Three dimensional structure of the antibiotic bacitracin A complexed to two different subtilisin proteases: novel mode of enzyme inhibition.

Abstract

The three dimensional crystal structures of thermitase-bacitracin (TMTBAC), Savinase- bacitracin (SAVBAC) and Savinase-zinc/bacitracin (SAVBAC/ZN) have been determined by X-ray diffraction to 2.2 angstroms, 2.2 angstroms and 1.95 angstroms resolution, respectively. The multifunctional dodecapeptide bacitracin A secreted by Bacillus licheniformis is well known as an antibiotic against gram-positive bacteria but also as an inhibitor for different proteases. The bacteriocidal activity requires the presence of divalent metal cations such as zinc or nickel. It also could be shown that bacitracin A is bound to subtilisin in the Bacillus licheniformis. This complex is stable throughout the purification by chromatography. Therefore the subtilisin proteases thermitase and Savinase were used for cocrystallization with bacitracin A and zinc/bacitracin A. The complexes are formed from two enzyme molecules and two bacitracin A molecules. All three complexes show the same novel mode of enzyme inhibition. Each bacitracin A chain binds non-covalently to two protease molecules: to the catalytic side of one and to the substrate recognition side of the second protease molecule. In that way the two bacitracin A molecules link two subtilisin molecules together to form a dimer. Despite this common feature we found some important differences in the conformations of bacitracin A in the three complex structures which were analysed and described in detail in this paper. An examination of the solvent structure of the complexes shows water molecules in the region around the bacitracin A molecules are not conserved and play a different role in the stabilization of the bacitracin A conformation.