Structure-function relationships in monotopic phosphoglycosyl transferases
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2019
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| Format: | Thesis |
| Language: | eng |
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Massachusetts Institute of Technology
2019
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| Online Access: | https://hdl.handle.net/1721.1/122206 |
| _version_ | 1826203535574827008 |
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| author | Entova, Sonya. |
| author2 | Barbara Imperiali. |
| author_facet | Barbara Imperiali. Entova, Sonya. |
| author_sort | Entova, Sonya. |
| collection | MIT |
| description | Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2019 |
| first_indexed | 2024-09-23T12:38:34Z |
| format | Thesis |
| id | mit-1721.1/122206 |
| institution | Massachusetts Institute of Technology |
| language | eng |
| last_indexed | 2024-09-23T12:38:34Z |
| publishDate | 2019 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/1222062019-09-20T03:03:41Z Structure-function relationships in monotopic phosphoglycosyl transferases Entova, Sonya. Barbara Imperiali. Massachusetts Institute of Technology. Department of Biology. Massachusetts Institute of Technology. Department of Biology Biology. Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2019 Cataloged from PDF version of thesis. Includes bibliographical references. Complex glycans play essential roles in prokaryotic and eukaryotic biology. While this ubiquitous post-translational modification takes a diversity of forms, many glycoconjugate biosynthesis pathway across domains of life follows a common logic. Glycan assembly is initiated by a phosphoglycosyl transferase (PGT) that transfers a phosphosugar from a nucleotide donor to a polyprenol phosphate (PrenP) chain embedded in the membrane. The PrenPP-sugar product is elaborated by downstream glycosyltransferases, transferred across the membrane and ultimately appended to various acceptor molecules. The PGTs initiating glycan assembly adopt diverse membrane architectures. An extensive superfamily of PGTs, elucidated in part by this thesis, is exemplified by PglC from the Gramnegative pathogen, Campylobacterjejuni. PglC comprises a globular cytosolic domain and an N-terminal membrane-resident domain. Recent structural and biochemical analyses determined that this domain forms a helix-break-helix motif, termed the reentrant membrane helix (RMH), that enters and exits on the same face of the membrane, resulting in a monotopic topology. The RMH anchors the PglC fold in the membrane in a manner not previously observed among other monotopic membrane proteins. This thesis focuses on structure-function relationships in the RMH and associated domains. Two conserved motifs are shown to drive formation of a reentrant topology for PglC, and to exemplify common principles of topology determination among diverse monotopic proteins. These principles are further applied to the identification of reentrant domains in an extensive superfamily of monotopic lipid A acyltransferases previously thought to be membrane-spanning. The next section of the thesis explores the highly conserved role of PrenP in complex glycan biosynthesis. The significance of PrenP geometry in mediating substrate binding and modulating the local membrane environment is presented. Additionally, a conserved proline residue in the PglC RMH is determined to drive PrenP binding and specificity. Molecular insights from this study shed new light on the roles of PrenP in facilitating diverse glycoconjugate biosynthesis pathways. Finally, a cell-free methodology for expression of PglC directly into model membrane lipid Nanodiscs is described. This system has valuable applications for the study of interactions between PglC and downstream glycosyltransferase enzymes, and for further structural characterization of PglC in a membrane environment. by Sonya Entova. Ph. D. Ph.D. Massachusetts Institute of Technology, Department of Biology 2019-09-17T16:29:38Z 2019-09-17T16:29:38Z 2019 2019 Thesis https://hdl.handle.net/1721.1/122206 1117709735 eng MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission. http://dspace.mit.edu/handle/1721.1/7582 218 pages application/pdf Massachusetts Institute of Technology |
| spellingShingle | Biology. Entova, Sonya. Structure-function relationships in monotopic phosphoglycosyl transferases |
| title | Structure-function relationships in monotopic phosphoglycosyl transferases |
| title_full | Structure-function relationships in monotopic phosphoglycosyl transferases |
| title_fullStr | Structure-function relationships in monotopic phosphoglycosyl transferases |
| title_full_unstemmed | Structure-function relationships in monotopic phosphoglycosyl transferases |
| title_short | Structure-function relationships in monotopic phosphoglycosyl transferases |
| title_sort | structure function relationships in monotopic phosphoglycosyl transferases |
| topic | Biology. |
| url | https://hdl.handle.net/1721.1/122206 |
| work_keys_str_mv | AT entovasonya structurefunctionrelationshipsinmonotopicphosphoglycosyltransferases |