Expression, purification, NMR structure, and molecular dynamics studies of Ost4 and Ost4V23D: A critical subunit of yeast oligosaccharyltransferase
Abstract
N-linked glycosylation is an essential and highly conserved protein modification reaction that takes place in all eukaryotes and some prokaryotes. This reaction is catalyzed by the enzyme complex, oligosaccharyltransferase (OST). In the central step of the N-linked glycosylation reaction, the pre-assembled high mannose oligosaccharide moieties are transferred from dolichol linked donors to the side chain of a specific asparagine residue in the Asn-X-Thr/Ser (where X does not equal proline) sequence of the nascent protein. Genetic defects in the N-linked glycosylation in humans results a group of disorders known as congenital disorders of glycosylation (CDG) that include but are not limited to mental retardation, developmental delay, hypoglycemia etc. The complete loss of N-linked glycosylation is lethal to all organisms. In Saccharomyces cerevisiae, the functional OST is composed of eight of the nine non-identical integral membrane protein subunits. The subunits Wbp1, Swp1, Ost1, Ost2, and Stt3p are essential for the viability of cells. The subunits Ost3 / Ost6 and Ost5 are non-essential but are required for optimal OST function. The subunit Ost4 is essential for the growth of cells at 37o C, but not at 25o C. Ost4 is the smallest subunit critical for the OST activity and the stability of the Stt3-Ost4-Ost3 sub-complex. Any mutation of the residues from 18 to 24 to a charged residue results in the destabilization of the sub-complex and impairs cell growth and in vitro OST activity. Mutation of valine (V) at position 23 in Ost4 to aspartate (D) causes defects in the N-linked glycosylation process. To understand the structure, function and role of Ost4 in N-linked glycosylation, characterizations of Ost4 and its functionally important mutant/s are critical. My doctoral dissertation is focused on the following three parts: (1) production and biophysical characterization of Ost4V23D, (2) 3D structure determination of the Ost4 and Ost4V23D in DPC micelles by solution-state NMR and molecular dynamics (MD) simulation, and (3) 3D structure determination of Ost4 and Ost4V23D in bilayer by solid state NMR followed by MD simulation. A comparison of the structure of the V23D mutant protein to its wild-type to reveal how the mutation affects the overall structure and function of the enzyme. Additionally, we have shed light on the molecular basis of why a point mutation of certain hydrophobic residues to charged residues destabilizes the catalytic sub-complex rendering the OST enzyme dysfunctional.
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