The pyrophosphate cage: the structure of the nisin/lipid II complex provides a blueprint for novel antibiotics

lanthionine or meso-lanthionine D-alanine moiety

'3-methyllanthionine or (2S, 3S, 6R)-3-methyllanthionine

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(hydroxy-oxido-phosphoryl) hydrogen phosphate

The nisin-lipid II complex reveals a pyrophosphate cage that provides a blueprint for novel antibiotics.

Nisin is an example of type-A lantibiotics that contain cyclic lanthionine rings
and unusual dehydrated amino acids. Among the numerous pore-forming
antimicrobial peptides, type-A lantibiotics form an unique family of
post-translationally modified peptides. Via the recognition of cell wall
precursor lipid II, nisin has the capacity to form pores against Gram-positive
bacteria with an extremely high activity in the nanomolar (nM) range. Here we
report a high-resolution NMR spectroscopy study of nisin/lipid II interactions
in SDS micelles as a model membrane system in order to elucidate the mechanism
of molecular recognition at residue level. The binding to lipid II was studied
through (15)N-(1)H HSQC titration, backbone amide proton temperature
coefficient analysis, and heteronuclear (15)N[(1)H]-NOE relaxation dynamics
experiments. Upon the addition of lipid II, significant changes were monitored
in the N-terminal part of nisin. An extremely low amide proton temperature
coefficient (Delta delta/Delta T) was found for the amide proton of Ala3 (>
-0.1 ppb/K) in the complex form. This suggests tight hydrogen bonding and/or
isolation from the bulk solvent for this residue. Large chemical shift
perturbations were also observed in the first two rings. In contrast, the
C-terminal part of nisin was almost unaffected. This part of the molecule
remains flexible and solvent-exposed. On the basis of our results, a multistep
pore-forming mechanism is proposed. The N-terminal part of nisin first binds to
lipid II, and a subsequent structural rearrangement takes place. The C-terminal
part of nisin is possibly responsible for the activation of the pore formation.
In light of the emerging antibiotic resistance problems, an understanding of
the specific recognition mechanism of nisin with lipid II at the residue
specific level may therefore aid in the development of novel antibiotics.

Hsu ST, Breukink E, de Kruijff B, Kaptein R, Bonvin AM, van
Nuland NA.
Biochemistry. 2002 Jun 18;41(24):7670-6.

Mapping the targeted membrane pore formation mechanism by solution NMR: the nisin Z and lipid II interaction in SDS micelles.

The peptidoglycan layers surrounding bacterial membranes are essential for
bacterial cell survival and provide an important target for antibiotics. Many
antibiotics have mechanisms of action that involve binding to Lipid II, the
prenyl chain-linked donor of the peptidoglycan building blocks. One of these
antibiotics, the pore-forming peptide nisin uses Lipid II as a receptor
molecule to increase its antimicrobial efficacy dramatically. Nisin is the
first example of a targeted membrane-permeabilizing peptide antibiotic.
However, it was not known whether Lipid II functions only as a receptor to
recruit nisin to bacterial membranes, thus increasing its specificity for
bacterial cells, or whether it also plays a role in pore formation. We have
developed a new method to produce large amounts of Lipid II and variants
thereof so that we can address the role of the lipid-linked disaccharide in the
activity of nisin. We show here that Lipid II is not only the receptor for
nisin but an intrinsic component of the pore formed by nisin, and we present a
new model for the pore complex that includes Lipid II.

Breukink E, van Heusden HE, Vollmerhaus PJ, Swiezewska E, Brunner L, Walker S,
Heck AJ, de Kruijff B.
J Biol Chem. 2003 May 30;278(22):19898-903.

Lipid II is an intrinsic component of the pore induced by nisin in bacterial membranes.

The sample is disolved in pure DMSO so no pH was measured