CgPrfH, a Novel Bifunctional Rescue Factor for Both rRNA-damage Translation and Non-stop Translation And Structural Studies of Human ISOC1, a Potential Target of the Anti-cancer Agent Dihydromotuporamine C (dhMotC)

There are two independent studies in this thesis. The first part is the Capnocytophaga gingivalis peptide-chain release factor H (CgPrfH), a novel bifunctional rescue factor for ribosome stalling. Rescue factors play essential roles in the quality control of protein synthesis. Our previous study identified the first rescue factor, the E. coli PrfH, to rescue the rRNA-damage translation (RDT). Here, we demonstrate the first bifunctional rescue factor, the CgPrfH, which can rescue both RDT and non-stop translation (NST). The cryo-EM is utilized to determine the complex structures of 70S ribosome/CgPrfH in both rescue states at the resolution of 2.65 Å (RDT) and 3.0 Å (NST), respectively. The structural data and peptide release assays are combined to reveal that the CgPrfH utilizes two independent mechanisms to specifically recognize the RDT and NST complexes, while it commonly depends on the GGQ motif to catalyze the failed peptide release. The bifunctional study of CgPrfH further inspires us to the evolutional connection between the non-stop and rRNA-damage rescue factors. Moreover, the rRNA rescue and repairing pathways in bacteria are the potential drug target for developing next-generation antibiotics.

The second part is the structural study of the human isochorismatase domain-containing 1 (hISOC1) which was identified as the cellular target for the anti-cancer agent dihydromotuporamine (dhMotC) in our previous work. However, the structure of hISOC1 and the binding mechanism of hISOC1/dhMotC are still unknown. Here, I utilized X-ray crystallography to determine the crystal structures of the apo-hISOC1 and the hISOC1 complexed with dhMotC at the resolution of 2.5 Å and 2.2 Å, respectively. The structural data reveal that the hISOC1 is a homo-tetramer, which is further demonstrated by the cryo-EM map at the resolution of 2.7 Å. hISOC1 contains the potential functional domain, the isochorismatase domain (ISO-domain). The putative active center is rightly bound with the dhMotC molecule, which suggests the dhMotC competes with the native substrate for the active pocket. Further functional investigation shows that hISOC1 is not an isochorismatase. The structural data gives the reason that the lack of binding region for isochorismate and the oversized pocket formed by the tetrameric assembly cannot stabilize the substate in the active center. On the other side, the hISOC1 retains the conserved catalytic residues and cis-peptide group in the putative active center, which suggests that the hISOC1 may function as a hydrolase but with different substrate specificity.

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