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

National Heart, Lung, And Blood Institute

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Abstract

Transport mechanism and activity regulation in potential drug transporters in the brain The blood-brain barrier presents a major challenge to the delivery of small-molecule drugs to the brain. Therefore, it is critical to study the polyspecific transporters in the brain and assess their potential as drug transporters. Several SLC transporter families, including the SLC22 transporters, monocarboxylate transporters (MCTs) from the SLC16 family, and organic anion-transporting polypeptides (OATPs) from the SLCO family, play pivotal roles in drug transport and drug-drug interactions across various tissues, including the brain. In collaboration with Pengxiang Huang’s group at Baylor College of Medicine, we conducted structural and mechanistic studies on MCT8 and OATP1C1, focusing on their roles in thyroid hormone transport and substrate polyspecificity. Both MCT8 and OATP1C1 facilitate the transport of thyroid hormones across the blood-brain barrier into the central nervous system. Mutations in MCT8 lead to Allan-Herndon-Dudley syndrome (AHDS), an X-linked disorder that causes neurodevelopmental impairments and peripheral hyperthyroidism, while deficiencies in OATP1C1 are associated with brain hypometabolism and progressive neurodegenerative diseases. These findings underscore the critical role of both transporters in maintaining thyroid hormone homeostasis, which is essential for proper brain development and function. Using our established composite masking method for cryo-EM data processing, we determined the cryo-EM structures of human MCT8 and OATP1C1 in complex with the active thyroid hormone triiodothyronine (T3) and the prohormone thyroxine (T4) at resolutions of 2.9 Å and 2.3 Å, respectively. These high-resolution structures revealed distinct mechanisms of substrate recognition and transport, which are key to their exceptional specificity for thyroid hormones, and provided insights into disease-associated mutations in MCT8 linked to AHDS. Additionally, we solved the structure of OATP1C1 bound to the natural hormone estrone 3-sulfate (E1S) and the synthetic analog estrone 3-glucuronide (E1G), uncovering an unexpected extracellular allosteric regulatory site, highly conserved across the OATP family. Notably, the substrate binding sites of both MCT8 and OATP1C1 are large enough to potentially accommodate other small molecules, suggesting their capacity to transport additional small molecule drugs. Structural and regulatory mechanisms in the SLC4 family The transporters in the SLC4 family play essential roles in maintaining acid-base homeostasis by regulating pH across organelles, the cytosol, and extracellular fluids. They are crucial for human health, as their dysfunction can lead to severe diseases and disorders, including blindness, short stature, cognitive impairments, cerebral calcification, metabolic acidosis, anemia, and hearing loss. NBCe1 (SLC4A4), a sodium-bicarbonate cotransporter, is predominantly expressed on the basolateral membrane of renal proximal tubule cells. It is responsible for transporting bicarbonate from the cell into the blood, contributing to the reabsorption of filtered bicarbonate and the maintenance of acid-base balance. Mutations in NBCe1 can result in proximal renal tubular acidosis. Given NBCe1’s pivotal role in the proximal tubule, a region rich in drug transporters, and its potential interactions with these transporters, we are particularly interested in the structural and functional characterization of NBCe1 and other SLC4 family members. In our recent study, we determined the cryo-EM structures of human NBCe1 in both apo and substrate-bound states at near-atomic resolution (1.9-2.1 Å). Complementary MD simulations reveal the precise binding of sodium and bicarbonate ions at the transporter’s substrate-binding site and supports the substrate ion stoichiometry during co-transport. These findings resolve long-standing ambiguity regarding NBCe1’s transport stoichiometry and advancing our understanding of its electrogenic transport mechanism. This work also provides a molecular framework for interpreting NBCe1’s role in both health and disease, while contributing to broader principles of substrate recognition and coupling within the SLC4 transporter family. While most SLC4 family members transport bicarbonate, some are borate transporters (BORs). Our structural study of Arabidopsis thaliana borate transporter Bor1 (AtBor1) aimed to uncover both common and distinct transport mechanisms within the SLC4 family. However, the focus soon shifted to the activity regulation of AtBor1. By combining high-resolution cryo-EM (2.2 Å), mutagenesis, and functional assays, we discovered that AtBor1’s activity is regulated by two distinct mechanisms: an autoinhibitory domain at the carboxyl terminus, which blocks the substrate pathway via conserved salt bridges, and phosphorylation of Thr410, which activates borate transport through interactions with a positively charged pocket. Interestingly, we observed that the short β1 strand of the TM10-β1-β2-TM11 region in AtBor1 undergoes a fold-switch between a β-sheet and α-helix conformation during the transition from the inward-open to the occluded state. This fold-switching, similar to the β2 switch found in my previous study of the related SLC4 transporter anion exchanger 1 (AE1, SLC4A1), suggests that such conformational transitions in the TM10-β1-β2-TM11 region may be a common feature in the SLC4 family. These findings clarify the molecular basis of AtBor1’s activity regulation and emphasize its role in the rapid regulation of boron levels in plants. Moreover, they offer new insights into the elevator transport mechanism in the SLC4 family. This discovery also underscores the importance of activity regulation in other SLC4 transporters, such as NBCe1, whose regulation by IRBIT binding remains to be fully understood.