Structural and functional studies of human ABCB6 using anti-helix antibodies

Abstract

The human ATP binding cassette superfamily B member 6 (ABCB6) that is expressed in broad organisms and transport diverse substrates such as porphyrin and heavy metal is one member of ABC transporter. Drug resistance is related to the overexpression of ABCB6 in several cancer cells. Only the nucleotide binding domain (NBD) structure of ABCB6 is revealed. The structural and functional information of ABCB6 is insufficient to understand the molecular mechanism. Structural study of membrane protein is a challenging inquiry. Therefore, many researchers use antibodies that are indispensable tools in protein engineering and structural biology. Antibodies suitable for structural studies should recognize the three dimensional conformations of target proteins. Generating such antibodies and characterizing their complexes with antigens take a significant amount of time and effort. Here, I show that I can expand the application of well-characterized antibodies by “transplanting” the epitopes they recognize to proteins with completely different structures and sequences. Previously, several antibodies have been shown to recognize the alpha helical conformation of antigenic peptides. I demonstrate that these antibodies can be made to bind to a variety of unrelated “off-target” proteins by modifying amino acids in the pre-existing alpha helices of such proteins. Using X-ray crystallography, I determined the structures of the engineered protein-antibody complexes. All the antibodies bound to the epitope-transplanted proteins forming accurately predictable structures. Furthermore, I showed that binding of these anti-helix antibodies to the engineered target proteins can modulate their catalytic activities by trapping them in selected functional states. The method is simple, efficient and will have applications in protein X-ray crystallography, electron microscopy, and nanotechnology. Additionally, I determined that the ABCB6 without TMD0 shows functions that are interaction with the substrate by pull-down assay and drug resistance by cytotoxicity assay with overexpression of ABCB6. I established single particle cryo-EM structure including the transmembrane domain (TMD) and NBD in 8.2 Å. Moreover, grid screening results will facilitate further high-resolution structural study.