Oral Presentation 11th Australian Peptide Conference 2015

Macrocyclic-peptide ligands that inhibit transporter function and improve protein crystallization (#59)

Chris J Hipolito 1 , Hiroaki Suga 2
  1. University of Tsukuba, Tsukuba, Japan
  2. Department of Chemistry, University of Tokyo, Tokyo, Japan

Cocrystallization of membrane proteins using selected high-affinity ligands has granted us insight into the structural information of intractable transmembrane proteins. In the past, macrocyclic-peptide ligands have commonly been selected for therapeutic application. Due to their high-affinity for their protein target, macrocyclic peptides are now also being used as cocrystallization ligands for membrane proteins. Following the successful cocrystallization of Pyrococcus furiosus multidrug and toxic compound extrusion (PfMATE) transporter1, in vitro selections against transporters of other families were pursued. One of the most successful in vitro selections resulted in the identification of a macrocyclic-peptide ligand, aCAP, which can bind to an ABC family multidrug transporter from Cyanidioschyzon merolae (CmABCB1)2. Similar to the PfMATE-binding macrocyclic peptide MaD3S, aCAP is capable of improving crystallization by stabilizing transmembrane helices and reducing overall flexibility. aCAP is also capable of inhibiting CmABCB1’s basal and substrate-stimulated ATPase activity. Unlike MaD3S, however, aCAP does not bind to a substrate-binding pocket like a small molecule. aCAP's ability to immobilize of the transmembrane helices, whose movements are coupled to ATPase activity, allows aCAP to function as an allosteric inhibitor without the need to access the substrate-binding pocket. The surface that aCAP binds to is not concave and the interaction between CmABCB1 and aCAP is more similar to a protein-protein interaction. The varied methods of binding of the functional macrocyclic peptides identified from the in vitro selections against PfMATE and CmABCB1 suggest that in vitro selection is capable of identifying macrocyclic peptides that can bind to a concave pocket like a small molecule or to a flat surface via protein-protein-like interactions like an antibody. Here we review the successful in vitro selection of macrocyclic-peptide ligands of transporters and their use in structural biology and therapeutic medicine.

  1. Tanaka Y., Hipolito C. J., Maturana A. D., Ito K., Kuroda T., Higuchi T., Katoh T., Kato H. E., Hattori M., Kumazaki K., Tsukazaki T., Ishitani R., Suga H., Nureki O., Nature, , 496, 247–251 (2013).
  2. Kodan A., Yamaguchi T., Nakatsu T., Sakiyama K., Hipolito C. J., Fujioka A., Hirokane R., Ikeguchi K., Watanabe B., Hiratake J., Kimura Y., Suga H., Ueda K., Kato H., Proc. Natl. Acad. Sci. U. S. A., 111, 4049–4054 (2014).