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The solution structure of the lantibiotic immunity protein NisI and its interactions with nisin
(2015)
Many Gram-positive bacteria produce lantibiotics, genetically encoded and posttranslationally modified peptide antibiotics, which inhibit the growth of other Gram-positive bacteria. To protect themselves against their own lantibiotics these bacteria express a variety of immunity proteins including the LanI lipoproteins. The structural and mechanistic basis for LanI-mediated lantibiotic immunity is not yet understood. Lactococcus lactis produces the lantibiotic nisin, which is widely used as a food preservative. Its LanI protein NisI provides immunity against nisin but not against structurally very similar lantibiotics from other species such as subtilin from Bacillus subtilis. To understand the structural basis for LanI-mediated immunity and their specificity we investigated the structure of NisI. We found that NisI is a two-domain protein. Surprisingly, each of the two NisI domains has the same structure as the LanI protein from B. subtilis, SpaI, despite the lack of significant sequence homology. The two NisI domains and SpaI differ strongly in their surface properties and function. Additionally, SpaI-mediated lantibiotic immunity depends on the presence of a basic unstructured N-terminal region that tethers SpaI to the membrane. Such a region is absent from NisI. Instead, the N-terminal domain of NisI interacts with membranes but not with nisin. In contrast, the C-terminal domain specifically binds nisin and modulates the membrane affinity of the N-terminal domain. Thus, our results reveal an unexpected structural relationship between NisI and SpaI and shed light on the structural basis for LanI mediated lantibiotic immunity.
Intrinsically disordered protein (IDP) duplexes composed of two IDP chains cross-linked by bivalent partner proteins form scaffolds for assembly of multiprotein complexes. The N-terminal domain of dynein intermediate chain (N-IC) is one such IDP that forms a bivalent scaffold with multiple dynein light chains including LC8, a hub protein that promotes duplex formation of diverse IDP partners. N-IC also binds a subunit of the dynein regulator, dynactin. Here we characterize interactions of a yeast ortholog of N-IC (N-Pac11) with yeast LC8 (Dyn2) or with the intermediate chain-binding subunit of yeast dynactin (Nip100). Residue level changes in Pac11 structure are monitored by NMR spectroscopy, and binding energetics are monitored by isothermal titration calorimetry (ITC). N-Pac11 is monomeric and primarily disordered except for a single α-helix (SAH) at the N terminus and a short nascent helix, LH, flanked by the two Dyn2 recognition motifs. Upon binding Dyn2, the only Pac11 residues making direct protein-protein interactions are in and immediately flanking the recognition motifs. Dyn2 binding also orders LH residues of Pac11. Upon binding Nip100, only Pac11 SAH residues make direct protein-protein interactions, but LH residues at a distant sequence position and L1 residues in an adjacent linker are also ordered. The long distance, ligand-dependent ordering of residues reveals new elements of dynamic structure within IDP linker regions.