Higher-Order Structure of Polymyxin B:  The Functional Significance of Topological Flexibility

Abstract

The higher order structure of antibacterial polymyxin B (PxB), an N-acylated pentacationic (4−10)-cyclic decapeptide, is determined from NMR data by simulated annealing calculations. The antibacterial selectivity of PxB against Gram-negative organisms suggests that PxB must participate in specific microscopic interactions with these organisms, and the structure of PxB provides insights into these interactions. Significance of the topological flexibility of certain parts of the structure in relation to the membrane-mimetic environment is developed to suggest the presence of two distinct and specific 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 structure in aqueous TFE and on anionic vesicles. Docking and energy minimization 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 the ring changes residues 4 and 5 on one side and residues 8 and 9 on the other side. The structural flexibility within the NMR constraints permits occupancy of the sites individually or simultaneously, in a 10 to 14 Å 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 remarkably, the two sites could also simultaneously accommodate the 1,4‘-diphosphodiglucosamine of lipid A backbone of a lipopolysaccharide. The observed combination of both fixed and flexible regions of PxB, referred to as higher order structure, accounts for its ability to perform a range of microscopically distinct functions guided by the local environment at the bacterial cell surface.

Polymyxin B–lipid interactions in Langmuir–Blodgett monolayers of Escherichia coli lipids: A thermodynamic and atomic force microscopy study

“…The structure of PxB in membrane-membrane contacts (PxBc) is not known at the molecular level, but CD spectra show a similar structure in aqueous trifluoroacetic acid (TFE) 42 and on anionic vesicles. 16 This structure has been characterized by two-dimensional (2D) NMR in combination with simulated annealing and molecular modeling calculations.…”

“…16 This structure has been characterized by two-dimensional (2D) NMR in combination with simulated annealing and molecular modeling calculations. 42 According to these studies, PxB adopts a planar structure with a combination of fixed conformations for residues Phe-6, Leu-7, and Thr-10, and structural flexibility for residues Dab-4, Dab-8, and Dab-9. Docking of phosphodiester groups to the minimized structure showed that PxB contained two sites with potential binding affinity to such groups: Dab-1-Dab-3-Dab-5 (site A), and Dab-8 -Dab-9 -Thr-10 (site B).…”

Structure−Activity Relationships of Polymyxin Antibiotics

“…The fact that polymyxin B nonapeptide (PMBN (193) , i.e., PMB minus the N -terminal fatty acyl chain and Dab1 residue), lacks antibacterial activity highlights the importance of both the electrostatic and hydrophobic interactions for the mechanism of polymyxin action. Following insertion, polymyxins are purported to transit the OM via a self-promoted uptake mechanism 23–25,27,29–31. Subsequently, the polymyxin molecule inserts and disrupts the physical integrity of the phospholipid bilayer of the inner membrane via membrane thinning by straddling the interface of the hydrophilic head groups and fatty acyl chains or transient poration 23–25,27,29–31.…”

Polymyxins and Analogues Bind to Ribosomal RNA and Interfere with Eukaryotic Translation in Vitro

“…[4b,4c,5] These observations have led to the hypotheses that polymyxins act on the IM by permeating the phospholipids bilayer or by pore formation. [4b,4d,6] An alternative hypothesis suggests that polymyxins may facilitate phospholipid exchange, which could create osmotic imbalance, leading to bacteriolysis. [7] …”