Structural basis of lantibiotic recognition by the nisin resistance protein from Streptococcus agalactiae

Lantibiotics are potent antimicrobial peptides. Nisin is the most prominent member and contains five crucial lanthionine rings. Some clinically relevant bacteria express membrane-associated resistance proteins that proteolytically inactivate nisin. However, substrate recognition and specificity of these proteins is unknown. Here, we report the first three-dimensional structure of a nisin resistance protein from Streptococcus agalactiae (SaNSR) at 2.2 Å resolution. It contains an N-terminal helical bundle, and protease cap and core domains. The latter harbors the highly conserved TASSAEM region, which lies in a hydrophobic tunnel formed by all domains. By integrative modeling, mutagenesis studies, and genetic engineering of nisin variants, a model of the SaNSR/nisin complex is generated, revealing that SaNSR recognizes the last C-terminally located lanthionine ring of nisin. This determines the substrate specificity of SaNSR and ensures the exact coordination of the nisin cleavage site at the TASSAEM region.


Cloning, expression and purification of SaNSR
The primers were designed in such a way that the first 30 amino acids encoding for the transmembrane helix were not present in the construct. This allowed soluble expression and included an 8xhis--tag at the N--terminus for purification purposes. SaNSR was expressed and purified via two--step purification protocol. A single transformed colony was inoculated into 20 ml LB media containing 30 µg ml --1 kanamycin. The culture was grown for 14 h at 310 K with shaking at 200 rev min --1 . 2 L LB media with 30 µg ml --1 kanamycin was inoculated with the overnight culture at an OD600 of 0.05 and grown at 310 K with shaking at 170 rev min --1 till OD600 of 0.3 was reached. The temperature was lowered to 291 K and the cells were further grown till OD600 of 0.8 before induction with 1 mM IPTG. The cells were further grown for 15 h.
The cells were harvested by centrifugation at 8000 rev min --1 for 20 min at 277 K. The harvested cell pellet was stored at 253 K till further use. The stored cell pellet was thawed and resuspended in 10 ml of buffer A (50 mM Tris pH 8.0, 50 mM NaCl and 10% glycerol) and 10 mg of DNase (Deoxyribonuclease I from bovine pancreas, Sigma Aldrich) was added.
The cells were lysed five times using a cell disruptor (Constant Cell Disruption Systems, United Kingdom) at 37709 psi (1kbar = 14.50 psi). The lysate was centrifuged at 42000 rev min --1 for 60 min using a Ti60 rotor to remove unlysed cells and debris.
Histidine was added to the cleared lysate at a final concentration of 1 mM. The lysate was then applied to a Ni 2+ loaded HiTrap HP Chelating column (GE Healthcare) pre--equilibrated with buffer B (20 mM Tris pH 8.0, 250 mM NaCl and 1 mM Histidine) at a flow rate of 1 ml min --1 . The column was washed with six column volumes of buffer B. The protein was then eluted with increasing concentrations of Histidine from 1 mM to 120 mM, in form of a linear gradient spanning 60 min with a flow rate of 2 ml min --1 . The fractions containing the protein of interest were pooled and concentrated up to 12 mg ml --1 in an Amicon centrifugal filter concentrator with a 10 kDa cut--off membrane (Millipore). The concentrated protein was then further purified by size exclusion chromatography using Superose 12 GL 10/300 column (GE Healthcare), equilibrated with buffer C (25 mM MES pH 6.0, 150 mM NaCl).
The protein eluted as a single homogeneous peak and the concerned fractions were pooled and concentrated to 8.6 mg ml --1 as mentioned before. The purity of the protein was analyzed with SDS--PAGE and colloidal coomassie stain.
To determine the oligomeric state of SaNSR protein in solution, we used conventional size-exclusion chromatography (SEC) and high performance liquid chromatography coupled to multi angle light scattering detection (HPLC--MALS). SEC was performed as described previously 1 and the size--exclusion column was standardized with a gel filtration markers kit (Sigma).

Crystallization
Crystallization screening was performed at 285 K using NT8 robot (Formulatrix) and

Expression, purification and crystallization of selenomethionine--substituted SaNSR
For selenomethionine substitution, E. coli BL834 (DE3) cells were grown according to manufacturer's protocol in M9 minimal media (Molecular Dimensions) supplemented with 50 μg ml --1 of L--seleno--methionine. Expression and purification were identical to the native SaNSR 1 . Selenomethionine derivatized SaNSR was crystallized in a similar manner as the native protein, using the hanging drop vapor diffusion method with a protein concentration of 10 mg ml --1 .

Expression of SaNSR and its variants in L. lactis NZ9000
The plasmid encoding pNZ--SV--SaNSR and its variants were transformed into the nisin sensitive L. lactis strain NZ9000. As a control the empty vector was also transformed, termed NZ9000Erm. The strains expressing NZ9000SaNSR and its mutations were grown in GM17 media supplemented with 5 µg ml --1 erythromycin to an OD600 of 0.8. The expression was induced by the addition of nisin (at a final concentration of 1 ngml --1 ) and the cultures were further grown overnight. The cells were then diluted to an OD600 of 0.1 in fresh GM17 media supplemented with 5µg ml --1 erythromycin. These cells were then used for the assays described below.

Cloning, Overexpression and purification of nisin and its variants
Nisin was purified from commercially available powder as described 3 . The cloning, overexpression and purification of precursor nisin variants were performed as described previously 3,4 , excepting that the elution buffer of the cationic exchange chromatography of the various precursor nisin variants were changed to 50 mM HEPES--NaOH, pH 7.0, 1 M NaCl, and 10% glycerol. The concentrations of nisin and its variants were determined by using RP--HPLC and in order to activate the nisin variants, the leader peptide was cleaved off using the protease NisP as previously described, thereby 5 .

Molecular dynamics simulations
Structures of NSRApo, NSRTail, and NSRNisin,1--3 were prepared using LEaP 6 of the Amber 14 suite of programs 7 . First, missing hydrogen atoms were added by LEaP 6 , and histidine residues were assigned the HIE state. Second, counter ions were added to neutralize each system. Finally, systems were solvated using the TIP3P water model 8 . The obtained systems comprised ~ 60.000 atoms. Atomic partial charges for Dha33 (dehydroalanine) and rings D and E in nisin, which are treated as one "residue" in the Amber scheme, were obtained following the RESP procedure 9 using Gaussian09 10 . For the non--standard amino acid Dha33, force field parameters were adapted from ref. 11 . All other parameters were taken from the Amber ff99SB force field 12,13 . Structural relaxation, thermalization, and production runs of MD simulations were conducted with pmemd.cuda 14 of Amber 14 7 .
Two steps of energy minimization were performed to relax the systems. First, harmonic restraints with a force constant of 25 kcalmol --1 Å --2 were applied to all protein atoms while all other atoms were free to move during 50 cycles of steepest descent (SD) and 200 cycles of conjugate gradient (CG) minimization. Second, the force constant of the harmonic restraints was reduced to 5 kcalmol --1 Å --2 , and 50 cycles of SD and 200 cycles of CG minimization were performed. Subsequently, the systems were heated from 100 K to 299.9 K, 300 K, or 300.1 K during canonical (NVT) MD simulations of 50 ps length to setup three independent MD production simulations for NSRApo, NSRTail, and NSRNisin,1--3, respectively.
Afterwards, the density was adjusted to 1 g·cm--3 during 30 ps of isobaric--isothermal (NPT) MD simulations. During heating and density adaptation, positional restraints of 5 kcal·mol --1 ·Å --2 were applied to all protein atoms. Finally, these positional restraints were removed by gradually decreasing the force constant from 5 to 0 kcal·mol --1 ·Å --2 in six NVT--MD runs of 10 ps length each. For MD simulations, the particle mesh Ewald (PME) method 15--17 was employed to treat long--range electrostatic interactions. For short--range non-bonded interactions, we set a distance cutoff of 8 Å. The SHAKE algorithm 18 was applied to all bonds involving hydrogens, allowing a 2 fs time step for integrating Newton's equations of motion. Production MD simulations were performed in the NVT ensemble at 300 K for 500 ns. Coordinates were saved every 20 ps and used for analyses. This led to a total simulation time of 5 x 3 x 500 ns = 7.5 µs.

Supplementary Table 1 (a) IC 50 values of nisin and its variants against the NZ9000Erm and NZ9000-SaNSR strains
as well as the calculated "Fold of resistance". The values reported are the average over minimum triplicates ± SEM.  (a) For models NSR Nisin,1 , and NSR Nisin,2 distances were measured between the side chain carboxylate of Asp 110 and the guanidino group of Arg 275 . (b) Residue-wise α-helix probability for residues that compose helix α4. Mean distances and mean α-helix probabilities over the independent MD trajectories are shown in the legend.