Biochemical Analysis of the Binding Interaction between SpaI and its Cognate Lantibiotic, Subtilin

Lantibiotics are potent antimicrobial agents that are effective at inhibiting the growth of other Gram-positive bacteria.  To protect the host organism, most lantibiotic producing bacteria have immunity proteins. Bacillus subtilis has two immunity proteins, SpaI and SpaFEG, that confer protection against its cognate lantibiotic, subtilin. SpaI, is a peripheral membrane protein located on the extracellular cytoplasmic side that has been shown to bind subtilin to prevent its interaction with the lipid membrane. SpaI is also found free floating in the medium. To our knowledge, no crystal structure is currently available for SpaI and little is known about its structural interaction with subtilin. In addition, the thermodynamic properties of this interaction are unknown. Preliminary structural studies indicate that a membrane free SpaI forms dimeric and tetrameric states. The higher oligomerization could be due to a cysteine at the N-terminal end. To test this hypothesis we generated alanine and serine mutants that indicated the dominant oligomeric state was a dimer. This was observed in the presence and absence of subtilin. Further studies are needed to understand the role of these states in actively binding subtilin. Preliminary circular dichroism studies also suggest a high b-sheet character that is consistent with a recent study, however further studies are necessary to confirm this alone and in the presence of subtilin. The thermodynamic properties of SpaI and subtilin by isothermal titration calorimetry are also ongoing. Together these approaches will help elucidate the structural and functional role of SpaI as an immunity protein.