Membrane protein structure determination remains one of the great challenges in structural biology. Despite the fact that 30% of all genes encode membrane proteins and over 50% of drug targets are a class of membrane proteins called G protein-coupled receptors (GPCRs), only 0.2% of the PDB structures represent integral membrane proteins. Clearly, this field is still in its infancy and lags far behind the soluble proteins in the era of structural genomics and proteomics. Oligosaccharyltransferase (OST), a multi-subunit enzyme localized in the endoplasmic reticulum (ER). This integral membrane protein complex catalyzes the co-translational modification of proteins. In the central reaction, a carbohydrate moiety is transferred from a lipid-linked pyrophosphate donor to the side chain of an asparagine within the -Asn-Xaa-Ser/Thr- tripeptide recognition motif on nascent polypeptides. This modification affects a large number of secreted and membrane proteins and ensure proper folding of nascent proteins, impart protease resistance, dictate intracellular trafficking, cell-cell specific interactions and plays a role in growth regulation. Genetic defects in this modification pathway in humans cause a class of disorders known as congenital disorders of glycosylation (CDG). These conditions affect multiple organs with severe clinical manifestations including mental retardation, developmental delay, hypoglycemia, liver dysfunction, gastrointestinal disorders, dysmorphic features, and anorexia. Complete loss of this modification is lethal in all organisms. In S. cerevisiae, OST is composed of nine nonidentical membrane proteins. Stt3p protein is a key subunit of the OST complex. The C-terminal domain of the Stt3p subunit is shown to recognize the glycosylation site present in the consensus sequence on nascent protein. Primary goal of this R21 proposal is: (1) to develop efficient heterologus expression system for overexpression of this 31.3 kDa TM containing membrane protein domain, (2) purification to homogeneity, and (3) to characterize the molecular basis of substrate recognition at atomic level. Our hypothesis is that the signature sequence motif W516-Y521 at the catalytic site of this domain of Stt3p serves as the recognition element for substrate binding since mutation of any single residue results in impairment of N- glycosylation. We have subcloned this domain into the pET28c vector, overexpressed it in E. coli cells, purified the protein to homogeneity, demonstrated binding to acceptor peptide substrate, and started biophysical characterization. Solution NMR and X-ray diffraction methods will be used to understand the mechanisms of OST function using yeast OST as the model system. Since glycosylation pathways are conserved among eukaryotes, understanding the mechanisms of N-glycosylation in model organisms such as yeast will be instrumental in elucidating the molecular basis of CDG. Moreover, membrane proteins are notoriously difficult to study, and probably constitute next frontier to reach in terms of developing proper experimental tools for understanding their structural biology.

Public Health Relevance

The Oligosaccharyltransferase enzyme complex is at a pivotal point of protein processing and folding. This enzyme is responsible for the proper functioning of many secretory and membrane proteins. Thus, as far as human health is concerned, any problem with the proper functioning of this enzyme can cause multitude of disorders involving multiple organs with severe clinical problems such as mental retardation, developmental delay, liver dysfunction, gastrointestinal problems, hypoglycemia, anorexia etc. Thus understanding the molecular basis of function of this complex integral membrane protein is very critical for the treatment or for therapeutic intervention of the above diseases.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Exploratory/Developmental Grants (R21)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Mckeon, Catherine T
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Auburn University at Auburn
Schools of Arts and Sciences
Auburn University
United States
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