Na+-coupled acid–base transporters play essential roles in human biology. Their dysfunction has been linked to cancer, heart, and brain disease. High-resolution structures of mammalian Na+-coupled acid–base transporters are not available. The sodium-bicarbonate cotransporter NBCe1 functions in multiple organs and its mutations cause blindness, abnormal growth and blood chemistry, migraines, and impaired cognitive function. Here, we have determined the structure of the membrane domain dimer of human NBCe1 at 3.9 Å resolution by cryo electron microscopy. Our atomic model and functional mutagenesis revealed the ion accessibility pathway and the ion coordination site, the latter containing residues involved in human disease-causing mutations. We identified a small number of residues within the ion coordination site whose modification transformed NBCe1 into an anion exchanger. Our data suggest that symporters and exchangers utilize comparable transport machinery and that subtle differences in their substrate-binding regions have very significant effects on their transport mode.

Aminoacylase 3 (AA3) deacetylates N-acetyl-aromatic amino acids and mercapturic acids including N-acetyl-1,2-dichlorovinyl-L-cysteine (Ac-DCVC), a metabolite of a xenobiotic trichloroethylene.Previous studies did not demonstrate metal-dependence of AA3 despite a high homology with a Zn 2+ -metalloenzyme aminoacylase 2 (AA2). A 3D model of mouse AA3 was created based on homology with AA2. The model showed a putative metal binding site formed by His21, Glu24 and His116, and Arg63, Asp68, Asn70, Arg71, Glu177 and Tyr287 potentially involved in catalysis/ substrate binding. The mutation of each of these residues to alanine inactivated AA3 except Asn70 and Arg71, therefore the corrected 3D model of mouse AA3 was created. Wild type (wt) mouse AA3 expressed in E. coli contained ~0.35 zinc atoms per monomer. Incubation with Co 2+ and Ni 2+ activated wt-AA3. In the cobalt-activated AA3 zinc was replaced with cobalt. Metal removal completely inactivated wt-AA3, whereas addition of Zn 2+ , Mn 2+ or Fe 2+ restored initial activity. Co 2+ and to a lesser extent Ni 2+ increased activity several times in comparison with intact wt-AA3. Co 2+ drastically increased the rate of deacetylation of Ac-DCVC and significantly increased the toxicity of Ac-DCVC in the HEK293T cells expressing wt-AA3. The results indicate that AA3 is a metalloenzyme significantly activated by Co 2+ and Ni 2+ .

Anion exchanger 1 (AE1) is the major erythrocyte membrane protein that mediates chloride/bicarbonate exchange across the erythrocyte membrane facilitating CO2 transport by the blood, and anchors the plasma membrane to the spectrin-based cytoskeleton. This multi-protein cytoskeletal complex plays an important role in erythrocyte elasticity and membrane stability. An in-frame AE1 deletion of nine amino acids in the cytoplasmic domain in a proximity to the membrane domain results in a marked increase in membrane rigidity and ovalocytic red cells in the disease Southeast Asian Ovalocytosis (SAO). We hypothesized that AE1 has a flexible region connecting the cytoplasmic and membrane domains, which is partially deleted in SAO, thus causing the loss of erythrocyte elasticity. To explore this hypothesis, we developed a new non-denaturing method of AE1 purification from bovine erythrocyte membranes. A three-dimensional (3D) structure of bovine AE1 at 2.4 nm resolution was obtained by negative staining electron microscopy, orthogonal tilt reconstruction and single particle analysis. The cytoplasmic and membrane domains are connected by two parallel linkers. Image classification demonstrated substantial flexibility in the linker region. We propose a mechanism whereby flexibility of the linker region plays a critical role in regulating red cell elasticity.

SLC4 transporters play significant roles in pH regulation and cellular sodium transport. The previously solved structures of the outward facing (OF) conformation for AE1 (SLC4A1) and NBCe1 (SLC4A4) transporters revealed an identical overall fold despite their different transport modes (chloride/bicarbonate exchange versus sodium-carbonate cotransport). However, the exact mechanism determining the different transport modes in the SLC4 family remains unknown. In this work, we report the cryo-EM 3.4 Å structure of the OF conformation of NDCBE (SLC4A8), which shares transport properties with both AE1 and NBCe1 by mediating the electroneutral exchange of sodium-carbonate with chloride. This structure features a fully resolved extracellular loop 3 and well-defined densities corresponding to sodium and carbonate ions in the tentative substrate binding pocket. Further, we combine computational modeling with functional studies to unravel the molecular determinants involved in NDCBE and SLC4 transport.

Trichloroethylene (TCE) is one of the most widespread environmental contaminants, which is metabolized to N-acetyl-S-1,2-dichlorovinyl-L-cysteine (NA-DCVC) before being excreted in the urine. Alternatively, NA-DCVC can be deacetylated by aminoacylase 3 (AA3), an enzyme that is highly expressed in the kidney, liver, and brain. NA-DCVC deacetylation initiates the transformation into toxic products that ultimately causes acute renal failure. AA3 inhibition is therefore a target of interest to prevent TCE induced nephrotoxicity. Here we report the crystal structure of recombinant mouse AA3 (mAA3) in the presence of its acetate byproduct and two substrates: N α -acetyl-L-tyrosine and NA-DCVC. These structures, in conjunction with biochemical data, indicated that AA3 mediates substrate specificity through van der Waals interactions providing a dynamic interaction interface, which facilitates a diverse range of substrates.mercapturates | metalloprotein | X-ray structure A minoacylase 3 (AA3) is a member of the aminoacylase family of enzymes that deacylates a broad range of substrates including both N α -acetylated amino acids and S-cysteine conjugates of N-acetyl-L-cysteine (mercapturic acids) ( Fig. 1) (1). There are three types of aminoacylases: (i) aminoacylase 1 (AA1) deacetylates neutral aliphatic N-acyl-α-amino acids and mercapturic acids; (ii) aminoacylase 2 or aspartoacylase (AA2) has a strict specificity for N α -acetyl-L-aspartate (NAD); and (iii) aminoacylase 3 (AA3) preferentially deacetylates N α -acetylated aromatic amino acids and mercapturic acids that are usually not deacetylated by AA1 (1-6). Despite different substrate specificities, AA2 and AA3 have a high degree of sequence (42% identity) and structure homology but are both substantially different from AA1 (∼10% of sequence identity) (2, 6-10).AA3 is of particular interest for human health because it participates in mediating toxicity of the xenobiotic trichloroethylene (TCE). The United States produces in excess of 130,000 tons of TCE per year (11), making it the most widespread chemical contaminant in both soil and ground water. TCE is readily absorbed into the body where it can enter the glutathione conjugation detoxification pathway producing the mercapturic acid N-acetyl-S-1,2-dichlorovinyl-L-cysteine (NA-DCVC) for subsequent urinary excretion (12, 13). However, NA-DCVC can be deacetylated by AA3-which is highly expressed in the renal proximal tubule, liver, and brain-to generate S-1,2-dichlorovinyl-L-cysteine (DCVC) (2) and further transformed via β-lyases or flavin monooxygenases into lethal products capable of causing acute renal failure and toxicity to the liver and brain. (4, 13-22). Thus, inhibition of AA3 can decrease DCVC formation and ameliorate TCE toxicity, presenting a potential target for drug discovery.Generating specific inhibitors for AA3 would be greatly aided by high-resolution structural data, but structural studies of aminoacylases have been limited to AA1 (7) and AA2 (8, 9), which differ in both overall architecture and ac...