Structural and functional study of ABC peptide transporter using Cryo-EM and mini protein

Abstract

Part I: The lysosomal peptide transporter TAPL has a role as phosphatidylserine floppase TAPL is referred the ATP-binding cassette (ABC) transporter superfamily B member 9 (ABCB9). It hydrolysis ATP to translocate unwanted broad spectrum of polypeptides from the cytoplasm into the lysosomal lumen for degradation. Unlike other ABC transporters, the TAPL exhibited a higher ATP hydrolysis activity even in the absence of peptides. Here I present that TAPL exhibits an ATP dependent phospholipid floppase activity that is the possible cause of its high basal ATPase activity and the lack of coupling between ATP hydrolysis and peptide efflux. It presents the first that cryo-EM structures of mouse TAPL complexed with (1) phospholipid, (2) CHS and 9mer peptide (RRYQKSTEL), (3) ATP analog (ADP-BeF3). Structure of inward facing reveals that F449 residue protrudes into transport pathway and divides it into a large hydrophilic central cavity and sizable hydrophobic upper cavity. The peptide binds to TAPL within the central cavity, while phospholipid binds into the upper cavity. The results suggest that TAPL use difference mechanisms to function as a peptide translocase and phospholipid floppase.

Part II: Design mini protein of the transporter associated with antigen processing protein (TAP1/2) The TAP protein is a type of ABC transporter that utilizes ATP hydrolysis to transport viral peptides from the cytosol to the endoplasmic reticulum (ER). The transported viral peptides are then delivered to MHC class I by the peptide loading complex, which includes the TAP protein. After binding with the viral peptide, the MHC class I molecules are translocated to the cell surface. The presence of viral peptide on the cell surface enable cytotoxic T cell to identify and eliminate the infected cells, triggering further immune response. The TAP protein consists of TAP1 (ABCB2) and TAP2 (ABCB3), forming a heterodimer. Like other ABC transporter, TAP protein features two transmembrane domain (TMD) that create the peptide transport pathway and two nucleotide binding domain (NBD) responsible for ATP hydrolysis. To elucidate the structure of the TAP protein using cryo-EM, it is essential to differntiate between TAP1 and TAP2 during the 3D reconstruction process. Therefore, in this study, a miniprotein was designed to bind specially to the TAP1 NBD serving as a marker using RF Diffusion and AlphaFold. The generated miniprotein has an ipTM score of 0.87 and a calculated interaction site of pAE value (2.6Å) between the NBD and miniprotein. These scores indicate that the miniprotein binds adequately to the NBD. Additionally, the binding ability between NBD and miniprotein was confirmed experimentally.