<span class="paragraphSection"><div class="boxTitle">Abstract</div>The broadly conserved ParB protein performs crucial functions in bacterial chromosome segregation and replication regulation. The cellular function of ParB requires it to dimerize, recognize <span style="font-style: italic;">parS</span> DNA sequences, clamp on DNA, then slide to adjacent sequences through nonspecific DNA binding. How ParB coordinates nonspecific DNA binding and sliding remains elusive. Here, we combine multiple <span style="font-style: italic;">in vitro</span> biophysical and computational tools and <span style="font-style: italic;">in vivo</span> approaches to address this question. We found that five conserved lysine residues in the C-terminal domain (CTD) of ParB play distinct roles. While the two central rigid lysine residues provide the structural platform for the CTD, the three peripheral flexible lysine residues are implicated in efficient ParB sliding. Mutations in each individual lysine decreased ParB’s DNA compaction capabilities, indicating that all five lysine residues are critical for properly positioning the DNA along the ParB CTD surface. Importantly, the integrity of these five lysines is crucial for ParB’s <span style="font-style: italic;">in vivo</span> functions, including fluorescence foci formation and sporulation initiation. Many proteins with diverse cellular activities need to move along DNA while loosely bound. Our findings provide molecular insight into how the fast backbone dynamics of multiple basic residues enable DNA-binding proteins to efficiently slide along DNA.</span>