Antibiotics (AB) resistance is a major threat to global health, thus the development of novel AB classes is urgently needed. Lantibiotics (i.e. nisin) are natural compounds that effectively control bacterial populations, yet their clinical potential is very limited. Nisin targets membrane-embedded cell wall precursor-lipid II-via capturing its pyrophosphate group (PPi), which is unlikely to evolve, and thus represents a promising pharmaceutical target. Understanding of exact molecular mechanism of initial stages of membrane-bound lipid II recognition by water-soluble nisin is indispensable. Here, using molecular simulations, we demonstrate that the structure of lipid II is determined to a large extent by the surrounding water-lipid milieu. In contrast to the bulk solvent, in the bilayer only two conformational states remain capable of nisin binding. In these states PPi manifests a unique arrangement of hydrogen bond acceptors on the bilayer surface. Such a "pyrophosphate pharmacophore" cannot be formed by phospholipids, which explains high selectivity of nisin/lipid II recognition. Similarly, the "recognition module" of nisin, being rather flexible in water, adopts the only stable conformation in the presence of PPi analogue (which mimics the lipid II molecule). We establish the "energy of the pyrophosphate pharmacophore" approach, which effectively distinguishes nisin conformations that can form a complex with PPi. Finally, we propose a molecular model of nisin recognition module/lipid II complex in the bacterial membrane. These results will be employed for further study of lipid II targeting by antimicrobial (poly)cyclic peptides and for design of novel AB prototypes. The widespread misuse of antibiotics (AB) results in fast development of resistance by bacteria, which presents a growing threat to global health. Discovery of new classes of antibacterial agents with alternative mode of action along with grasp of these mechanisms present the primary scientific challenges. There is a class of lanthionine-containing antimicrobial peptides (AMPs) referred to as lantibiotics, which possesses medicinal potential. Nisin, the most studied lantibiotic, was known even before A. Fleming's penicillin discovery 1. Although nisin has never been used in clinical practice due to its poor pharmacokinetics, it has been widely utilized as a food preservative since 1953 with no evidence of any resistance development 2-4. Nisin is a 34-amino acid peptide produced by certain strains of Lactococcus Lactis. Following the ribosomal synthesis, nisin prepeptide undergoes significant post-translational modifications, including proteolytic removal of a leader peptide 5 and introduction of atypical amino acids, resulting in five lanthionine rings and three unsaturated residues (Fig. 1A) 6. NMR studies of nisin and its major degradation products under various conditions revealed that the molecule is highly flexible in aqueous solution and consists of two structural domains: an amphiphilic N-terminal fragment including lanthionine rings A, B a...