Structures of DPAGT1 explain glycosylation disease mechanisms and advance TB antibiotic design

Summary: Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic “lipid-altered” tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug

Human Dolichyl-Phosphate Alpha-N-Acetyl glucosaminyl transferase (DPAGT1); A Target Enabling Package

<p>The ER integral membrane enzyme dolichyl-phosphate alpha-N-acetyl glucosaminyl phosphotransferase (DPAGT1) catalyses the first step in the synthesis of the oligosaccharide-P-P-dolichol unit which provides the glycans structure for N-glycosylation of proteins. Mutations in DPAGT1 cause two muscle weakness conditions, limb-girdle congenital myasthenic syndrome (CMS) and congenital disorder of glycosylation type 1j (CDG1j). DPAGT1 overexpression has also been implicated in oral cancer. We have produced and solved structures of this integral membrane enzyme, DPAGT1 with the V264G mutation found in a patient with CMS, and complexes with a 50 nM inhibitor, tunicamycin. We have developed enzymatic activity and thermostability assays which have allowed us to assess the activity and stability of DPAGT1 mutants and the effect of small molecules. There are > 20 DPAGT1 associated missense variants in patients with CMS and CDG1j. We have mapped these mutations to the structure, and we will used the assays described here to assess how the activity and stability of DPAGT1 is affected by these missense variants.</p

Human Dolichyl-Phosphate Alpha-N-Acetyl glucosaminyl transferase (DPAGT1); A Target Enabling Package

<p>The ER integral membrane enzyme dolichyl-phosphate alpha-N-acetyl glucosaminyl phosphotransferase (DPAGT1) catalyses the first step in the synthesis of the oligosaccharide-P-P-dolichol unit which provides the glycans structure for N-glycosylation of proteins. Mutations in DPAGT1 cause two muscle weakness conditions, limb-girdle congenital myasthenic syndrome (CMS) and congenital disorder of glycosylation type 1j (CDG1j). DPAGT1 overexpression has also been implicated in oral cancer. We have produced and solved structures of this integral membrane enzyme, DPAGT1 with the V264G mutation found in a patient with CMS, and complexes with a 50 nM inhibitor, tunicamycin. We have developed enzymatic activity and thermostability assays which have allowed us to assess the activity and stability of DPAGT1 mutants and the effect of small molecules. There are > 20 DPAGT1 associated missense variants in patients with CMS and CDG1j. We have mapped these mutations to the structure, and we will used the assays described here to assess how the activity and stability of DPAGT1 is affected by these missense variants.</p

Human Dolichyl-Phosphate Alpha-N-Acetyl glucosaminyl transferase (DPAGT1); A Target Enabling Package

<p>The ER integral membrane enzyme dolichyl-phosphate alpha-N-acetyl glucosaminyl phosphotransferase (DPAGT1) catalyses the first step in the synthesis of the oligosaccharide-P-P-dolichol unit which provides the glycans structure for N-glycosylation of proteins. Mutations in DPAGT1 cause two muscle weakness conditions, limb-girdle congenital myasthenic syndrome (CMS) and congenital disorder of glycosylation type 1j (CDG1j). DPAGT1 overexpression has also been implicated in oral cancer. We have produced and solved structures of this integral membrane enzyme, DPAGT1 with the V264G mutation found in a patient with CMS, and complexes with a 50 nM inhibitor, tunicamycin. We have developed enzymatic activity and thermostability assays which have allowed us to assess the activity and stability of DPAGT1 mutants and the effect of small molecules. There are > 20 DPAGT1 associated missense variants in patients with CMS and CDG1j. We have mapped these mutations to the structure, and we will used the assays described here to assess how the activity and stability of DPAGT1 is affected by these missense variants.</p

Structural characterization of human urea transporters UT-A and UT-B and their inhibition

Abstract: In this study, we present the structures of human urea transporters UT-A and UT-B to characterize them at molecular level and to detail the mechanism of UT-B inhibition by its selective inhibitor, UTBinh-14. High-resolution structures of both transporters establish the structural basis for the inhibitor's selectivity to UT-B, and the identification of multiple binding sites for the inhibitor will aid with the development of drug lead molecules targeting both transporters. Our study also discovers phospholipids associating with the urea transporters by combining structural observations, native MS, and lipidomics analysis. These insights improve our understanding of urea transporter function at a molecular level and provide a blueprint for a structure-guided design of therapeutics targeting these transporters

Three-dimensional Structure and Enzymatic Function of Proapoptotic Human p53-inducible Quinone Oxidoreductase PIG3*

Tumor suppressor p53 regulates the expression of p53-induced genes (PIG) that trigger apoptosis. PIG3 or TP53I3 is the only known member of the medium chain dehydrogenase/reductase superfamily induced by p53 and is used as a proapoptotic marker. Although the participation of PIG3 in the apoptotic pathway is proven, the protein and its mechanism of action were never characterized. We analyzed human PIG3 enzymatic function and found NADPH-dependent reductase activity with ortho-quinones, which is consistent with the classification of PIG3 in the quinone oxidoreductase family. However, the activity is much lower than that of ζ-crystallin, a better known quinone oxidoreductase. In addition, we report the crystallographic structure of PIG3, which allowed the identification of substrate- and cofactor-binding sites, with residues fully conserved from bacteria to human. Tyr-59 in ζ-crystallin (Tyr-51 in PIG3) was suggested to participate in the catalysis of quinone reduction. However, kinetics of Tyr/Phe and Tyr/Ala mutants of both enzymes demonstrated that the active site Tyr is not catalytic but may participate in substrate binding, consistent with a mechanism based on propinquity effects. It has been proposed that PIG3 contribution to apoptosis would be through oxidative stress generation. We found that in vitro activity and in vivo overexpression of PIG3 accumulate reactive oxygen species. Accordingly, an inactive PIG3 mutant (S151V) did not produce reactive oxygen species in cells, indicating that enzymatically active protein is necessary for this function. This supports that PIG3 action is through oxidative stress produced by its enzymatic activity and provides essential knowledge for eventual control of apoptosis

Bilayer-Mediated Structural Transitions Control Mechanosensitivity of the TREK-2 K2P Channel

The mechanosensitive two-pore domain (K2P) K(+) channels (TREK-1, TREK-2, and TRAAK) are important for mechanical and thermal nociception. However, the mechanisms underlying their gating by membrane stretch remain controversial. Here we use molecular dynamics simulations to examine their behavior in a lipid bilayer. We show that TREK-2 moves from the "down" to "up" conformation in direct response to membrane stretch, and examine the role of the transmembrane pressure profile in this process. Furthermore, we show how state-dependent interactions with lipids affect the movement of TREK-2, and how stretch influences both the inner pore and selectivity filter. Finally, we present functional studies that demonstrate why direct pore block by lipid tails does not represent the principal mechanism of mechanogating. Overall, this study provides a dynamic structural insight into K2P channel mechanosensitivity and illustrates how the structure of a eukaryotic mechanosensitive ion channel responds to changes in forces within the bilayer