Titration calorimetric studies to elucidate the specificity of the interactions of polymyxin B with lipopolysaccharides and lipid A.
Lipopolysaccharide (LPS), the major cell wall constituent of Gram-negative bacteria, evokes a multitude of biological effects in mammals including pyrogenicity and toxic shock syndrome. Polymyxin B (PmB), a polycationic cyclic peptide, is known to neutralize most of its activities. The nature of the interaction of PmB with LPS and lipid A was investigated by isothermal titration calorimetry. PmB binds to LPS as well as lipid A stoichiometrically and non-co-operatively with micromolar affinity. These interactions are driven primarily by a favourable change in entropy (delta S) and are endothermic in nature. These positive changes in enthalpies decrease with increasing temperature, yielding a heat capacity change, delta Cp, of -2385 J.mol-1.degree-1 for PmB-LPS interactions while the binding of PmB to lipid A displays a delta Cp of -2259 J.mol-1.degree-1. The negative heat capacity changes provide strong evidence for the role of hydrophobic interactions as the driving force for the association of PmB with LPS and lipid A. A correlation of the energetics of these interactions with analyses of the molecular models of PmB suggests that a cluster of solvent-exposed non-polar amino acid side-chains that line one surface of the molecule, together with a ring of positively charged residues on its other surface, are responsible for its strong and stoichiometric binding to LPS.
Srimal
S
S
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
Surolia
N
N
Balasubramanian
S
S
Surolia
A
A
D000818:Animals; D001665:Binding Sites; D002151:Calorimetry; D004563:Electrochemistry; D004731:Endotoxins / Q000037:antagonists & inhibitors / Q000633:toxicity; D006801:Humans; D066298:In Vitro Techniques; D008050:Lipid A / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity*; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity*; D015394:Molecular Structure; D008970:Molecular Weight; D011112:Polymyxin B / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D013816:Thermodynamics
D004731:Endotoxins; D008050:Lipid A; D008070:Lipopolysaccharides; D011112:Polymyxin B