General mechanisms for ABC transporters revealed by the transporter associated with antigen processing.

摘要

Adaptive cell-mediated immunity requires recognition by the immune system of class I MHC-bound peptides derived from foreign proteins. These foreign proteins, perhaps of viral or bacterial origin, are degraded in the host cell cytosol, and the generated peptides are transported into the endoplasmic reticulum for loading onto nascent class I MHC molecules. Peptide transport is mediated by a member of the ABC transporter family, the transporter associated with antigen processing (TAP). TAP, a heterodimer of homologous TAP1 and TAP2 subunits, fuels transport by binding and hydrolyzing ATP.;It is shown here that each TAP subunit has three domains: an N-terminal accessory domain, a transmembrane domain (TMD) and a C-terminal nucleotide-binding domain (NBD). The accessory domain physically interacts with tapasin, a class I-specific chaperone, to enhance peptide loading, but this domain is not essential for transport. The TMDs and NBDs of TAP1 and TAP2 form a core functional unit sufficient for transport.;The power stroke for peptide transport is hypothesized to occur when the TAP NBDs bind ATP and tightly associate, with two ATP molecules sandwiched at the TAP1--TAP2 interface. By targeting putative electrostatic interactions across the TAP interface, this closure of the TAP NBDs within the transporter is demonstrated to occur at some stage during a catalytic cycle. Further, the two composite ATPase sites formed around each ATP are not equivalent. One ATPase site has consensus motifs for ATP hydrolysis, while the second site is degenerate and has non-consensus substitutions. Using the isolated TAP1-NBD as a scaffold for engineering different ATPase sites, it is demonstrated that the consensus site has comparatively higher ATPase activity and is the principal driver of ATP-dependent NBD closure. Mutagenesis in full-length TAP confirms that at least one consensus site is necessary for activity. Sequence analysis reveals that nearly half of the mammalian ABC transporters similarly have distinct ATPase sites, and a general mechanism is proposed that the consensus site is the 'workhorse' during transport, while the degenerate site possibly has a structural role.;Finally, the purification of over-expressed mammalian TAP from insect cells is presented, which will enable future functional and structural studies.