Biochemical characterization of nucleotide binding by SECR1 and SSBP from Synechococcus elongatus

Abstract

Cyclic-di-AMP is a second messenger involved in various bacterial stress responses important for cell survival [1]. Cyanobacteria, that are essential for oxygen production in our ecosystem, also produce c-di-AMP [2]. Although it is established that c-di-AMP mediates cell survival from nighttime stress [2] and DNA damage [3], the mechanisms are not known. This study aims to biochemically characterize interactions between two Synechococcus elongatus proteins and cyclic-di-AMP and other potential nucleotide ligands. Both of these proteins, SeCR1 and SsbP, have been shown to interact with cyclic-di-AMP in a previous study, however neither had KD values reported [4]. ☐ We expressed SeCR1 and SsbP in E. coli C41(DE3) cells and purified them through multiple column chromatographic steps. We determined through peptide mass fingerprinting that SeCR1 becomes truncated during expression, an issue that could not be alleviated at transcriptional level by reducing nonspecific nuclease activity, translational level through codon optimization or post-translational level by the induction of molecular chaperones. While SeCR1 consistently co-purified in a complex with its truncated form , our MST experiments indicated that this complex, and not the individual SeCR1-WYL domain, binds cyclic-di-AMP in vitro. This implicates N-terminal regions of SeCR1 that contains a AAA domain in binding a novel ligand c-di-AMP. ☐ Our MST results showed interactions between SsbP and multiple ligands including single-stranded DNA, cyclic-di-AMP, and ATP. We determined for the first time that binding of ATP to SsbP increased the binding affinity of SsbP for ssDNA, which could be a part of the mechanism of SsbP dissociation from ssDNA during RecA -mediated homologous recombination.