Molecular recognition is at the core of all biological processes whereby protein-protein interactions (PPI) relay messages to drive signaling events. However, many regulatory responses are driven by weak or transient PPI. These interactions are difficult to study using structural biology techniques because they are labile and result in heterogeneous populations. Moreover, interactions reconstituted using peptides are difficult to interpret because they lack context. In this thesis, I characterized key signaling complexes implicated in the replication checkpoint response (Dbf4-Rad53-Cdc7 complex), mitosis (Dbf4-Cdc5 complex), and DNA mismatch repair (clamp-MutL complex). I solved the crystal structures of the Dbf4-Rad53 and clamp-MutL weak complexes by generating fusions of the binding partners. The structures revealed that Dbf4 and MutL undergo subtle conformational movements upon engaging their binding partners, which were sufficient to alter both interfaces. Overall, the structures offer insight as to how Rad53 could inhibit Dbf4-Cdc7 during the replication checkpoint and how the clamp could activate MutL during mismatch repair. Acquiring the Dbf4-Cdc5 co-crystal structure required optimization the Dbf4 peptide. The Dbf4-Cdc5 and Dbf4-Rad53 complexes were relatable because both interactions were phosphorylation-independent even though Rad53 and Cdc5 are known to recognize phosphorylated targets. Dbf4 engaged a binding site on Cdc5 located opposite to the phosphoepitope binding pocket, which is reminiscent to its interaction with Rad53. Collectively, the structures of Dbf4 and its binding partners reveal that Rad53 and Cdc5 function beyond phosphoepitope recognition whereby they utilize additional binding surfaces to engage substrates. / Thesis / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/24282 |
| Date | 11 1900 |
| Creators | Almawi, Ahmad |
| Contributors | Guarne, Alba, Biochemistry and Biomedical Sciences |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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