Identification of SPOR domain amino acids important for septal localization, peptidoglycan binding, and a disulfide bond in the cell division protein FtsN.

SPOR domains are about 75 amino acids long and probably bind septal peptidoglycan during cell division. We mutagenized 33 amino acids with surface-exposed side chains in the SPOR domain from an Escherichia coli cell division protein named FtsN. The mutant SPOR domains were fused to Tat-targeted green fluorescent protein ((TT)GFP) and tested for septal localization in live E. coli cells. Lesions at the following 5 residues reduced septal localization by a factor of 3 or more: Q251, S254, W283, R285, and I313. All of these residues map to a β-sheet in the published solution structure of FtsN(SPOR). Three of the mutant proteins (Q251E, S254E, and R285A mutants) were purified and found to be defective in binding to peptidoglycan sacculi in a cosedimentation assay. These results match closely with results from a previous study of the SPOR domain from DamX, even though these two SPOR domains share <20% amino acid identity. Taken together, these findings support the proposal that SPOR domains localize by binding to septal peptidoglycan and imply that the binding site is associated with the β-sheet. We also show that FtsN(SPOR) contains a disulfide bond between β-sheet residues C252 and C312. The disulfide bond contributes to protein stability, cell division, and peptidoglycan binding.