Structural studies of membrane proteins using high-resolution cryo electron microscopy

National Heart, Lung, And Blood Institute

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Abstract

(1) High-resolution structure determination of SLC drug transporters using cryoEM The SLC family includes transporters of a broad spectrum of organic solutes, including cations, anions, or zwitterions. They play a pivotal role in secretion and homeostasis of organic ions in various tissues, including kidney, liver, brain, placenta, intestine, and lung. Their substrates include not only endogenous metabolites such as monoamine neurotransmitters, choline, carnitine, alpha-ketoglutarate, urate, or steroid hormones, but also a large number of therapeutic drugs. A few SLC transporters, such as organic cation transporters (OCTs), organic anion transporters (OATs), and organic anion transporting polypeptide (OATPs), have been recommended for in vitro tests for transporter-mediated drug-drug interactions during new drug developments, while the structural details of these transporters are still unknown. Structure determination of the SLC drug transporters using cryoEM is very challenging due to their structural flexibilities and small molecular mass (<70 kDa). There are two clear hurdles: a) the small sizes of these transporters lead to insufficient signals in cryoEM images for data processing; b) the disordered detergent/lipid micelle that binds to each transporter particle in cryoEM study causes a large amount of noise that is generally irrelevant to the size of the transporter. The combination of both issues severely reduces signal-to-noise ratio in cryoEM images of transporters, leading to poor results in cryoEM 3D reconstructions of transporters. We have developed a method of using special composite masks in cryoEM data processing that utilizes the low-resolution structural features of the micelle as a global constrain and the high-resolution signals from the ordered protein structure to gain precise particle alignment and classification. We have determined several high-resolution structures (better than 3 ) of the drug transporter with the help from the above mentioned cryoEM data processing method. This progress demonstrates the potential of cryoEM in the structural study of drug transporters and will allow us to reveal the mechanisms of substrate binding with poly-specificity and of drug transport. (2) The substrate and inhibitor binding mechanism of polyspecific transporter OAT1 OATs of the SLC22 family play critical roles in the transport of organic anions, including metabolites and therapeutic drugs, and in the transporter-mediated drug-drug interactions. In the kidneys, OATs facilitate the elimination of metabolic waste products and xenobiotics. However, their transport activities can also lead to the accumulation of certain toxic compounds within cells, potentially causing kidney damage. Moreover, OATs are important drug targets, as their inhibition can modulate the elimination or retention of substrates linked to diseases. Despite extensive research on OATs, the molecular basis of substrate and inhibitor binding remains poorly understood due to the lack of structural information. We have used single particle cryoEM to determine the structures of rat OAT1 (SLC22A6) and its complexes with substrate para-aminohippuric acid and inhibitor probenecid at 2.1, 2.8, and 2.9 resolution, respectively. Our findings reveal a highly conserved mechanism for substrate and inhibitor binding in the SLC22 transporters, wherein four aromatic residues form a cage to accommodate the polyspecific binding of diverse compounds.