Molecular Recognition of Lipid II by Lantibiotics: Synthesis and Conformational Studies of Analogues of Nisin and Mutacin Rings A and B

Abstract

In response to the growing threat posed by antibiotic-resistant bacterial strains, extensive research is currently focused on developing antimicrobial agents that target lipid II, a vital precursor in the biosynthesis of bacterial cell walls. The lantibiotic nisin and related peptides display unique and highly selective binding to lipid II. A key feature of the nisin–lipid II interaction is the formation of a cage-like complex between the pyrophosphate moiety of lipid II and the two thioether-bridged rings, rings A and B, at the N-terminus of nisin. To understand the important structural factors underlying this highly selective molecular recognition, we have used solid-phase peptide synthesis to prepare individual ring A and B structures from nisin, the related lantibiotic mutacin, and synthetic analogues. Through NMR studies of these rings, we have demonstrated that ring A is preorganized to adopt the correct conformation for binding lipid II in solution and that individual amino acid substitutions in ring A have little effect on the conformation. We have also analyzed the turn structures adopted by these thioether-bridged peptides and show that they do not adopt the tight α-turn or β-turn structures typically found in proteins.

A Chemical Biology Approach to Understanding Molecular Recognition of Lipid II by Nisin(1–12): Synthesis and NMR Ensemble Analysis of Nisin(1–12) and Analogues

“…Previously, we have reported the synthesis and average solution phase conformations (calculated in XPLOR‐NIH) of the individual lipid II‐binding rings of nisin and mutacin I . We therefore sought to compare the NAMFIS solutions to these previously calculated structures to determine whether the presence of a second ring significantly affects conformation.…”

“…Another factor to consider,e specially in the interesto fd evelopingm ore stable antibiotics based on the structureo f nisin, [28][29][30][31] is the effect of residue mutation within lantibiotic binding rings on either solutionc onformation or antibacterial activity.S ignificant efforts have been directed towards understanding the effects of dehydro residue replacement,a st hese residues contribute to the metabolic instability of these pep-tides, however no clear picture has yet emerged. Palmer et al have shown that substitution of Dha5 for Ala in nisin ring A leads to significant conformational change of the isolated ring A, [32] conversely,o ur NMR studies [25] comparingi solated ring A structures of nisina nd mutacin Iw ith saturated analogues of mutacin Ir ing Ai ndicated that the replacement of Dha5 by either Ser or Ala did not significantly affectt he overall conformation of the Leu4-Xaa5-Leu6 portion of ring A. This is supported by mutation studies.…”

“…[41,42] We have developed very effective solid-phase peptides ynthesis methodology which we and others have appliedtothe synthesis of individual rings of lantibiotics, [43,44] overlapping rings [45] and to the total synthesis of complete lantibiotics. [46][47][48][49] We have previously reported the solid-phase synthesis of the individual Aa nd Br ings of nisin and of the related lantibiotic, mutacin I, [25] and have investigated the conformationalp roperties of these isolated rings ands ynthetic analogues by NMR. We have now extended this work to prepares ynthetic analogues of WT nisin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12), which can be compared with WT nisin(1-12) itself (1)( Figure 2).…”

“…In both analogues 2 and 3,w ea lso sub-stitutedL an for the naturally-occurring MeLani nr ing B, as our previouss tudies had indicatedt hat replacement of Lan for MeLan did not significantly change the backbonec onformation. [25] Our route to (Thr2, Ser5) analogue (2)startedfrom the previously reported [25] resin-bound cyclic ring Bp eptide 4.W ei nitially attempted to couple the next (Teoc, TMSE/Fmoc) Lan monomer 5 [45] to the free -NH 2 group of 4 to give resin-bound intermediate 6,a nd then to build up the linear precursor to ring A, using standard Fmoc SPPS coupling conditions (HOAt and PyAOP) (Scheme 1). However, only trace amounts of product with extensive impurities were obtained.…”

“…Previously,w eh ave reported the synthesis and average solution phase conformations( calculated in XPLOR-NIH [62,63] )o ft he individual lipid II-binding rings of nisin and mutacin I. [25] We therefore sought to compare the NAMFIS solutions to these previously calculated structurest od etermine whether the presenceo fasecond ring significantly affects conformation. Firstly,i solated nisin ring B( 15)a nd aL an analogue ( 16)w ere compared to the corresponding regionso ft he nisin(1-12) (1) and (Thr2, Ser5) analogue (2)N AMFIS solutions( Figure 9A and B).…”