Part:BBa_K1365006

NisR and NisK

Usage and Biology

This BioBrick contains the coding parts for the genes NisR and NisK. NisK is a membrane-associated protein kinase that phosphorylates NisR in response to nisin in the environment. NisR is a regulatory protein, regulating promoters like PNisA and PNisI.

Together with the genes NisA, NisB, NisC, NisP they are responsible for producing the lantibiotic nisin in Lactococcus lactis, see the figure below. The NisA protein is first modified and then transported out of the cell. The serines and threonines of NisA are dehydrated by NisB and then the precursor is cyclized by NisC. After this process, the precursor is transported out of the cell (2). Here, the lead peptide is cut off by NisP (3) and the mature nisin is formed.¹

Nisin is a lantibiotic, an bacteriocidal peptide. Nisin inhibits the growth of a broad range of Gram positive bacteria, of which many are spoilage bacteria or pathogens. Nisin is therefore extensively used in the food industry as a preservative. Nisin forms pores in the membrane of the bacteria it kills and inhibits the peptidoglycan synthesis.²

Sequence and features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Cheigh, C. I. and Pyun, Y.R. (2005) Nisin biosynthesis and its properties. Biotechnol. Lett. 27: 1641-1648
2. Zhou, H. et al. (2014) Mechanisms of nisin resistance in Gram-positive bacteria. Ann. Microbiol. 64: 413-420

Contribution by Team IISER_Kolkata 2021

Group: iGEM21_IISER_Kolkata
Author: Mahapatra Anshuman Jaysingh, Shubhamay Das
Summary: This year our team focuses on one segment of this part which is nisA. NisA gene is responsible for the production of the 57-amino acid long pre-peptide. This 57-amino acid comprises a leader peptide, cleavage of which produces a 34 amino acid long Nisin A protein. Nisin Resistance Protein (NSR) is a protein that is expressed on the cell surface of certain pathogenic strains like Streptococcus agalactiae, Streptococcus uberis. NSR is responsible for inactivating nisin by proteolytically cleaving the peptide bond between MeLan28 and Ser29 resulting in a truncated nisin (nisin1–28). NisinPV is a synthetically designed mutant of NisinA with the Mutation of Serine to proline at position 29 (S29P) combined with isoleucine to valine at position 30 (I30V). Nisin PV (BBa_K379904) shows enhanced antimicrobial activity against Streptococcus uberis by inhibiting biofilm formation and decreasing its viability. In the section below we have shown a comparative analysis of the stability of NisinA and NisinPV under the interaction of Nisin Resistence protein.

Interaction with Nisin Resistance Protein:The data in this section is adapted from the simulations by Field, Des et al. They carried out MD simulations to predict the difference in molecular dynamics of the NSR protein in its interaction with the nisin C-terminus (residues 22–34) for nisin-A and nisin PV models.
Molecular docking:We tried to replicate the setup used by Des et al. For the NSR (Nisin Resistance protein) molecule, the PDB file 4Y68 was used and for the Nisin the 1WCO PDB file was used. NisinA structure was mutated using PyMol and a PRO-VAL mutation was added to the 29th and 30th residue respectively. To determine a starting configuration for the NSR-nisin complex a docking program Autodock for ligand and protein binding was used where the Nisin (A/PV) was set as ligand and NSR was set as the receptor. We first performed the docking of NisinA with the tunnel region of NSR and selected the lowest energy stage as the starting configuration. The docs location was observed to be consistent with the ligand-binding site of Nisin as discussed in the literature. The ligand-binding site for NisinA was noted and NisinPV was docked using the same coordinates.
The free energies of the configurations obtained from docking are as follows:

• molecular docking of N-terminus region of Nisin A (cyan) and NisinPV (magenta) to NSR
• 

  NisinA docking

• 

  NisinPV docking

The 1st position is the best docking position for both molecules. Subsequent docking positions were also found by this study. This shows both Nisin A and Nisin PV docks similarly to NSR protein, which indicates that binding is not the mechanism by which NSR cleaves Nisin A.

RMSD Analysis

Initially, both models quickly deviate from their reference structure by approximately 3 Å (this happens over the course of the first 0.3 ns during the heating, equilibration, and the start of the production phase of the simulation). From then on in the simulation, the nisin A model continues to deviate until it reaches approximately 5 angstroms. In contrast, the nisin PV model exhibits different behavior. Although it too initially deviates further to almost 5 angstroms early in the simulation it then returns to just under 4 angstroms deviation from its reference structure and remains virtually constant for the rest of the simulation.

This evidence of greater structural change in the nisin A model that is less apparent in the nisin PV model supports the findings of the binding energy analyses for both bond and total energies. Indeed, it is clear that the nisin PV model is more durably bound to the NSR protein and is certainly more robust than the nisin A model.

Hydrogen bond occupancy
These simulations revealed the hydrogen bond occupancy (defined as whether a hydrogen bond is present or absent at every frame over the duration of simulation) for all nisin residues greater than 20%. It is evident that the nisin PV model exhibits higher occupancy values than the nisin-A model for residues corresponding to the region 27-29. In particular, there is a hydrogen bond with 47.65% occupancy between the sidechain oxygen of proline in the nisin-PV model and the hydrogen from the nitrogen sidechain on residue 265 (asparagine) in NSR that may play a role in the inhibition of proteolytic cleavage for nisin PV.
Free energy per residue

Analysis of the total binding energies for each residue in both nisin A and nisin PV models reveals that at the critical residue 29, the total energy for proline is seven times greater than serine (4.6292 kcal/mol for PRO and 0.8775 kcal/mol for SER).

Key takeaways
Considering the binding of Nisin(A/PV) to NSR, there is compelling evidence that NSR has a much more difficult time cleaving the bond at the 29th in the case of NisinPV.

References
[1] D. Field et al., “Bioengineering nisin to overcome the nisin resistance protein,” Mol. Microbiol., vol. 111, no. 3, pp. 717–731, 2019, doi: 10.1111/mmi.14183.