Higher-order structure of polymyxin B: The functional significance of topological flexibility

The higher order structure of antibacterial polymyxin B (PxB), an N-acylate d pentacationic (4-10)-cyclic decapeptide, is determined from NMR data by s imulated annealing calculations. The antibacterial selectivity of PxB again st Gram-negative organisms suggests that PxB must participate in specific m icroscopic interactions with these organisms, and the structure of PxB prov ides insights into these interactions. Significance of the topological flex ibility of certain parts of the structure in relation to the membrane-mimet ic environment is developed to suggest the presence of two distinct and spe cific phosphoester binding sites per PxB. Although disordered in water, PxB remains in a monomeric form and adopts a well-defined structure in aqueous trifluoroethanol (TFE). Circular dichroism results show a comparable struc ture in aqueous TFE and on anionic vesicles. Docking and energy minimizatio n calculations show that the two phosphoester binding sites are essentially on the same face of the structure. The topology of the ring is locked in a fixed relationship between residues 6, 7, and 10. However, the pucker of t he ring changes residues 4 and 5 on one side and residues 8 and 9 on the ot her side. The structural flexibility within the NMR constraints permits occ upancy of the sites individually or simultaneously, in a 10 to 14 Angstrom range for the phosphorus-to-phosphorus distance between the two sites. Thus , for interactions at the Gram-negative cell surface, a PxB molecule could not only bind to the headgroup of one or two phosphatidylglycerols, but rem arkably, the two sites could also simultaneously accommodate the 1,4'-dipho sphodiglucosamine of lipid A backbone of a lipopolysaccharide. The observed combination of both fixed and flexible regions of PxB, referred to as high er order structure, accounts for its ability to perform a range of microsco pically distinct functions guided by the local environment at the bacterial cell surface.