Exploiting topological constraints to reveal buried sequence motifs in the membrane-bound N-linked oligosaccharyl transferases
The central enzyme in N-linked glycosylation is the oligosaccharyl transferase (OTase), which catalyzes glycan transfer from a polyprenyldiphosphate-linked carrier to select asparagines within acceptor proteins. PglB from Campylobacter jejuni is a single-subunit OTase with homology to the Stt3 subun...
| Main Authors: | , |
|---|---|
| Other Authors: | |
| Format: | Article |
| Language: | en_US |
| Published: |
American Chemical Society (ACS)
2012
|
| Online Access: | http://hdl.handle.net/1721.1/73497 https://orcid.org/0000-0002-5749-7869 |
| _version_ | 1826210249104687104 |
|---|---|
| author | Imperiali, Barbara Jaffee, Marcie Beth |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Imperiali, Barbara Jaffee, Marcie Beth |
| author_sort | Imperiali, Barbara |
| collection | MIT |
| description | The central enzyme in N-linked glycosylation is the oligosaccharyl transferase (OTase), which catalyzes glycan transfer from a polyprenyldiphosphate-linked carrier to select asparagines within acceptor proteins. PglB from Campylobacter jejuni is a single-subunit OTase with homology to the Stt3 subunit of the complex multimeric yeast OTase. Sequence identity between PglB and Stt3 is low (17.9%); however, both have a similar predicted architecture and contain the conserved WWDxG motif. To investigate the relationship between PglB and other Stt3 proteins, sequence analysis was performed using 28 homologues from evolutionarily distant organisms. Since detection of small conserved motifs within large membrane-associated proteins is complicated by divergent sequences surrounding the motifs, we developed a program to parse sequences according to predicted topology and then analyze topologically related regions. This approach identified three conserved motifs that served as the basis for subsequent mutagenesis and functional studies. This work reveals that several inter-transmembrane loop regions of PglB/Stt3 contain strictly conserved motifs that are essential for PglB function. The recent publication of a 3.4 Å resolution structure of full-length C. lari OTase provides clear structural evidence that these loops play a fundamental role in catalysis [Lizak, C.; (2011) Nature474, 350−355]. The current study provides biochemical support for the role of the inter-transmembrane domain loops in OTase catalysis and demonstrates the utility of combining topology prediction and sequence analysis for exposing buried pockets of homology in large membrane proteins. The described approach allowed detection of the catalytic motifs prior to availability of structural data and reveals additional catalytically relevant residues that are not predicted by structural data alone. |
| first_indexed | 2024-09-23T14:46:29Z |
| format | Article |
| id | mit-1721.1/73497 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T14:46:29Z |
| publishDate | 2012 |
| publisher | American Chemical Society (ACS) |
| record_format | dspace |
| spelling | mit-1721.1/734972022-09-29T10:27:11Z Exploiting topological constraints to reveal buried sequence motifs in the membrane-bound N-linked oligosaccharyl transferases Imperiali, Barbara Jaffee, Marcie Beth Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Chemistry Imperiali, Barbara Imperiali, Barbara Jaffee, Marcie Beth The central enzyme in N-linked glycosylation is the oligosaccharyl transferase (OTase), which catalyzes glycan transfer from a polyprenyldiphosphate-linked carrier to select asparagines within acceptor proteins. PglB from Campylobacter jejuni is a single-subunit OTase with homology to the Stt3 subunit of the complex multimeric yeast OTase. Sequence identity between PglB and Stt3 is low (17.9%); however, both have a similar predicted architecture and contain the conserved WWDxG motif. To investigate the relationship between PglB and other Stt3 proteins, sequence analysis was performed using 28 homologues from evolutionarily distant organisms. Since detection of small conserved motifs within large membrane-associated proteins is complicated by divergent sequences surrounding the motifs, we developed a program to parse sequences according to predicted topology and then analyze topologically related regions. This approach identified three conserved motifs that served as the basis for subsequent mutagenesis and functional studies. This work reveals that several inter-transmembrane loop regions of PglB/Stt3 contain strictly conserved motifs that are essential for PglB function. The recent publication of a 3.4 Å resolution structure of full-length C. lari OTase provides clear structural evidence that these loops play a fundamental role in catalysis [Lizak, C.; (2011) Nature474, 350−355]. The current study provides biochemical support for the role of the inter-transmembrane domain loops in OTase catalysis and demonstrates the utility of combining topology prediction and sequence analysis for exposing buried pockets of homology in large membrane proteins. The described approach allowed detection of the catalytic motifs prior to availability of structural data and reveals additional catalytically relevant residues that are not predicted by structural data alone. National Institutes of Health (U.S.) (GM039334) 2012-10-01T15:01:57Z 2012-10-01T15:01:57Z 2011-08 2011-07 Article http://purl.org/eprint/type/JournalArticle 0006-2960 1520-4995 http://hdl.handle.net/1721.1/73497 Jaffee, Marcie B., and Barbara Imperiali. “Exploiting Topological Constraints To Reveal Buried Sequence Motifs in the Membrane-Bound N-Linked Oligosaccharyl Transferases.” Biochemistry 50.35 (2011): 7557–7567. © 2011 American Chemical Society https://orcid.org/0000-0002-5749-7869 en_US http://dx.doi.org/10.1021/bi201018d Biochemistry Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf American Chemical Society (ACS) Prof. Imperiali via Erja Kajosalo |
| spellingShingle | Imperiali, Barbara Jaffee, Marcie Beth Exploiting topological constraints to reveal buried sequence motifs in the membrane-bound N-linked oligosaccharyl transferases |
| title | Exploiting topological constraints to reveal buried sequence motifs in the membrane-bound N-linked oligosaccharyl transferases |
| title_full | Exploiting topological constraints to reveal buried sequence motifs in the membrane-bound N-linked oligosaccharyl transferases |
| title_fullStr | Exploiting topological constraints to reveal buried sequence motifs in the membrane-bound N-linked oligosaccharyl transferases |
| title_full_unstemmed | Exploiting topological constraints to reveal buried sequence motifs in the membrane-bound N-linked oligosaccharyl transferases |
| title_short | Exploiting topological constraints to reveal buried sequence motifs in the membrane-bound N-linked oligosaccharyl transferases |
| title_sort | exploiting topological constraints to reveal buried sequence motifs in the membrane bound n linked oligosaccharyl transferases |
| url | http://hdl.handle.net/1721.1/73497 https://orcid.org/0000-0002-5749-7869 |
| work_keys_str_mv | AT imperialibarbara exploitingtopologicalconstraintstorevealburiedsequencemotifsinthemembraneboundnlinkedoligosaccharyltransferases AT jaffeemarciebeth exploitingtopologicalconstraintstorevealburiedsequencemotifsinthemembraneboundnlinkedoligosaccharyltransferases |