MksEF Accessory Proteins Inhibit MksB ATPase Activity and Modulate DNA Substrate Binding

Chromosome organization and segregation are fundamental processes across all domains of life. In bacteria, the mechanisms governing nucleoid organization remain poorly understood. This study investigates the function of an alternative structural maintenance of chromosomes (SMC) complex, MksBEF, in <i>Mycobacterium smegmatis</i>. We show that MksB, the SMC subunit of the complex, binds DNA and plays a crucial role in local chromosome organization, a function distinct from that of other condensins. We successfully reconstituted the MksBEF complex and determined its stoichiometry as MksB₂E₄F₂ using gel filtration and ultracentrifugation. Gel shift assays and isothermal titration calorimetry reveal that the accessory proteins MksE and MksF interact with MksB and significantly enhance its DNA-binding affinity─an effect not observed in SMC or MukB-associated accessory proteins. Furthermore, ANS-based fluorescence experiments indicate that DNA binding induces structural rearrangements in both MksB alone and the MksBEF complex. Notably, although MksEF enhances the DNA-binding affinity of MksB, it also markedly suppresses its ATPase activity, a unique regulatory mechanism distinct from other SMC complexes. These findings provide mechanistic insights into how MksE and MksF modulate MksB activity, advancing our understanding of chromosome dynamics in mycobacteria.