Project 1 Our goal is to generate recombinant monoclonal antibodies (rAbs) that bind to the multiple conformations of ATP Binding Cassette (ABC) transporter family members and to use these antibodies to determine the various structures of this multi protein complex as it progresses through the transport cycle. The ABC transporter family is an important class of drug targets. For example, ABC transporters are responsible for multidrug resistance to a diverse structural range of anti-cancer drugs. Genetic diseases associated with ABC transporters include cystic fibrosis and age-related macular degeneration. The transporters undergo conformational changes but direct structural evidence for the entire transport cycle is lacking. Antibodies that target domains and are selective for certain conformations would accelerate structural and mechanistic studies and in some cases facilitate formation of diffraction quality crystals for X-ray structure determination. In other cases, the antibodies could bypass the need for crystallization allowing subnanometer resolution structure determination by single particle cryoEM. Furthermore, the antibodies can selectively deliver probes to the transporter protein enabling rapid measurement of key interatomic distances on purified protein or in cells. Finally, we expect that some of the antibodies we generate will inhibit transporter activity and be useful therapeutic lead molecules. The Craik lab has a record of generating recombinant antibodies to a variety of proteins including ABC transporters and has demonstrated the utility of recombinant antibody fragments (Fabs) as fiducial markers for single particle cryoEM studies of protein. Given the availability of Fabs to TmrA/B and other ABC transporters, similar cryoEM studies will be performed to determine structures of different conformations. High-resolution structures could result from rigid Fab-membrane protein complexes.
Our aims are to 1) Design and produce locked-in transporters, genetic variants and mutants. Different conformations of the transporter complexes will be locked-in by disulfide crosslinking mutagenesis, by addition of nucleotide analogs, inhibitory viral factors or chemical inhibitors. Where available, mutants will be used for epitope characterization. 2) Conduct functional transport studies on variant and mutant locked-in transporters. ABC transporter function will be determined for the wild type and all cross-linked and variant transporters, with and without bound Fab, so that conformation can be linked to function. 3) Measure distances between transporter domains in different conformational states to generate structural models of different transporter conformations in the transport cycle. The Fabs generated in Core C will be labeled with fluorescent tags using a variety of linkers and dyes that are optimal for single molecule distance measurements. Distance measurements will be made using single-molecule localization techniques.
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