Abstract
Oligosaccharyl transferase (OT) is a multisubunit enzyme that catalyzes N-linked glycosylation of nascent polypeptides in the lumen of the endoplasmic reticulum. In the case of Saccharomyces cerevisiae, OT is composed of nine integral membrane protein subunits. Defects in N-linked glycosylation cause a series of disorders known as congenital disorders of glycosylation (CDG). The C-terminal domain of the Stt3p subunit has been reported to contain the acceptor protein recognition site and/or catalytic site. We report here the subcloning, overexpression, and a robust but novel method of production of the pure C-terminal domain of Stt3p at 60-70 mg/L in Escherichia coli. CD spectra indicate that the C-terminal Stt3p is highly helical and has a stable tertiary structure in SDS micelles. The well-dispersed two-dimensional (1)H-(15)N HSQC spectrum in SDS micelles indicates that it is feasible to determine the atomic structure by NMR. The effect of the conserved D518E mutation on the conformation of the C-terminal Stt3p is particularly interesting. The replacement of a key residue, Asp(518), located within the WWDYG signature motif (residues 516-520), led to a distinct tertiary structure, even though both proteins have similar overall secondary structures, as demonstrated by CD, fluorescence and NMR spectroscopies. This observation strongly suggests that Asp(518) plays a critical structural role, in addition to the previously proposed catalytic role. Moreover, the activity of the protein was confirmed by saturation transfer difference and nuclear magnetic resonance titration studies.