Search for: All records

PDB Code(s): 7UO6 

Abstract Asparagine-linked glycosylation, also known as N-linked glycosylation, is an essential and highly conserved co- and post-translational protein modification in eukaryotes and some prokaryotes. In the central step of this reaction, a carbohydrate moiety is transferred from a lipid-linked donor to the side-chain of a consensus asparagine in a nascent protein as it is synthesized at the ribosome. Complete loss of oligosaccharyltransferase (OST) function is lethal in eukaryotes. This reaction is carried out by a membrane-associated multisubunit enzyme, OST, localized in the endoplasmic reticulum. The smallest subunit, Ost4, contains a single membrane-spanning helix that is critical for maintaining the stability and activity of OST. Mutation of any residue from Met18 to Ile24 of Ost4 destabilizes the enzyme complex, affecting its activity. Here, we report solution nuclear magnetic resonance structures and molecular dynamics (MD) simulations of Ost4 and Ost4V23D in micelles. Our studies revealed that while the point mutation did not impact the structure of the protein, it affected its position and solvent exposure in the membrane mimetic environment. Furthermore, our MD simulations of the membrane-bound OST complex containing either WT or V23D mutant demonstrated disruption of most hydrophobic helix–helix interactions between Ost4V23D and transmembrane TM12 and TM13 of Stt3. This disengagement of Ost4V23D from the OST complex led to solvent exposure of the D23 residue in the hydrophobic pocket created by these interactions. Our study not only solves the structures of yeast Ost4 subunit and its mutant but also provides a basis for the destabilization of the OST complex and reduced OST activity. 

Asparagine-linked glycosylation is an essential and highly conserved protein modification reaction that occurs in the endoplasmic reticulum of cells during protein synthesis at the ribosome. In the central reaction, a pre-assembled high- mannose sugar is transferred from a lipid-linked donor substrate to the side-chain of an asparagine residue in an -N-X-T/S- sequence (where X is any residue except Proline). This reaction is carried by a membrane-bound multi-subunit enzyme complex, Oligosaccharyltransferase (OST). In humans, genetic defects in OST lead to a group of rare metabolic diseases collectively known as congenital disorders of glycosylation (CDG). Certain mutations are lethal for all organisms. In yeast, the OST is composed of nine non-identical protein subunits. The functional enzyme complex contains eight subunits with either Ost3 or Ost6 at any given time. Ost4, an unusually small protein, plays a very important role in the stabilization of the OST complex. It bridges the catalytic subunit Stt3 with Ost3 (or Ost6) in the Stt3-Ost4-Ost3 (or Ost6) sub-complex. Mutation of any residue from M18-I24 in the trans-membrane helix of yeast Ost4 negatively impacts N-linked glycosylation and the growth of yeast. Indeed, mutation of valine23 to an aspartate impairs OST function in vivo resulting in a lethal phenotype in yeast. To understand the structural mechanism of Ost4 in the stabilization of the enzyme complex, we have initiated a detailed investigation of Ost4 and its functionally important mutant, Ost4V23D. Here, we report the backbone 1H, 13C and 15N resonance assignments for Ost4 and Ost4V23D in DPC micelles. 

Lepidopteran male moths have an extraordinarily sensitive olfactory system that is capable of detecting and responding to minute amounts of female-secreted pheromones over great distances. Pheromone-binding proteins (PBPs) in male antennae ferry the hydrophobic ligand across the aqueous lymph to the olfactory receptor neuron triggering the response. PBPs bind ligands at physiological pH of the lymph and release them at acidic pH near the receptor while undergoing a conformational change. InAnthereae polyphemusPBP1, ligand binding to the hydrophobic pocket and its release is regulated by two biological gates: His70 and His95 at one end of the pocket and C-terminus tail at the other end. Interestingly, in Asian corn borerOstrinia furnacalisPBP2 (OfurPBP2), critical residues for ligand binding and release are substituted in both biological gates. The impact of these substitutions on the ligand binding and release mechanism in OfurPBP2 is not known. We report here overexpression of soluble OfurPBP2 and structural characterization at high and low pH by circular dichroism (CD) and NMR. Ligand binding and ab initio model development were carried out with fluorescence and small-angle X-ray scattering (SAXS) respectively. OfurPBP2 in solution at pH 6.5 is homogeneous, well-folded and has a compact globular shape.