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 under-
stood. This study investigates the function of an alternative structural
maintenance of chromosomes (SMC) complex, MksBEF, in
Mycobacterium smegmatis. 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 ultracentrifuga-
tion. 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.