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

National Heart, Lung, And Blood Institute

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Abstract

(1) Study of the structures and activity regulation of the solute carrier 4 (SLC4) family of transporters The transporters in the SLC4 family play critical roles in acid-base homeostasis to regulate pH in organelles, the cytosol, and the extracellular fluid. They are important in human biology as their functional loss leads to severe diseases or disorders including blindness, short stature, abnormal cognitive function, cerebral calcification, metabolic acidosis, anemia, and hearing abnormalities. The SLC4 transporters work in the Na+-independent or Na+-coupled manner with their transport mode controlled by a small number of key residues within the ion coordination site. We have been working on three representative transporters in the SLC4 family: 1) human anion exchanger 1 (AE1; also known as band 3), an electroneutral transporter that exchanges Cl and HCO3 across the cytoplasmic membrane with 1:1 ratio; 2) human NBCe1, an electrogenic sodium-coupled HCO3 transporter. 3) plant Bor1, a borate transporter in plant roots whose transport action has not been fully elucidated. We have determined the structures of all three transporters at high resolution using cryoEM. The cryoEM structures and biochemical data provide us with an unprecedented opportunity to compare these structures in different conformations to understand their similarities and differences, the transport mechanism of the SLC4 family, and activity regulation. (2) Structural mechanism of multi-substrate selectivity in bacterial urea/amide channel (UAC) The pH homeostasis in some bacteria is regulated using the urea/amide channel. Helicobacter pylori, a pathogenic bacterium that lives in the highly acidic environment in the stomach, uses the urea channel UreI to import urea from the gastric juice and breaks it down into ammonia that maintains pH at about 6.0 in the periplasmic space. Loss of UreI function abolishes the survival of H. pylori at low pH, suggesting UreI to be a potential drug target to specifically kill this pathogen in the stomach. The structures of UreI determined by X-ray crystallography and cryoEM show a permeation pathway that is always blocked in the middle, which has difficulties to explain how the urea channel works. We use UAC from Bacillus cereus, that is known to permeate few amide molecules, including urea and nicotinamide, as the model system to understand its simple yet sophisticated mechanism of substrate binding and permeation, with a goal to find implications in pathogenesis of H. pylori and other bacteria. Using cryoEM, we have determined the structures of UAC in the apo form and in complex with its substrates at atomic resolution. The best resolution is higher that 2 , allowing us to observe very small conformational changes during the process of substrate handling. (3) Structure study of the poly-specific drug and organic ion transporters in the solute carrier 22 (SLC22) family The SLC22 family includes over 30 members that transport 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, -ketoglutarate, urate, or steroid hormones, but also more than 60 therapeutic drugs such as metformin and quinidine. An important and interesting feature of the SLC22 transporters is that they are poly-specific in substrate selectivity, i.e. an SLC22 transporter is able to transport structurally different compounds. This associates some SLC22 transporters to several drug side effects or drug-drug interactions. Defects in SLC22 transporters are linked to several diseases, such as primary carnitine deficiency, hyperuricemia, and Crohns disease. The structure study of the SLC22 transporters has been extraordinarily challenging due to its structural flexibilities and small molecular mass for cryoEM. We have obtained the cryoEM structure of rat organic anion transporter 1 (OAT1) at an intermediate resolution. The structure unambiguously reveals that OAT1 is related to the major facilitator superfamily (MFS). The combination of cryoEM structure study and structure prediction using the latest algorithms provides a new path to understand the mechanism of transport and functional regulation of the SLC22 transporters and its implications in drug metabolism and drug-drug interactions.