the presence of ATP or ADP is critical for maintaining the structural stability of TAP complexes at physiological temperatures (6), and TAP mutants that are defective in nucleotide binding at the TAP2 site lose their ability to bind peptide at 37°C but not at lower temperatures (7). ATP hydrolysis is critical for peptide translocation across the endoplasmic reticulum membrane (8), and a peptide-stimulated ATPase activity has been described for TAP complexes (9).The role of each NBD during peptide translocation is not well understood. Mutations in both NBD have been shown to affect peptide translocation efficiency, although mutations on TAP2 NBD generally have more severe consequences. Based upon mutagenesis studies at structurally analogous residues on TAP1 and TAP2, experiments from several labs have resulted in the postulation of functional distinctions between a TAP1 and a TAP2 NBD during t...

Functional Non-equivalence of ATP-binding Cassette Signature Motifs in the Transporter Associated with Antigen Processing (TAP)

“…In summary, we concluded that these single site mutations do not influence ATP binding to TAP. This is in agreement with studies on single TAP subunits and TAP complexes, all showing similar affinities for ATP in the micromolar range (33).…”

“…Accordingly, mutations in ATP-binding site I (formed by Walker A and B motifs of TAP1 and C-loop of TAP2) are tolerated to a certain extent, whereas TAP complexes with the same mutations in site II are inactive. These findings are in harmony with studies on TAP complexes containing Walker A mutations in TAP1 (site I), which are still able to transport peptides although with very low efficiency, whereas equivalent mutations at site II fully disrupt peptide translocation (15,33). Notably, all C-loop mutants are active in peptide and ATP binding, indicating that these mutations influence the ATPase activity of TAP.…”

Antigen processing and presentation: TAPping into ABC transporters

“…However, at some stage during the transport cycle, TAP2 binds and hydrolyzes ATP, which is critical for driving conformational changes associated with transport [46] -TAP2 forms the main contacts to the ATP hydrolyzed in the consensus ATPase site [39]. While it has been suggested that TAP2 only displays a preference for ADP when isolated from TAP1 and that the physiological TAP1/2 heterodimer has no such nucleotide preferences [47], another possibility is that the nucleotide specificity of TAP2 is regulated during a transport cycle. Using chimeras between the TAP1 and TAP2 proteins, it was observed that a short stretch of amino acids near the C-terminus is responsible for the unique nucleotide specificities [48].…”

TAP and TAP-like — Brothers in arms?

“…The apparent dissociation constant of Mg•ATP of 90± 12 μM for TAPL was derived. This value is in the same range as that of other ABC transporters (Horn et al 2003;Lapinski et al 2003;Qu et al 2003). A lower K m(ATP) value than the K D for TAP indicates that the ATP-binding step is followed by intermediates before final hydrolysis occurs (Fersht 1997).…”