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Supplemental material, sj-pdf-1-jbx-10.1177_24725552211026261 for Probing the SAM Binding Site of SARS-CoV-2 Nsp14 In Vitro Using SAM Competitive Inhibitors Guides Developing Selective Bisubstrate Inhibitors by Kanchan Devkota, Matthieu Schapira, Sumera Perveen, Aliakbar Khalili Yazdi, Fengling Li, Irene Chau, Pegah Ghiabi, Taraneh Hajian, Peter Loppnau, Albina Bolotokova, Karla J. F. Satchell, Ke Wang, Deyao Li, Jing Liu, David Smil, Minkui Luo, Jian Jin, Paul V. Fish, Peter J. Brown and Masoud Vedadi in SLAS Discovery

The evolutionarily well-conserved SecA is essential for bacterial post-translational translocation. SecA uses the energy of ATP to drive preproteins through the membrane pore. The functional oligomeric state of SecA and the molecular basis for recognition of unfolded polypeptides by SecA are major unresolved questions that must be addressed to understand preprotein targeting and the molecular mechanics of SecA-mediated translocation. This thesis will address three aspects of these questions. First, the role of unstructured termini in the oligomerization and function of various SecA constructs was elucidated. By re-examining the tetramerization of a truncated SecA construct (SecA-N68), it was shown that the unstructured polypeptides at its termini are mediating its oligomerization. In turn, by removal of the first 14 N-terminal residues of the functional SecA-N95 construct, dimerization was drastically weakened. Although the weakened dimerization did not significantly affect the solution ATPase activity of SecA-N95 in vitro, it was shown that SecA-N95ΔN is not functional in vivo. Second, the interaction of unfolded polypeptides with SecA protein was investigated. It was revealed that preproteins contain sequences that can bind SecA in vitro though the binding site(s) located on the nucleotide binding domains and/or the stem region of the preprotein cross-linking domain. Both nucleotides and the N-terminal segment of SecA affected these interactions. Moreover, the sequences of several high affinity SecA binding peptides were analyzed and a possible “motif” on preproteins for interaction with SecA was identified. Finally, to acquire high-resolution insight into preprotein targeting mechanisms, we aimed at obtaining and improving the crystals of E. coli SecA-N95 and M. tuberculosis SecB (mtSecB). By removal of the unstructured termini of these proteins, larger crystals were obtained with higher frequency. Although the high-resolution structures of these molecules were not resolved, the advances made in the crystallization of these proteins paves the way for future efforts. In conclusion, this thesis has shown that SecA is able to interact with the unstructured polypeptides in vitro, and the presence of unstructured polypeptides at the termini of SecA have a profound affect on its function both in vivo and in vitro.