Structural Basis of the Membrane Association by the Conserved RocS Membrane‐Targeting Sequence in <i>Streptococcus</i>

Chromosome segregation in the human pathogen Streptococcus pneumoniae relies on the membrane-binding protein RocS, which links the chromosomal DNA to the membrane. Beside the ability of RocS to bind DNA and interact with the chromosome partitioning protein ParB, little is known about how C-terminal membrane anchor of RocS contributes to chromosome segregation. More precisely, the molecular basis of membrane targeting remains unresolved. This study addresses the interplay between RocS C-terminal region and the lipid membrane. Combined magic-angle spinning NMR and wide-line NMR reveal that the membrane anchoring peptide folds into a short membrane-inserted kink-helix motif connected to an extended linker, with membrane insertion locally perturbing lipid packing. The fluidity of the lipid bilayer, modulated by temperature, in turn influences the anchor-membrane interactions. At the mesoscale, AFM imaging shows that the anchor selectively associates with lipid nanodomains, clustering into discrete foci. Mutational studies further reveals that a single glycine mutation in the C-terminal significantly perturbs chromosome segregation and alters lipid membrane properties. These findings reveal a mechanism for nanodomain association of a highly conserved membrane-targeting motif in Streptococci, highlighting the kink-helix anchor as a conserved element for membrane targeting across bacteria and beyond.