Poster Presentation The 48th Lorne Conference on Protein Structure and Function 2023

The Cryo-EM Structure of Human OCT1 in Monomeric and Pseudo-Dimeric Forms (#109)

Yi C Zeng 1 2 , Simon HJ Brown 3 , James I Vandenberg 1 2 , Renae M Ryan 4 , Alastair G Stewart 1 2
  1. Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia
  2. St Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Darlinghurst, NSW, Australia
  3. Molecular Horizons, University of Wollongong, Wollongong, NSW, Australia
  4. School of Medical Science, Faculty of Medicine and Health, the University of Sydney, Camperdown, NSW, Australia

The SLC22 family of organic cation and anion transporters mediate the transfer of a range of endogenous and xenobiotic compounds across the cell membrane. These transporters are pivotal in the absorption and excretion of a number of drug compounds, however, a structural understanding of their function and substrate selectivity is lacking. Here, we describe the initial progress towards the structural characterisation of one such member, human organic cation transporter 1 (OCT1). OCT1 was recombinantly expressed and purified from HEK293 cells to yield monomeric and pure OCT1 solubilized in detergent. Optimization of grid-freezing conditions enabled high-resolution cryo-EM reconstructions of monomeric OCT1 in the inward-open state revealing a unique folded extracellular loop and a hydrophobic/negatively-charged internal binding pocket. Interestingly, we also observed a pseudo-dimeric population of OCT1 particles present when the sample was applied to gold foil girds that was resolved to low resolution. The present progress towards structural characterisation of OCT1 will facilitate future work towards uncovering the molecular basis of specificity in promiscuous SLC22 transporters.