A multiscale structural understanding of organic anion transporting polypeptide transporters (OATPs) functions

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OATPs are a family of Solute Carrier Transporters (SLC) that mediates sodium independent uptake of endogenous and xenobiotic molecules in more than 40 animal species. OATP2A1, also called prostaglandins transporter (PGT), is an ubiquitously expressed OATP that is responsible for the cellular intake (influx) and secretion (efflux) of prostaglandins bioactive lipids. PGT plays a central role in prostaglandins physiological functions by regulating prostaglandins receptors extracellular accessibility to their ligands. Some OATPs have been shown to oligomerize using conserved structural motifs that regulate their cellular functions. However, because of the absence of a tridimensional structure and the technical challenge to probe membrane proteins oligomerization, the structure-function mechanism of OATPs remains poorly characterized. The overarching long-term goal of this research program is to understand the structural mechanism of OATPs cellular functions, with short-term focus to study PGT as a prototypical OATP. More specifically, we propose to establish the methodological grounds that will lead to the determination of a high-resolution X-ray crystal structure of PGT, and to investigate the role of OATPs oligomerization on PGT-mediated prostaglandin cellular transport. Theme I aim at defining the molecular mechanism of prostaglandins transport by PGT at an atomic level. Using a molecular model as a guide, will develop PGT overexpression, purification, and optimization procedures, to enable the structural biology study of the transporter using X-ray crystallography. The model will also serve to identify important structural features in PGT regulating the transport function. The results obtained will be used to derive an understanding of solute transport by OATPs at an atomic level. With the help of the tools developed above, we aim at obtaining diffracting crystals that will allow us to solve the high-resolution structure of PGT and launch a larger scale structural biology program on OATPs. Theme II aim at further characterizing the role of OATP oligomerization on their cellular functions. We will use Bioluminescence Resonance Energy Transfer (BRET)-based approaches to determine the oligomerization map of human OATPs in cells and probe the regulatory role of oligomerization in PGT transport functions. In addition to provide a better multiscale understanding of prostaglandins regulation, this program on OATPs will have a high impact in many research areas beyond structural biology such as cell biology, ecology, and veterinary sciences. Students and postdoctoral fellows from my laboratory will develop strong expertise in protein engineering, expression and purification, molecular modelling, BRET cellular assays, X-ray crystallography, synchrotron experimentation, and pharmacology. Such training is currently in high demand, and trainees will therefore fulfill highly needed qualified positions in life sciences.