Substrate Specificity of the Secreted Nisin Leader Peptidase NisP

Nisin (NisA) is an antimicrobial peptide produced by <i>Lactococcus lactis</i> and belongs to the class of lanthipeptides, more specifically to the class of lantibiotics. They are ribosomally synthesized as a precursor peptide and are comprised of an N-terminal leader peptide and a C-terminal core peptide. The core peptide is post-translationally modified and contains dehydrated amino acids in addition to five (methyl)-lanthionine rings, which are crucial for its activity. The leader peptide serves as a signal sequence and ensures that NisA remains inactive but secretion-competent within the cell. After translocation into the extracellular space, the leader peptide is cleaved by the leader peptidase NisP, resulting in active nisin. NisP is an extracellular subtilisin-like serine protease, which recognizes the cleavage site GASPR|IT located at the C-terminal end of the leader peptide. Here, we present the biochemical characterization of secreted and purified NisP (NisP_s) with its natural substrate, the fully modified NisA (mNisA). Furthermore, we determined the kinetic parameters of NisP_s in the presence of NisA containing different modification states. Additionally, <i>in vitro</i> data revealed that NisP_s can efficiently cleave the leader peptide of mNisA. However, it is strictly dependent on the modification state of the core peptide. Thus, NisP_s has a sequence-based cleavage activity, and the presence of at least one lanthionine ring is crucial for optimal substrate recognition and subsequent cleavage

Structure of NsrR-RD.

<p>Cartoon representation of the helices (α1 – α5) and β-sheets (β1 - β5). Structural areas with the highest variations to the receiver domains of DrrB (pink, 1P2F), MtrA (grey, 2GWR), and PhoB (blue, 1B00) are marked in separate boxes.</p

Human BSEP and MDR3 expression in <i>Pichia pastoris.</i>

<p><b>A</b> 5 µg of membranes derived from <i>P. pastoris</i> cells carrying the empty expression plasmid pSGP18 (neg ctrl), BSEP or BSEP-GFP were subjected to SDS-PAGE and immunoblotting (lanes from left to right). The negative control (left lane) did not react with the monoclonal antibody (F-6), while BSEP (middle lane) and BSEP-GFP (right lane) could be detected by the same antibody. <b>B</b> Identical samples were probed with a monoclonal GFP antibody. The negative control (left lane) as well as BSEP (middle lane) showed no signal with anti-GFP antibody, while BSEP-GFP could be detected (right lane). <b>C</b> In case of MDR3 the negative control (left lane) showed no signal with the monoclonal antibody C219; MDR3 (middle lane) as well as MDR3-GFP (right lane) could be detected with the monoclonal antibody C219. <b>D</b> Identical MDR3 samples were probed with a monoclonal GFP antibody. The negative control (left lane) as well as MDR3 (middle lane) showed no signal with anti-GFP antibody, while MDR3-GFP could be detected (right lane). The position of the molecular weight markers are shown on the left.</p

Characterization of purified human MDR3 in Fos-choline-16.

<p><b>A</b> Coomassie Brilliant Blue-stained SDS-PAGE and immunoblot using an anti-MDR3 antibody of purified MDR3 wild-type and the MDR3 EQ/EQ-mutant via TAP. Molecular weight markers are shown on the left. <b>B</b> Normalized ATPase activity of MDR3 wild-type (black) and of an ATPase deficient mutant (E558Q E1207Q, white) in FC-16 without and with different phospholipids. The ATPase activity of three independent MDR3 purifications was determined ± SD (n = 3).</p

Purification and nucleotide binding of human BSEP.

<p><b>A</b> Coomassie Brilliant Blue-stained SDS-PAGE of purified BSEP solubilized in Fos-choline-16 or in β-DDM and Cymal5, which were exchanged after solubilization. Molecular weight markers are indicated on the left. <b>B</b> Purified BSEP in all three detergents was incubated with ATP-agarose and bound protein was eluted in SDS sample buffer and examined with immunoblotting with a monoclonal antibody (F-6). BSEP signals could be detected in β-DDM and Cymal5, but not in Fos-Choline-16, indicating only binding to ATP in maltosides.</p

NisC Binds the FxLx Motif of the Nisin Leader Peptide

Nisin is a model system for lantibiotics, a class of peptides displaying antimicrobial activity against various Gram-positive bacteria. After ribosomal synthesis, the precursor peptide is modified in two steps, of which the last one involves consecutive cyclization reactions mediated by the cyclase NisC. Here, we present a detailed <i>in vitro</i> study of the interaction between NisC and the nisin precursor peptide. Our results unravel a specific interaction of NisC with the leader peptide independent of the maturation state. Furthermore, mutagenesis studies identified a specific binding sequence within the leader. Two amino acids (F_–18 and L_–16) within the highly conserved -FNLD- box of class I lantibiotics are essential for binding. They represent a potential general binding motif between leader peptides of a group of lantibiotics with their cyclase family. In summary, these <i>in vitro</i> data provide a new perception on the complexity of the lantibiotic modification machineries

Structure of the C-terminal effector domain of NsrR.

<p>Cartoon representation of the C-terminal effector domain of NsrR (green; recognition helix in cyan). The structural areas with the highest variations compared to the effector domains of DrrB (pink, 1P2F), MtrA (grey, 2GWR), and PhoB (blue, 1GXQ) are marked. The transactivation loop of MtrA is missing in the structure, therefore, the two termini are connected by a dashed line.</p

Detergent screening utilizing FSEC.

<p>FSEC analysis of BSEP-GFP (<b>A</b>) in five representative detergents and MDR3-GFP in five detergents (<b>B</b>). The arrows indicate the estimated elution position of the void volume and free GFP. Additional FSEC profiles are summarized in the supplementary material.</p

Fluorescence microscopy of BSEP-GFP and MDR3-GFP expressing <i>Pichia pastoris</i> cells.

<p><i>P. pastoris</i> cells expressing GFP, BSEP-GFP or MDR3-GFP were harvested 48 h after induction and examined for GFP fluorescence (upper row) by confocal LSM. BSEP-GFP as well as MDR-GFP was located in the plasma membrane of <i>P. pastoris</i> cells in contrast to soluble GFP, which was homogenously distributed within the cell. Bottom row: merge of the GFP fluorescence and the Differential Interference Contrast (DIC) scans.</p

Data collection, phasing, and refinement statistics for the receiver and effector domains of NsrR.

<p>Data collection, phasing, and refinement statistics for the receiver and effector domains of NsrR.</p