Abstract 1490 Identification of flexible hinge region residues in bacterial topoisomerase IA

Topoisomerase IA enzymes are ubiquitous in all domains of life and can catalyze topological changes for both DNA and RNA during vital cellular processes. Bacterial type IA topoisomerases are promising targets for novel antibiotics to treat drug-resistant infections including MDR-TB. Human type IA topoisomerase TOP3B may be a host factor for the replication of positive sense RNA viruses including dengue. However, unanswered questions remain regarding the dynamic conformations of type IA topoisomerases during catalysis. According to the proposed enzyme mechanism, the active core of topoisomerase IA undergoes large conformational changes defined as "gate opening" and "gate closing" due to domain rearrangements. These conformational changes control DNA entry and lead to the relaxation of negatively supercoiled DNA or decatenation of DNA. For this project, our goal is to identify the flexible hinge residues of topoisomerase IA which initiate and control domain rearrangements. Our findings on mycobacterial topoisomerase IA will shed light on the nature and location of the possible hinge region. Our first approach was to use the online PACKMAN server to predict possible hinges from the crystal structure of Mycobacterium tuberculosis topoisomerase I (PDB 8CZQ). The predicted region from P506 to L526 with a p-value <0.05 was then studied as a potential hinge since these residues are in the region that connects domains D2 and D4, which is one of the attributes of hinge regions in the protein. Next, two highly conserved polar residues, R516 and E519 in the predicted hinge region were substituted with alanine. In the assay of complementation of topoisomerase I mutation in E. coli AS17 cells, the mutant R516A showed more than 10-fold loss in activity and E519A showed almost no activity. This suggests the E519 may be required for conformational change after DNA binding. The residue R516 is in a flexible loop of domain D2 and interact with the DNA inside the toroidal cavity as well as maintains inter-domain interaction with E207 of D4 and D691 of D5 domains. We further studied these interactions in biochemical and biophysical experiments after purifying the mutant proteins with alanine substitutions. The results showed significant loss in DNA relaxation after mutating E207 and R516 consistent with their proposed role as hinge residues in domain rearrangements. The retention of DNA substrate binding through DNA binding assay and protein melting temperatures measured in thermal shift assays ruled out the effect of the mutations on protein folding and supported the loss in activity to be due to alteration of local charge and flexibility. Our findings can be utilized in structure-based drug design and in silico screening of inhibitors which are still dependent on single conformation crystal structures despite proteins having function-related dynamics and conformational changes. This research is supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM139817.