Peptides Induce ATP Hydrolysis at Both Subunits of the Transporter Associated with Antigen Processing

Abstract

The transporter associated with antigen processing (TAP) plays a key role in the adaptive immune response by pumping antigenic peptides into the endoplasmic reticulum for subsequent loading of major histocompatibility complex class I molecules. TAP is a heterodimer consisting of TAP1 and TAP2. Each subunit is composed of a transmembrane domain and a nucleotide-binding domain, which energizes the peptide transport. To analyze ATP hydrolysis of each subunit we developed a method of trapping 8-azido-nucleotides to TAP in the presence of phosphate transition state analogs followed by photocross-linking, immunoprecipitation, and high resolution SDS-PAGE. Strikingly, trapping of both TAP subunits by beryllium fluoride is peptide-specific. The peptide concentration required for half-maximal trapping is identical for TAP1 and TAP2 and directly correlates with the peptide binding affinity. Only a background level of trapping was observed for low affinity peptides or in the presence of the herpes simplex viral protein ICP47, which specifically blocks peptide binding to TAP. Importantly, the peptide-induced trapped state is reached after ATP hydrolysis and not in a backward reaction of ADP binding and trapping. In the trapped state, TAP can neither bind nor exchange nucleotides, whereas peptide binding is not affected. In summary, these data support the model that peptide binding induces a conformation that triggers ATP hydrolysis in both subunits of the TAP complex within the catalytic cycle.Cytotoxic T-lymphocytes recognize peptides as a "non-self " signal presented by MHC 1 class I molecules at the cell surface. These antigenic peptides, which are derived from endogenous proteins, are degraded within the proteasomal pathway in the cytosolic compartment. The transporter associated with antigen processing (TAP) translocates these peptides into the ER lumen, where loading of peptides on MHC class I molecules takes place (1). Subsequently, fully assembled MHC⅐peptide complexes can pass the ER quality control and traffic to the cell surface, where they are inspected by cytotoxic T-lymphocytes.TAP belongs to the ABC transporter superfamily, whose members share the common structural organization of four domains: two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs) (2). The TMDs are generally formed by 5-10 transmembrane helices, whereas the NBDs are defined by three consensus motifs: Walker A, Walker B, and C-signature sequences. Functional TAP is a heterodimer consisting of TAP1 and TAP2, each of which contains one Nterminal TMD followed by a C-terminal NBD (3). The transmembrane domains determine the peptide specificity and form a putative translocation pore across the membrane, whereas the nucleotide-binding domains catalyze the ATP hydrolysis and provide the energy for peptide translocation.TAP binds peptides with a preferential length of 8 -16 amino acids (4). Substrate selectivity is determined by the three amino-terminal residues and the carboxyl-terminal residue of the peptide (5). Peptide binding...

Intracellular peptide transporters in human – compartmentalization of the “peptidome”

“…TAP chimeras with two canonical C-loops showed the highest transport rate compared to chimeras with two degenerate C-loops showing the lowest transport rate, demonstrating that the ABC signature motifs control peptide transport efficiency [10]. Markedly, mutation of leucine or glycine in TAP1 (LSGGQ) fully abolished peptide transport; whereas, equivalent mutations in TAP2 still showed residual transport activity [10]. In summary, the functional asymmetry of both TAP subunits suggests that one NBD hydrolyzes ATP to provide energy for the transport process, whereas, the other site presumably acts as a regulatory unit for the ATPase activity.…”

“…The C-loop in TAP2 is degenerated to LAAGQ compared to the canonical signature motif LSGGQ. TAP chimeras with two canonical C-loops showed the highest transport rate compared to chimeras with two degenerate C-loops showing the lowest transport rate, demonstrating that the ABC signature motifs control peptide transport efficiency [10]. Markedly, mutation of leucine or glycine in TAP1 (LSGGQ) fully abolished peptide transport; whereas, equivalent mutations in TAP2 still showed residual transport activity [10].…”

Structure and function of ABC transporters: the ATP switch provides flexible control

“…High affinity for propafenone is also restored at this step but, in contrast, high affinity for vinblastine is not recovered until phosphate is released from the NBDs. It therefore seems that the conformational changes in the TMDs, at different steps in the catalytic cycle, may have different consequences for the different drug-binding sites for P-gp [68,69], TAP [70] and the histidine permease [71]. For some ABC transporters, the two pockets hydrolyse ATP non-simultaneously, for example, P-gp [22,52,63,72], MutS [36], and the bacterial histidine [50,61] and maltose [73] transporters.…”

Nucleotide-Binding Sites of the Heterodimeric LmrCD ABC-Multidrug Transporter of Lactococcus lactis Are Asymmetric

“…These data demonstrate that the defective ATPase activity of the mutants bearing the LmrD E587Q mutation is not due to an impaired nucleotide-binding activity but is caused by a catalytic defect. (20,21). We have used these trapping agents to determine if both nucleotide-binding sites on LmrCD are capable of the hydrolysis of the -γ bond of ATP.…”