scholarly journals Cryo-EM structure of the sodium-driven chloride/bicarbonate exchanger NDCBE

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Weiguang Wang ◽  
Kirill Tsirulnikov ◽  
Hristina R. Zhekova ◽  
Gülru Kayık ◽  
Hanif Muhammad Khan ◽  
...  
Keyword(s):  
Sodium Carbonate ◽  
Sodium Transport ◽  
Ph Regulation ◽  
Binding Pocket ◽  
Extracellular Loop ◽  
Substrate Binding Pocket ◽  
Functional Studies ◽  
Carbonate Ions ◽  
Molecular Determinants ◽  
Transport Modes

AbstractSLC4 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.

scholarly journals Crystal structure of steroid reductase SRD5A reveals conserved steroid reduction mechanism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yufei Han ◽  
Qian Zhuang ◽  
Bo Sun ◽  
Wenping Lv ◽  
Sheng Wang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Biochemical Characterization ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Transmembrane Segments ◽  
Therapeutic Molecules ◽  
Binding Cavity ◽  
Immune System Regulation ◽  
Mediated Reduction

AbstractSteroid hormones are essential in stress response, immune system regulation, and reproduction in mammals. Steroids with 3-oxo-Δ4 structure, such as testosterone or progesterone, are catalyzed by steroid 5α-reductases (SRD5As) to generate their corresponding 3-oxo-5α steroids, which are essential for multiple physiological and pathological processes. SRD5A2 is already a target of clinically relevant drugs. However, the detailed mechanism of SRD5A-mediated reduction remains elusive. Here we report the crystal structure of PbSRD5A from Proteobacteria bacterium, a homolog of both SRD5A1 and SRD5A2, in complex with the cofactor NADPH at 2.0 Å resolution. PbSRD5A exists as a monomer comprised of seven transmembrane segments (TMs). The TM1-4 enclose a hydrophobic substrate binding cavity, whereas TM5-7 coordinate cofactor NADPH through extensive hydrogen bonds network. Homology-based structural models of HsSRD5A1 and -2, together with biochemical characterization, define the substrate binding pocket of SRD5As, explain the properties of disease-related mutants and provide an important framework for further understanding of the mechanism of NADPH mediated steroids 3-oxo-Δ4 reduction. Based on these analyses, the design of therapeutic molecules targeting SRD5As with improved specificity and therapeutic efficacy would be possible.

Characterization of the Acyl Substrate Binding Pocket of Acetyl-CoA Synthetase†

Biochemistry ◽  
2006 ◽  
Vol 45 (38) ◽  
pp. 11482-11490 ◽  
Author(s):  
Cheryl Ingram-Smith ◽  
Barrett I. Woods ◽  
Kerry S. Smith
Keyword(s):  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Acetyl Coa

scholarly journals N-glycosylation critically regulates function of oxalate transporter SLC26A6

2016 ◽  
Vol 311 (6) ◽  
pp. C866-C873 ◽  
Author(s):  
R. Brent Thomson ◽  
Claire L. Thomson ◽  
Peter S. Aronson
Keyword(s):  
Plasma Membrane ◽  
Intestinal Secretion ◽  
Integral Membrane Proteins ◽  
Extracellular Loop ◽  
Transport Activity ◽  
Intact Cells ◽  
Functional Studies ◽  
And Function ◽  
Oxalate Transport

The brush border Cl−-oxalate exchanger SLC26A6 plays an essential role in mediating intestinal secretion of oxalate and is crucial for the maintenance of oxalate homeostasis and the prevention of hyperoxaluria and calcium oxalate nephrolithiasis. Previous in vitro studies have suggested that SLC26A6 is heavily N-glycosylated. N-linked glycosylation is known to critically affect folding, trafficking, and function in a wide variety of integral membrane proteins and could therefore potentially have a critical impact on SLC26A6 function and subsequent oxalate homeostasis. Through a series of enzymatic deglycosylation studies we confirmed that endogenously expressed mouse and human SLC26A6 are indeed glycosylated, that the oligosaccharides are principally attached via N-glycosidic linkage, and that there are tissue-specific differences in glycosylation. In vitro cell culture experiments were then used to elucidate the functional significance of the addition of the carbohydrate moieties. Biotinylation studies of SLC26A6 glycosylation mutants indicated that glycosylation is not essential for cell surface delivery of SLC26A6 but suggested that it may affect the efficacy with which it is trafficked and maintained in the plasma membrane. Functional studies of transfected SLC26A6 demonstrated that glycosylation at two sites in the putative second extracellular loop of SLC26A6 is critically important for chloride-dependent oxalate transport and that enzymatic deglycosylation of SLC26A6 expressed on the plasma membrane of intact cells strongly reduced oxalate transport activity. Taken together, these studies indicated that oxalate transport function of SLC26A6 is critically dependent on glycosylation and that exoglycosidase-mediated deglycosylation of SLC26A6 has the capacity to profoundly modulate SLC26A6 function.

scholarly journals The Hydrophobic Hinge Region of Rat DNA Polymerase β Is Critical for Substrate Binding Pocket Geometry

2005 ◽  
Vol 280 (31) ◽  
pp. 28388-28393 ◽  
Author(s):  
Daniela Starcevic ◽  
Shibani Dalal ◽  
Joachim Jaeger ◽  
Joann B. Sweasy
Keyword(s):  
Dna Polymerase ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Hinge Region ◽  
Dna Polymerase Β ◽  
Substrate Binding Pocket

scholarly journals Structural basis of AdoMet-dependent aminocarboxypropyl transfer reaction catalyzed by tRNA-wybutosine synthesizing enzyme, TYW2

2009 ◽  
Vol 106 (37) ◽  
pp. 15616-15621 ◽  
Author(s):  
Masataka Umitsu ◽  
Hiroshi Nishimasu ◽  
Akiko Noma ◽  
Tsutomu Suzuki ◽  
Ryuichiro Ishitani ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Binding Pocket ◽  
Structural Basis ◽  
Binding Modes ◽  
Substrate Binding Pocket ◽  
Methyl Group ◽  
Group Transfer ◽  
Canonical Class ◽  
Functional Analyses

S-adenosylmethionine (AdoMet) is a methyl donor used by a wide variety of methyltransferases, and it is also used as the source of an α-amino-α-carboxypropyl (“acp”) group by several enzymes. tRNA-yW synthesizing enzyme-2 (TYW2) is involved in the biogenesis of a hypermodified nucleotide, wybutosine (yW), and it catalyzes the transfer of the “acp” group from AdoMet to the C7 position of the imG-14 base, a yW precursor. This modified nucleoside yW is exclusively located at position 37 of eukaryotic tRNAPhe, and it ensures the anticodon-codon pairing on the ribosomal decoding site. Although this “acp” group has a significant role in preventing decoding frame shifts, the mechanism of the “acp” group transfer by TYW2 remains unresolved. Here we report the crystal structures and functional analyses of two archaeal homologs of TYW2 from Pyrococcus horikoshii and Methanococcus jannaschii. The in vitro mass spectrometric and radioisotope-labeling analyses confirmed that these archaeal TYW2 homologues have the same activity as yeast TYW2. The crystal structures verified that the archaeal TYW2 contains a canonical class-I methyltransferase (MTase) fold. However, their AdoMet-bound structures revealed distinctive AdoMet-binding modes, in which the “acp” group, instead of the methyl group, of AdoMet is directed to the substrate binding pocket. Our findings, which were confirmed by extensive mutagenesis studies, explain why TYW2 transfers the “acp” group, and not the methyl group, from AdoMet to the nucleobase.

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