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

National Heart, Lung, And Blood Institute

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Abstract

(1) Method developments for high-resolution cryoEM structure determination of SLC transporters While overall the single-particle cryoEM technology has been making an exceptional progress in structural biology in the past decade, cryoEM structural studies of transporters are lagging due to the size limit from cryoEM. There are two clear hurdles: a) the small sizes of transporters (usually <100 kDa) 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 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. This method has helped us solve several structures of transporters that are small and/or flexible to high resolution with the best one at 1.65 A. (2) CryoEM study of SLC drug transporters 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. We have used cryoEM to obtain two high-resolution structures 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.