E26 transformation specific family transcriptional repressor that self-associates by its PNT domain to facilitate cooperative DNA binding. Chromosomal translocations frequently generate constitutively active oncoproteins with the ETV6 PNT domain fused to the kinase domain of one of many protein tyrosine kinases. Although an attractive target for therapeutic intervention, the propensity of the ETV6 PNT domain to polymerize via the tight head-to-tail association of two relatively flat interfaces makes it challenging to identify suitable small molecule inhibitors of this protein–protein interaction. Herein, we provide a comprehensive biophysical characterization of the ETV6 PNT domain interaction interfaces to aid future drug discovery efforts and help define the mechanisms by which its self-association mediates transcriptional repression. Using NMR spectroscopy, X-ray crystallography, and molecular dynamics simulations, along with amide hydrogen exchange measurements, we demonstrate that monomeric PNT domain variants adopt very stable helical bundle folds that do not change in conformation upon self-association into heterodimer models of the ETV6 polymer. Surface plasmon resonance–monitored alanine scanning mutagenesis studies identified hot spot regions within the self-association interfaces. These regions include both central hydrophobic residues and flanking salt-bridging residues. Collectively, these studies indicate that small molecules targeted to these hydrophobic or charged regions within the relatively rigid interfaces could potentially serve as orthosteric inhibitors of ETV6 PNT domain polymerization.ETV6 is an  E26 transformation specific) family for which head-to-tail polymerization of its PNT (or SAM) domain facilitates cooperative binding to tandem DNA sites by its ETS domain  or to the DNA-binding domain from one of several transcription factors . Known rvia the tight head-to-tail association (KD ∼ nM) of two relatively flat interfaces  fusion oncoprotein, the introduction of monomerizing mutations blocked the ability of EN to polymerize, to activate its PTK, and to transform NIH3T3 cells . FurtherUnderstanding the mechanisms of PNT domain polymerization will yield insights in the transcriptional repression properties of ETV6 and the oncogenic properties of ETV6 fusions. In particular, defining structural and thermodynamic differences between monomeric and heterodimeric forms of the PNT domain and determining “hot spot” regions for self-association are both important for delineating the mechanisms underlying polymerization and for developing strategies to inhibit this process. Herein, using NMR spectroscopy, X-ray crystallography, and molecular dynamics (MD) simulations, we demonstrate that the structures of monomeric ETV6 PNT domain variants do not change significantly upon mutation or self-association into heterodimers. Amide hydrogen exchange (HX) measurements confirmed that the monomeric PNT domain is very stable in solution. Protection against exchange increased upon heterodimerization for amides clustering near the self-association interfaces. Complementary alanine scanning mutagenesis revealed several hot spot regions within these ML- and EH-surfaces. These residues partake in both hydrophobic and electrostatic interactions and can serve as starting points for targeted rational drug design.15N-heteronuclear single quantum coherence (HSQC) spectra to monitor the titration of a 15N-labeled protein with an unlabeled, and hence NMR silent, ligand such as another protein . However, the latter showed some propensity to self-associate, and improved spectra were obtained at a sample pH value of 8.0. Presumably, this reflects the deprotonation of E112, which may have an anomalously high pKa value when buried at the polymer interface  and bound (heterodimeric) forms of the labeled protein were observed. This is consistent with the previously reported KD value of ∼2 nM for the high-affinity binding equilibrium (15N-labeled V112E-PNT domain (B). Although the oligomerization states of the PNT domains in these experiments were not directly determined, the results are entirely consistent with previous studies showing that the A93D-PNT and V112E-PNT domains are monomeric when separated and heterodimeric when combined . These coincide well with the four α-helices  identified in the X-ray crystal structures of the self-associated ETV6 PNT domains (PDB: 1JI7 and 1LKY) by PDBsum . Most importantly, these analyses demonstrated that the A93D-PNT and V112E-PNT domains have very similar secondary structures in their monomeric and heterodimeric forms. Thus, the proteins in solution do not undergo any significant conformational changes upon mutation or association into the structures previously characterized by X-ray crystallography.Through a combination of scalar and NOE correlation experiments, NMR signals were assigned from most main chain partners . Utilizipartners , the secpartners C. These y PDBsum . Matchindynamics ) indicatdynamics C. Most i1HN-15N CSPs resulting from PNT domain dimerization were readily calculated. Of note, residues in the 15N-labeled A93D-PNT and V112E-PNT domains that experienced the greatest spectral perturbations cluster within the EH- and ML-surfaces, respectively  . Subsequently, they determined the structure of a heterodimer composed of a A93D-PNT domain bound to a V112R-PNT domain via their complementary wild-type interfaces   . Despitesymmetry A. SubseqP6522 with two monomers in the asymmetric unit (B). Most importantly, the presence of both monomerizing mutations prevented any intermonomer interactions within the crystal lattice via the ML- and EH-surfaces. Rather, nearest neighbor contacts were via alternative interfaces that are not relevant to polymerization of the wild-type PNT domain.The NMR spectroscopic studies presented above indicate that the secondary structures and binding interfaces of the A93D-PNT and V112E-PNT domains in solution closely resemble those observed by X-ray crystallography. However, the PNT domain in a complexed form may have subtle structural differences relative to a monomeric form. To examine this possibility, a variant with both A93D and V112 E mutations was generated and found to crystallize in 2.8 M sodium acetate at pH 7.0. Its structure was solved to 1.86 Å resolution using molecular replacement . The A93ric unit B. Most iC). For example, residues N-terminal to helix H1 have variable conformations. This is consistent with their RCI-S2 scores indicating a degree of flexibility (C). Not unexpectedly, several surface residues adopted different side chain rotamer conformations, whereas residues within the interior hydrophobic core of the PNT domain superimposed well. Most importantly, the local structural features of the ML- (including residue 93) and EH- (including residue 112) surfaces do not differ despite the presence or absence of monomerizing mutations or their association upon heterodimer or polymer formation. Thus, interactions of the ETV6 PNT domain do not contribute to any discernible conformational changes between its monomeric, heterodimeric, or polymeric forms.Regardless of differing crystallization conditions and mutations, the structure of the A93D-V112E-PNT domain closely resembles those previously determined for other ETV6 PNT domain variants. Using the DALI server to compare one subunit from this structure with a A93D-PNT domain subunit from PBD entry 1LKY, a total of 77 residues were aligned with a root mean squared deviation (RMSD) value of 0.7 Å and Z-score of 16.8 . HoweverFig. 3xibility C. Not un1H-detected NMR, and thus, its signal will disappear from an 15N-HSQC spectrum. The rate at which it disappears is determined by its structural features , as well as the experimental conditions . To account for the latter, the observed exchange rate constant can be compared with the predicted rate constant for a random coil polypeptide with the same sequence and under the same conditions. The ratio of the predicted versus observed rate constants is the protection factor (PF).Amide HX is a useful technique to characterize protein structure, stability, and dynamics, as well as identifying ligand-binding interfaces , 28, 29.A), it is immediately obvious that more amides were protected from HX in the heterodimeric versus monomeric species. Exchange rate constants for most amides were determined by fitting their time-dependent 1HN-15N signal intensities to single exponential decays. These values were converted into the PFs summarized in B and C and tabulated in To gain further insights into the ETV6 PNT domain dynamics, NMR spectroscopy was used to measure the amide PFs of its monomeric and heterodimeric forms. By comparing the spectra recorded after 3 days of exchange A, it is 2 values (C). Conversely, amides within or near the four α-helices showed substantial protection from HX. In particular, W69 and L70 in helix H1 and L116 and L117 in helix H4 of both proteins exchanged very slowly, with several of these residues having log(PF) values >7. Under the commonly observed EX2 conditions, with pH-dependent bimolecular kinetics, PFs reflect the residue-specific free energy changes, ΔGHX = 2.303RTlog(PF), governing local or global conformational equilibria leading to exchange . Indeed, several amides in both proteins did not exchange significantly even after 3 months in D2O buffer and thus have log(PF) > 8.5 (and ΔGHX > 48 kJ/mol). Although global stabilization upon heterodimer formation is expected, many of the residues with at least a 100-fold increase in HX protection localized around the complementary interfacial regions of the two PNT domains (A and B). These are exemplified by amides within or near the wild-type ML-surface of the V112E-PNT domain  and the wild-type EH-surface of the A93D-PNT domain . Given that the structures of the A93D-PNT and V112E-PNT domains do not change significantly upon heterodimerization, the increased protection of interfacial residues against HX may result from their local stabilization against conformational fluctuations allowing exchange. Alternatively, if exchange occurs predominantly through transient monomers, then the increased protection of amides in the heterodimer would reflect the equilibrium population distribution of these two species  was used to characterize the binding interaction as this technique is rapid and uses only small quantities of bacterially expressed proteins.D value of 7.5 nM, and the V112E-PNT domain analyte bound the A93D-PNT domain ligand with a fit KD value of 5.1 nM. These results agreed well with previously reported KD values of 1.7 to 4.4 nM for the PNT domain interactions as measured by SPR . Although more difficult to interpret than KD values, these mutations acted to varying degrees by slowing the association rate constants, kon, and/or increasing the dissociation rate constants, koff. The higher proportion of hotspot interfacial residues on the V112E-PNT domain, including the three most detrimental of the entire set of alanine mutants, suggests that the features of this interface contribute most significantly to PNT domain polymerization.Six out of 18 on the A93D-PNT domain and seven out of 14 tested residues on the V112E-PNT domain had large detrimental effects on binding (ΔΔG >6 kJ/mol) and can be classified as hot spots A. AlthouB, L96 protrudes out of the V112E-PNT domain as a “bump” to fit into the “hole” between residues F77, V112, E115, and H119 on the EH-surface of the A93D-PNT domain. Thus, in addition to contributing to heterodimerization through the hydrophobic effect, L96 partakes in favorable van der Waals interactions at the protein–protein interface. Other hydrophobic residues that are important include L97 and M89 on the ML-surface and F77, L79, and V112 on the EH-surface.Many of the hydrophobic residues at the center of the heterodimer interfaces are hotspots for binding. In particular, the L96 A mutation on the ML-surface of the V112E-PNT domain resulted in an ∼1000-fold decrease in affinity. As shown in Figure 6B). However, an important exception is the peripheral K99 on the A93D-PNT domain EH-surface. This residue forms an intermolecular salt bridge with D101 on the ML-surface of the V112E-PNT domain. Alanine mutations of K99 or D101 resulted in decreased binding affinities of 930 nM or 400 nM, respectively. Thus, consistent with their salt bridge pairing, either mutation weakened binding relative to the wild-type by a factor of ∼100. Mutation of K99 has been shown to interfere with EN oncogenic cellular transformation, confirming the functional importance of this salt bridge . Importantly, the PNT domain subunits remained associated with each other, indicating that the heterodimer can be modeled in silico. The transition occurring around 150 ns in the monomer trajectory corresponds to a rearrangement of its N-terminal residues. Indeed, the RMSD values of these ETV6 truncations (residues 47–123) are dominated by motions of their termini. Restricting the analyses to residues 57 to 120 reduced the backbone RMSD values to ∼1 Å. This further demonstrates that, regardless of mutation or association state, the PNT domain core helical bundle is relatively rigid.MD simulations were run on the A93D-V112E-PNT domain monomer, reported herein, and the PNT domain heterodimer, comprising an A93D-PNT domain and V112R-PNT domain from PBD: 1LKY. Both the monomer and the heterodimer remained structurally stable throughout the 1 μs simulations with no drift in the RMSDs of backbone atoms after equilibration A. ImportB). This is consistent with the RCI-S2 values presented in Figure 1C. Principal component analysis of the trajectories was also performed to explore low frequency conformational motions. In each case, the proportion of the motion (variance) was largely confined to the first few principal components  of each residue over the trajectories are similar for the monomeric and heterodimeric PNT domains, again showing low mobility in the core and higher mobility at the termini B. This imponents . Inspectmponents  shows thB shows that the RMSF values of residues near the termini of the A93D-PNT domain in the heterodimer are lower than those of both its V112R-PNT domain partner and the monomeric A93D-V112E-PNT domain. This can be better seen in a plot of the difference in RMSF values between corresponding residues in the two components of the heterodimer (C). When mapped onto the structure of the heterodimer, residues with the largest ΔRMSD values clustered near the interface (D). That is, over the course of the MD simulations, residues near or at the wild-type ML-surface of the V112R-PNT domain exhibited smaller fluctuations than the corresponding residues of the mutated unbound ML-surface of the A93D-PNT domain. Similarly, residues near or at the wild-type EH-surface of the A93D-PNT domain, which includes its termini, exhibited smaller fluctuations than the corresponding residues of the mutated EH-surface of the V112R-PNT domain. Thus, although overall relatively rigid in MD simulations, to some degree heterodimerization reduced the mobility of residues at the bound interfacial regions of the heterodimeric PNT domains.A closer look at the data presented in Figure 7erodimer C. When mnterface D. That ii.e., with log(PF) > 3). However, several additional hydrogen-bonded amides in loop regions or the N-terminal segments of the PNT domain constructs exchanged more rapidly, with log(PF) values less than an upper measurable limit of ∼3. Thus, in contrast to the very stable α-helices, these regions of the PNT domain undergo more facile local conformational fluctuations, including hydrogen bond breakage, and hence have reduced protection from HX.We also explored whether the dynamics simulations could be correlated with the experimental HX PFs. Despite time scales differing by many orders of magnitude, such a correlation between hydrogen bond persistence in MD simulations with PFs, measured with NMR spectroscopy, was observed in a short helical peptide . PresumaOur biophysical studies of the ETV6 PNT domain show that its tertiary structure is very stable in the monomeric and heterodimeric forms and is not perturbed by the presence of monomerizing mutations. There are no substantial conformational differences between these species as seen both by X-ray crystallography and by NMR spectroscopy . Furthermore, despite differences in contact surfaces within crystal lattices, the X-ray crystallographic structure of a monomeric A93D-V112E-PNT domain closely resembles that of the heterodimers. Similarly, the 1 μs MD simulations did not show any evidence for large conformational fluctuations and only small changes in the mobility of interfacial residues upon heterodimer formation. Thus, the well-characterized ETV6 PNT domain structure is retained both before and after polymerization. In this respect, the PNT domain can be viewed as a rigid “lego block”, poised to self-assemble without any requisite change in shape.It is noteworthy that upon association of the A93D-PNT and V112E-PNT domains, amides exhibiting chemical shift perturbations and substantially increased HX protection localized to the wild-type ML- and EH-surfaces and not to the mutated surfaces. This confirms that only the former participate in heterodimer formation. Furthermore, along with a lack of any structural differences between the crystallographically characterized variants, this demonstrates that the “head” and “tail” interaction surfaces of a PNT domain are not allosterically coupled. Thus, the initial formation of the wild-type ETV6 PNT homodimer is unlikely to change the binding affinity of subsequent monomers to a growing polymer chain.D ∼ nM) because of residues partaking in electrostatic, van der Waals, and hydrophobic interactions. Although it has been reported that hotspots are generally not enriched in electrostatic interactions , including these charge pairs, adopt similar well-ordered side chain conformations in all known X-ray crystallographic structures, regardless of intermolecular packing within the crystal lattice. In contrast, residues less critical for self-association adopt more variable side chain conformations.Detailed SPR-monitored alanine scanning mutagenesis studies revealed that the ETV6 PNT domains heterodimerize with high affinity , several hydrophobic residues with the EH- and ML-surfaces are also hotspots for heterodimerization. In particular, L96 on the ML-surface plays a critical role in binding as removal of its side chain resulted in ∼1000-fold weaker affinity. The leucine makes contacts with several residues on the reciprocal EH-interface for which alanine substitutions also reduced the binding affinity, albeit each to a lesser extent. Targeting a molecule to bind near these EH-surface residues may exclude L96 and thereby inhibit PNT polymerization.Alanine scanning mutagenesis and amide HX experiments provide complementary insights into the self-association of the ETV6 PNT domain. That is, alanine scanning reveals the effect of removing the side chain of a given residue on the affinity for heterodimer formation, whereas HX experiments show how dimerization changes the conformational fluctuations of backbone amide hydrogens leading to exchange with water. In general, many of the hydrophobic hotspot residues within the core regions of the EH- and ML-surfaces, including L79, L96, L97, T98, Y104, V112, Y114, and L116, also exhibited enhanced HX protection upon heterodimerization. This is consistent with their burial, and likely dampened dynamics, within the heterodimer relative to the monomeric species. In contrast, although partaking in important intermolecular interactions, K99 and R105 underwent fast amide HX in both the monomeric and heterodimeric PNT domains, whereas their salt bridge partners D101 and D111 exhibited increased HX protection. This is also consistent with the location of the amides of these residues at the periphery of the dimer interface.Drosophila Yan  or salt-bridging  residues may competitively prevent PNT domains from self-associating. However, designing or identifying such small molecules is challenging as the polymerization occurs with high affinity, and there does not appear to be any structural differences between the PNT domains in their monomeric and heterodimeric states that could be exploited for potential binding sites. One strategy that could be implemented for screening assays is to utilize an alanine mutant that weakens binding. This way, it may be easier to identify an initial compound that inhibits a micromolar affinity interaction, as opposed to the nanomolar interaction of the wild-type PNT domains. Once discovered, such a lead molecule could be optimized for higher affinity binding. As previously shown, mutations of K99 weaken PNT domain polymerization and alter the oncogenic properties of EN  is ∼10 min. The lifetime of polymeric forms will be longer since dissociation must occur at multiple interfaces to completely monomerize. If the dissociation of the PNT domain polymer is slow in the context of a PNT–PTK fusion oncoprotein, then a prospective small molecule inhibitor that would have the greatest effect may likely need to act on newly translated PNT–PTK fusion oncoproteins, before they polymerize.As a closing comment, SPR experiments demonstrated that the lifetime of the PNT domain heterodimer , preceded by a thrombin-cleavable N-terminal Hisnity tag , was ininity tag . The monnity tag . Thus, EEscherichia coli BL21 (λDE3) cells grown at 37 °C to an OD600 ∼ 0.6 and induced with 1 mM IPTG overnight. Unlabeled proteins were produced in lysogeny broth (LB) media, and M9 minimal media was supplemented with either 1 g/L of 15NH4Cl for 15N-labeled proteins or 1 g/L of 15NH4Cl and 3 g/L 13C6-glucose for 13 C/15N-labeled proteins. In all cases, 35 mg/L kanamycin was included for plasmid selection. After continued growth at 37 °C overnight, the cells were harvested by centrifugation  and frozen at −80 °C. The cell pellet was thawed for purification and resuspended in denaturing buffer  and lysed by sonication on ice. The lysate was then cleared by centrifugation , and the resulting supernatant was passed through either a 0.45 or 0.8 μm pore size filter and loaded onto a 5 ml Ni+2-NTA HisTrap HP column  pre-equilibrated with binding buffer . After washing with several column volumes of binding buffer, the protein was eluted using a 120 ml linear gradient of elution buffer .PNT domain-containing proteins were expressed in 2+ column. Previous studies demonstrated that proteins from these fractions had the same masses yet showed small differences in their 15N-HSQC spectra  with ∼1 unit of thrombin (Millipore) per 20 ml of collected fractions. The cleaved protein was separated from any His6-tagged species by passage through the Ni+2-NTA HisTrap HP column and purified further using size-exclusion chromatography . This also served to exchange the protein in a buffer optimized for NMR experiments (noted below). The concentration of each protein sample was determined by ultraviolet absorbance at 280 nm based on its predicted molar absorptivity  added for signal locking. Unless otherwise noted, experiments involving the A93D-PNT domain were conducted at pH 7.0, and those involving the V112E-PNT domain were conducted at pH 8.0 because of its propensity to self-associate under lower sample pH conditions. In the case of the heterodimer species, the unlabeled partner protein was added to its isotopically labeled partner in a 1.1 M excess. The heterodimer containing an isotopically labeled V112E-PNT domain was studied at pH 7.5, whereas that containing an isotopically labeled A93D-PNT domain was studied at pH 7.0.NMR spectra were recorded with cryoprobe-equipped Bruker Avance III 500, 600, and 850 MHz spectrometers. All data acquired were processed and analyzed with NMRPipe  and NMRF1H, 13C, and 15N nuclei of the 13C/15N-labeled PNT domain constructs were assigned using standard heteronuclear 1H-13C-15N scalar correlation experiments  and 15N (ΔδN) signals of the corresponding amides in the dimer versus monomer species were calculated as Δδ = [(ΔδH)2 + (0.14ΔδN)2)]1/2 2)]1/2 .15N-labeled A93D-PNT domains and 7.5 for those containing the 15N-labeled V112E-PNT domain. Initial reference 15N-HSQC spectra of the proteins in H2O buffer were recorded. Subsequently a 450 μl aliquot was lyophilized, resuspended with the same volume of D2O, and immediately put into the NMR spectrometer to begin data recording within 4 to 7 min. Initially, a series of ∼ 5-min 15N-HSQC spectra were acquired with two scans/free induction decay to characterize amides exchanging on the minutes timescale. Then ∼20-min 15N-HSQC spectra with eight scans/free induction decay were collected back-to-back for ∼3 h, followed by a 20-min spectrum every hour for ∼24 h, and then intermittent 20-min spectra over a period up to 3 months. After the first week, the sample was removed from the spectrometer and stored at ambient room temperature between recording spectra. Upon completion of data recording, the pH∗ (pH meter reading uncorrected for the deuterium isotope effect) of each sample was measured as 7.3 (monomeric A93D-PNT domain), 7.6 (monomeric V112E-PNT domain), 7.4 (15N-labeled A93D-PNT domain in complex with V112E-PNT domain), and 7.5 (15N-labeled V112E-PNT domain in complex with A93D-PNT domain).Protium–deuterium HX experiments for the PNT domains in the absence (monomeric) and presence of their unlabeled partner (heterodimeric) were conducted on a Bruker Avance 600 MHz spectrometer at 21 °C (matching ambient room temperature). The initial pH values were 7.0 for the samples containing the 2O, the pseudo-first order exchange rate constant kobs was obtained by fitting with NMRFAM-Sparky the 1HN-15N peak intensity It, scaled for number of acquisitions/free induction decay, to the equation for a single exponential decay:I0 is the fit initial intensity extrapolated to t = 0. The protection factor (kpred) in an unstructured polypeptide of the same amino acid sequence versus its experimentally measured kobs. The kpred values were determined with the program Sphere  were identified using the online Solvent accessibility-based Protein-Protein Interface iDEntification and Recognition server [SPPIDER ]. AlaninE. coli BL21 (λDE3) with the pET21a-BirA plasmid, which produces biotin ligase. Selection of both plasmids was maintained by including kanamycin (35 mg/L) and ampicillin (100 mg/L) in all media. Overnight seed cultures were used to inoculate LB media, supplemented with 0.05 mM biotin, and grown at 37 °C to an OD600 ∼ 0.6. Protein expression was induced with 0.5 mM IPTG, and the cells were grown at 30 °C overnight. The cells were collected by centrifugation and cell pellets were frozen at −80 °C.Each plasmid encoding either the biotinylated A93D-PNT and V112E-PNT domain was co-transformed into 6-tag by passage through a Ni+2-NTA HisTrap HP column. The final protein samples were exchanged into 20 mM MOPS, 50 mM NaCl, 0.5 mM EDTA buffer, at pH 8.0, and concentrated to ∼1 ml with an Amicon 3K MWCO centrifugal filter. The protein samples were then snap frozen in liquid nitrogen and stored at −80 °C before SPR analysis. The proteins were ∼50 to 95% biotinylated as judged by MALDI-ToF mass spectrometry.Protein purification was carried out fully as described above with minor modifications. The final size exclusion purification step was omitted after thrombin cleavage and removal of the HisSPR experiments were performed at 25 °C on a Biacore X100 instrument using the streptavidin Sensor Chip SA to capture the biotinylated PNT domain “ligand”. The ligand was diluted to 50 μg/ml in HBS-EP+ buffer  and immobilized on the chip using the Biacore X100 control software immobilization wizard. The regeneration scouting software wizard was used to determine the regeneration step involving flowing 0.2% SDS over the chip for 30 s.on) and dissociation (koff) rate constants and the equilibrium dissociation constant (KD) for binding of the analyte with the immobilized ligand were determined using the Biacore X100 kinetics/affinity assay software wizard. The positive and negative binding controls were analytes with the complementary wild-type PNT domain interface or with the same monomerizing substitution, respectively. For the experimental runs, the analyte protein sample was initially diluted in HBS-EP+ buffer to 0.2, 2, 20, 40, and 60 nM. If weakened binding (KD > 60 nM) was observed, the analyte protein sample was re-run in HBS-EP+ buffer at 20, 200, 2000, 4000, and 6000 nM.The association (kA construct of ETV6 spanning residues 40 to 125 with the A93D and V112 E substitutions (A93D-V112E-PNT domain) was purified as described above. Crystals were grown by sitting drop vapor diffusion at room temperature with 2 μl drops prepared with a 1:1 mixture of ∼9.3 mg/ml protein  and reservoir solutions from the Hampton Index reagent crystallization screen (Hampton Research). Several conditions yielded protein crystals and those grown in 2.8 M sodium acetate (pH 7.0) were used for data collection. These crystals were cryoprotected by brief soaking in reservoir buffer supplemented with 30% (v/v) glycerol, followed by flash freezing in liquid nitrogen.P6522 with two protein molecules in the asymmetric unit. Phase determination using molecular replacement was performed with a PNT domain monomer from PDB: 1LKY using the AutoSol program in Phenix (Diffraction data were collected at the CLS (Canadian Light Source) on beamline 08B1-1 . The datn Phenix . Model bn Phenix . Eight oMD simulations were based on coordinate files, encompassing residues S47 to Q123, from the crystal structure of the monomeric A93D-V112E-PNT domain (determined herein) and a dimer of the A93D-PNT domain and V112R-PNT domain from PDB: 1LKY.The MD simulations were performed on the University of Bristol High Performance Computer BlueCrystal using GROMACS (Version 5.1.2) . The sysThe X-ray structure (coordinates and structure factor files) has been submitted to the PDB under accession number 7JU2. The NMR chemical shifts have been submitted to the BMRB under accession numbers 50430 (Monomeric V112E-PNT domain), 50431 (Complexed V112E-PNT domain), 50432 (Complexed A93D-PNT domain), and 50433 (Monomeric A93D-PNT domain). All other data described here are available within the manuscript and supporting information.The authors declared no conflicts of interest with respect to the research, authorship, and/or publication of this article.


Cytochrome P450 (CYP) heme monooxygenases require two electrons for their catalytic cycle. For mammalian microsomal CYPs, key enzymes for xenobiotic metabolism and steroidogenesis and important drug targets and biocatalysts, the electrons are transferred by NADPH-cytochrome P450 oxidoreductase (CPR). No structure of a mammalian CYP–CPR complex has been solved experimentally, hindering understanding of the determinants of electron transfer (ET), which is often rate-limiting for CYP reactions. Here, we investigated the interactions between membrane-bound CYP 1A1, an antitumor drug target, and CPR by a multiresolution computational approach. We find that upon binding to CPR, the CYP 1A1 catalytic domain becomes less embedded in the membrane and reorients, indicating that CPR may affect ligand passage to the CYP active site. Despite the constraints imposed by membrane binding, we identify several arrangements of CPR around CYP 1A1 that are compatible with ET. In the complexes, the interactions of the CPR FMN domain with the proximal side of CYP 1A1 are supplemented by more transient interactions of the CPR NADP domain with the distal side of CYP 1A1. Computed ET rates and pathways agree well with available experimental data and suggest why the CYP–CPR ET rates are low compared to those of soluble bacterial CYPs. Mukherjee, Nandekar and Wade investigate the structural arrangement of the complex between membrane-bound cytochrome P450 1A1 and NADPH-cytochrome P450 reductase. They find that upon binding to the reductase, the catalytic domain of cytochrome P450 1A1 reorients subject to the constraints of membrane binding, potentially explaining why the electron transfer rates between the proteins are low when compared to those of soluble bacterial cytochrome P450s. CYPs are widely used as biocatalysts for regio- and enantioselective C–H hydroxylation reactions. CYPs are heme monooxygenases that activate one molecule of dioxygen and oxidize the substrate by inserting one oxygen atom to form the product and reducing the other oxygen atom to water. Thus, drugs are transformed into more polar metabolites during the process of drug metabolism4.CYP enzymes constitute the cardinal xenobiotic-metabolizing enzyme superfamily and they are of considerable interest for the synthesis of novel drugs and drug metabolites, targeted cancer gene therapy, biosensor design, and bioremediation5. For the microsomal CYPs, the redox partner is CPR, a flavoprotein containing three cofactor binding domains: the FMN domain with the flavin mononucleotide (FMN) cofactor, the FAD domain with the flavin adenine dinucleotide (FAD) cofactor, and the NADP domain with the nicotinamide adenine dinucleotide phosphate (NADP) cofactor. Besides these three domains, CPR also contains an amino-terminal membrane-binding domain, which serves as a nonspecific membrane anchor, and a flexible tether region that connects this domain and the FMN domains6. The membrane-binding domain is necessary for efficient ET from CPR to CYP6 and its interactions with the membrane have been found to depend on cofactor binding and the oxidation state of CPR7. Cytochrome b5 (cyt b5) is another natural redox partner for some CYPs and can also allosterically modulate CYP catalysis11.In the catalytic cycle, CYP requires two electrons to be transferred sequentially from a redox partner to its heme cofactor (HEME), and the ET steps have been observed to be rate-limiting for the reaction in many cases12.All mammalian CYPs have three domains: a globular catalytic HEME-containing domain, an N-terminal transmembrane α-helical domain, and a flexible linker region connecting these two domains. In humans, both the CYP and the CPR are anchored in the endoplasmic reticulum (ER) membrane by a transmembrane helix (TM-helix). The N-terminal region of the CYPs not only anchors the protein in the ER membrane, but also influences their localization to different microdomains of the membrane14, atomic force microscopy and antibody mapping15. To date, there is no experimentally determined structure of a full CYP–CPR/cyt b5 complex available, although structures of the individual globular domains of the number of mammalian CYPs and CPRs have been determined by crystallography after removal of the N-terminal transmembrane domain to facilitate expression and crystallization2. Various modeling and simulation procedures have been developed to predict the orientation of full-length CYPs in phospholipid bilayers22. Notably, T. brucei CYP 51 simulated20 prior to the determination in 2014 of the crystal structure of yeast CYP 5123, the only eukaryotic CYP for which a full-length structure has been determined experimentally, indicated a very similar orientation of the protein with respect to the membrane. Experiments, such as solid-state NMR and mutagenesis, indicate that the association between a CYP and its CPR/cyt b5 redox partner is driven by electrostatic and hydrophobic interactions with positively charged residues on the proximal side of CYP interacting with negatively charged residues on the CPR FMN domain or cyt b529.Information about the orientation of the globular domain of CYPs, alone or in CYP–CPR/cyt b5 complexes in the membrane, has been gleaned from experiments such as linear dichroism measurements in NanodiscsBacillus megaterium cytochrome P450 BM3 (P450BM3)30, a non-stoichiometric complex of two CYP HEME domains and an FMN domain, as a template, for modeling the CYP 1A1–CPR complex but, despite extensive MD simulation, we did not succeed in obtaining ET-competent complexes. Indeed, considering the P450BM3 structure as a template for modeling mammalian CYP–CPR interactions has four major drawbacks: (i) The crystal structure of P450BM3 (PDB ID: 1BVY) is not consistent with measured ET rates due to the high redox center separation distance; (ii) The low sequence identity between P450BM3 and CYP/CPR of about 30% for the CYP and FMN domains; (iii) Unlike mammalian CYPs, the dimeric form of P450BM3 is catalytically functional and ET from FMN to HEME is proposed to occur in a trans fashion32; (iv) Mammalian CYP and CPR are membrane-anchored proteins, whereas P450BM3 is not and the membrane attachment may affect the binding of CPR to the CYP. Hence, it is preferable to employ a de novo protocol rather than a template-based method for modeling full-length CYP–CPR interactions in a membrane.We initially took the crystal structure of 34. Many of its substrates, such as benzo[o]pyrene, a carcinogen found in tobacco smoke, are polycyclic aromatic hydrocarbons. The standard reference substrate for CYP 1A1 is 7-ethoxyresorufin, which is O-dealkylated by the enzyme. Constitutive expression of CYP 1A1 is low but can be induced in response to environmental contaminants, for example in smokers’ epithelial lung cells. Moreover, engineered CYP 1A1 has potential as a biocatalyst36. Notably, CYP 1A1–CPR fusion proteins have been made to improve ET rates. A fusion between rat CYP 1A1 and rat CPR was found to exhibit four times higher monooxygenase activity than rat CYP 1A1 alone35, whereas a fusion between rat CYP 1A1 and yeast CPR had a similar ET rate to P450BM336. Hence, a detailed atomic-level understanding of CYP 1A1–CPR interactions and ET kinetics would provide the basis for the design of drugs targeting CYP 1A1, as well as the exploitation of CYP 1A1 as a biocatalyst. We therefore here describe the building and simulation of the full-length CYP 1A1–CPR complex in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer. POPC was chosen because phosphatidylcholine is the main component in the mammalian ER membrane37 and because it provides a simple representation, which is often used in in vitro studies of CYPs, of the disordered regions of the ER where CYP 1A1 has been observed to localize38.Here, we have carried out multiresolution simulations with the aim of achieving a detailed understanding of the structure and dynamics of the membrane-associated full-length CYP 1A1–CPR complex, and consequently of the determinants of the ET process between CYP 1A1 and CPR. CYP 1A1 is an extrahepatic human drug target protein that plays an important role in the bioactivation of procarcinogens and detoxification of carcinogens41. For example, the orientation of putidaredoxin in the predicted complex with cytochrome P450cam42 was similar (about 15° rotated) to that in the crystal structure of the complex published afterward by Tripathi et al.43. The six representative encounter complexes of the globular domains of CYP 1A1 and CPR generated by BD rigid-body docking simulations and selected for subsequent molecular dynamics (MD) simulations are listed in Table The multiresolution computational method used to model and simulate the membrane-associated full-length CYP 1A1–CPR complex is depicted in Fig. BM3 CYP–FMN domain complex30. In the encounter complexes, both the α1- and α3-helices of the FMN domain contribute to the interface whereas, in P450BM3, it is mainly the α1-helix of the FMN domain. The distance between the redox centers, DFe-N5, in the six representative encounter complexes ranges from 15.4–20.2 Å and is shorter than the distance (23.6 Å) observed in the crystal structure of P450BM3 even though the distance between the centers of mass of the globular domain of CYP 1A1 and the FMN domain, DCYP-FMN domain, is higher in the representative diffusional encounter complexes (34.6–37.9 Å) than in the P450BM3 crystal structure (31.7 Å).  values of the CYP globular domain and the FMN domain were 2.0–2.6 Å and 1.2–1.4 Å, respectively , decreases in ns Table , indicatns Table . Next, tα-RMSD plots , with identical initial coordinates but with different initial assigned velocities, was run for the D2 system. The structures of the individual protein domains were well maintained as shown in Cots Fig.  but therots Fig. .DCYP-mem, increased by 3–8 Å  and the center of mass (CoM) of the membrane for the D2 simulations. Thus, rearrangements of the highly flexible linker region between the TM-helix of CPR and the FMN domain affected the CPR orientation with respect to the membrane. However, this large rearrangement of the FMN domain towards the membrane had little effect on the interdomain distance, DCYP-FMN domain.For CPR, a greater extent of rearrangement of the FMN domain with respect to the membrane was observed in the D2 and D2′ simulations as indicated by the large drop in Due to the highly flexible linker hinge between the FMN and FAD domains of CPR, rearrangements of the FAD and NADP domains with respect to the membrane were observed Fig. . This doDCYP-FMN domain was stable at 33–36 Å. It remained longer than the interdomain distance in the crystal structure of P450BM3 (31.7 Å), possibly because the membrane prevents the two domains from coming as close as in the membrane-free P450BM3. Nevertheless, in all the CYP 1A1–CPR complexes, DFe-N5 was within the range (14–16 Å) for biological ET  of 47 nM45. The CPR NADP domain interactions with the distal side of CYP create an additional contact interface. The high contact area of the CYP 1A1–CPR complexes indicates the formation of a rather strongly bound transient complex between the proteins.No contacts between the TM-helices of CYP 1A1 and CPR were observed, which is consistent with the observations of Sundermann and Oostenbrink24 showed the same region of the FMN domain at the interface.The CYP–FMN domain interactions involve charge pairing of the positively charged proximal side of CYP 1A1 with the negatively charged surface of the FMN domain surrounding the cofactor. These electrostatic interactions are complemented by van der Waals and hydrophobic interactions. The interface residues in CYP 1A1 are located on the B, C, and L-helices, and the JK-loop and the loop structure near the HEME 51, CYP 1A2 (CPR)45, and P450BM3 (FMN domain)30, revealing the importance of positively charged residues for redox protein binding. Even though the residues at the FMN domain-binding interface of CYP 1A1 do not align completely with the interfacial residues of the other CYPs in a multiple sequence alignment , C-helix , CC′-loop , and loop near heme moiety  were found to interact with the FMN domain of CPR. In our simulations, we found that a similar, positively charged region of CYP 1A1 was involved in interactions with the FMN domain of CPR BM3, although with the position of FMN cofactor shifted by about 20° in angle θ  showed complexes with a similar arrangement of the FMN and CYP domains with approximately the same protein binding faces to that observed in the crystal structure of P450In the initial model for the D2 system, the NADP domain of CPR was relatively far from the membrane and from the globular domain of CYP 1A1 Fig. . During Km = 5.1 pM)53. Based on a combination of in vitro mutagenesis, in vivo screening, and spectral analysis, it was confirmed that K268 and R275 of rat CYP 1A1 are important for CPR binding55. However, residues 268 and 271 are in the G-helix and residues 275 and 279 are in the GH-loop, neither of which is on the proximal side of rat CYP 1A1  of the CPR-supported metabolism of 7-ethoxycoumarin and ethoxyresorufin was observed in K271I and K279I single-point mutants of rat CYP 1A1, accompanied by a reduction by a factor of about 2 and 9, respectively, of the Michaelis constant for the reductase  with the J-helix and the JK-loop of CYP 1A1 , only two major ET pathways were identified. These are FMN-I458-C457-HEME (56%) and FMN-K456-C457-HEME (35%) Fig. . Only thN-demethylase activity by about two-fold due to a decreased Km value, indicating slightly tighter binding of the substrate, possibly due to realignment of the heme in the mutant65. The I458P mutation had a smaller and opposite effect to the I458V mutation65. From a QM/MM study of CYP 3A4, it was concluded that the I443 backbone amide forms a hydrogen bond with the side chain of the heme-coordinating C442 and thus stabilizes the Fe–S bond and prevents the localization of the radical on the sulfur in the presence of substrate66. Furthermore, computational modeling of CYP 19A1 and the CPR FMN domain by Magistrato and co-workers revealed that C437, A438, K440, and Y441 are involved in ET67.  was found to be involved in the electron transfer pathway, with A442 and C443 of CYP 2D6 aligning with K456 and C457, the two residues in the electron transfer pathway in our D2′ model of the CYP 1A1–CPR complex.With respect to the second path, the S441P mutation in CYP 17A1  causes a complete loss of both 17α-hydroxylase and 17,20-lyase activitiesDFe-N5 and the θ angle . The maximum population of structures has log(kET) between 0.1 to 0.7. The calculated ET rates   has a region from 82° to 90° where no structures were sampled during the simulations as this arrangement of the CYP 1A1 C-helix and FMN domain α1-helix is disfavored as discussed above.To investigate the relation between ET rate and the structure of the complex, 2D histograms of the computed binding free energies between the CYP globular domain and the FMN domain in the complexes Fig. , DFe-N5 We analyzed the effects of CPR binding on the opening of ligand tunnels between the buried CYP active site and the protein surface in the conventional MD simulations described above Fig.  and in R72). In the D2′ simulation and the simulation of CYP 1A1 in a membrane in the absence of CPR, both tunnels were open, whereas, in the C3 simulation, only the solvent tunnel was present and, in the D2 simulation, only tunnel 2ac was present. These tunnel closures were due to BC-loop motion resulting in hydrogen bond formation closing tunnel 2ac  the2ac Fig.  and move2ac Fig. . A direc52. Our results indicate, however, that more extensive studies of substrate access and product release would be necessary to confirm the effect of CPR.The RAMD simulations, in which 7-ethoxyresorufin traveled from the active site to the protein surface, revealed five ligand egress routes in the CYP–CPR complexes  and three routes  in CPR-free CYP 1A1  was consistent with the high binding affinity of the CYP 1A1–CPR complex. In all the final model complexes, the CYP1A1 catalytic domain reoriented in the membrane. This reorientation affected ligand access and egress pathways between the active site and the membrane but there was little effect on interactions of the ligand in the catalytic site on the timescale of the simulations. CPR rearranged in the membrane and underwent a large conformational change from the open to the semi-open form, which enabled the formation of an additional interface between the distal side of CYP 1A1 and the CPR NADP domain. These data clearly indicate that, in presence of CPR, a rearrangement in the orientation of CYP occurs and vice versa.Upon binding CPR, the interaction between CYP 1A1 and the membrane became weaker as the CYP-FMN domain interface area increased. Although no TM-helix–TM-helix contacts between CYP and CPR were observed, the large interface area of the final complexes (ca. 2000 Å51. One of our model complexes (D2′) has a similar arrangement of the CPR FMN domain with CYP 1A1 to that observed in the crystal structure of P450BM3, although, with the position of the FMN cofactor shifted by about 20°. The computed ET rates and pathways from CPR to CYP 1A1 are in excellent agreement with available experimental data. The slower ET rates compared to soluble CYPs appear to result from the competing requirements of ET and membrane binding by both CYP and CPR, as well as sequence differences at the CYP-FMN domain interface. The importance of identifying ET pathways is highlighted by the fact that mutation of I458, a key residue in the dominant computed ET pathway, has been shown to affect prodrug activation65. The most common non-synonymous polymorphisms of CYP 1A1 have the mutations T461N and/or I462V, resulting in high-risk variants for non-small cell lung cancer in non-smokers74. We observed direct contact (<5 Å) of I462 with I458 only in our simulations in the presence of CPR, indicating that the I462V variant may have a role in the regulation of substrate metabolism. T461 interacted with the Lβ4-loop region of the CPR FMN domain in our simulations, indicating that alterations in CPR binding of the T461N mutant might contribute to the observed increased formation of the mutagenic diol epoxide of benzo[a]pyrene-7,8-dihydrodiol75. Thus, the present simulations give detailed atomistic insights into structural and functional aspects of CYP 1A1-mediated drug and carcinogen metabolism. Moreover, they provide insights into how CYP-redox partner interactions can vary, and provide a basis for further work to explore the interactions of different CYPs with CPR.The FMN domain binding face composition of CYP1A1 in the modeled CYP 1A1–CPR complexes is mostly similar to other experimentally determined FMN domain binding interfaces of CYPsThe computational workflow for building systems for simulations of full-length CYP–CPR complexes in a membrane bilayer is illustrated in Fig. 76, following refinement and rebuilding using the PDB-REDO server78 and addition of missing residues using Modeller81 v.10. All four chains (A–D) of the asymmetric unit were used in BD simulations. For the full protein, the TM-helix in CYP 1A1 was assigned to residues 6–27 and the flexible linker and TM-helix were modeled as described previously82. The structure of an open conformation of human CPR was modeled on the basis of the crystal structures of the N-terminally truncated rat CPR in an open conformation 62 and the closed form of the N-terminally truncated human CPR 83. Missing residues were modeled using VMD84 and Modeller.Modeling of CYP 1A1 was based on the crystal structure of the globular domain of human CYP 1A1 , in complex with the inhibitor α-naphthoflavone87. All cofactors were retained but the ligand was removed from the CYP. Docking was performed subject to electrostatic interaction, electrostatic desolvation, and non-polar desolvation forces87.BD rigid-body docking simulations of the four structures of the globular domain of CYP 1A1 and the N-terminally truncated CPR were carried out using the SDA 7 software71. Hence, the coordinates of diffusional encounter complexes were recorded subject to two distance constraints: (i) the cofactor–cofactor distance from FMN:N to HEME:Fe, DFe-N5 < 20 Å, and (ii) the center-to-center distance between the CYP globular domain and the CPR FMN domain, DCYP-FMN domain < 60 Å, to ensure close approach of the domains.A total of 240,000 independent trajectories were run starting with CPR at a random relative orientation and position at a center-to-center distance of 450 Å from the CYP, and they were all terminated when the inter-protein center-to-center separation reached 600 Å. ET in redox proteins typically occurs when the edges of the redox centers lie within 14–15 Å39. Thus, 40 representative encounter complexes were generated and named according to the chain identifier in the crystal structure of CYP 1A1 and the docking cluster number of CPR. E.g., C3 for chain C of CYP 1A1 and the third-largest docked cluster of CPR. These complexes were further filtered according to interaction energy, and to ensure that the clusters were structurally distinct and did not have orientations of CPR inconsistent with membrane anchoring if the CYP was assumed to be positioned in the membrane as observed in MD simulations of CYP 1A1 in a POPC bilayer. As a result, six structures of diffusional encounter complexes were selected for further studies.For each of the four CYP structures, the energetically ranked top 5000 docked encounter complexes were clustered into 10 clusters ranked by cluster size using a hierarchical method88.To account for protein flexibility, all-atom MD simulations in 150 mM aqueous solution (referred to as “soluble simulations”) were carried out for all six representative structures of CYP 1A1–CPR encounter complexes obtained from BD simulations. For computational efficiency, only the globular domain of CYP 1A1 and the FMN domain of CPR were simulated in the soluble simulations. After equilibration, all-atom MD production simulations were performed for about 50–140 ns using the NAMD v2.10 softwareAll the simulations with phospholipid bilayers, “membrane simulations”, were carried out for complete structures of full-length CYP and CPR. The initial configurations of the complexes of the globular domain of CYP and the FMN domain of CPR were obtained from the last frames of the soluble simulations.16. Hence, we superimposed the CYP domain of the CYPglobular-domain–CPRFMN-domain complexes onto a modeled structure of the membrane-bound full-length CYP 1A1 in a POPC membrane82. Next, we replaced the globular domain of CYP 1A1 with each CYPglobular-domain–CPRFMN-domain complex and connected the TM-helix and the linker region (residues 1–50) from the membrane-bound CYP 1A1 simulated in the absence of CPR with residue H51 of CYP 1A1 in the CYPglobular-domain–CPRFMN-domain complex89. A local structural refinement to optimize the covalent linkage between residues 50 and 51 was performed using the interactive energy minimization module of Maestro 89, resulting in complete initial structures of the membrane-bound CYPfull-length–CPRFMN-domain complex.We previously developed a protocol for inserting the full-length structure of a CYP into a membrane bilayer without predetermining the orientation or insertion depthfull-length–CPRFMN-domain complex was superimposed onto the FMN domain of truncated human CPR (residues 66–677) and residues 231–677 of CPR were extracted and connected with the FMN domain of the membrane-bound CYPfull-length–CPRFMN-domain complex. The amide bond between residues 230 and 231 was optimized using Maestro. The TM-helix region of CPR was predicted by using the Orientation of Protein in Membrane (OPM) (http://opm.phar.umich.edu/server.php)57 and the TMpred90 webservers and its structure was modeled using Modeller v 9.10. Next, the modeled TM-helix  was placed arbitrarily in the membrane so that there were no interactions between the CYP and CPR transmembrane helices. Then, the missing residues (1–25 of the membrane-binding domain and 51–65 of the flexible tether region) were added using Modeller to complete the full structure of the membrane-bound CYPfull-length–CPRfull-length complex.The FMN domain of CPR (residues 66–230) in each membrane-bound CYP+ and Cl− ions. Production simulations were performed for protein–phospholipid bilayer systems based on three encounter complexes (with one system simulated in duplicate) using the NAMD v2.10 software.Each CYP–CPR complex in a bilayer of POPC phospholipids was immersed in a periodic box of water molecules with 150 mM ionic strength maintained by adding Na92 protocol implemented in the AMBER 14 package  for snapshots collected at 100 ps intervals after completion of 350 ns simulation . Substrate access tunnels were computed using CAVER 3.093 for the last 500 snapshots of each trajectory collected at 100 ps intervals. Random acceleration molecular dynamics (RAMD)94 simulations, as implemented in NAMD 2.10, were performed to study the ligand egress pathways from the CYP 1A1 active site in the presence and absence of CPR. ET pathways and rates were computed using Beratan’s model with the VMD pathway plugin module95.CYP globular domain–FMN domain interaction free energies were estimated using the MMGBSAFurther information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting Summary


Staphylococcus epidermidis is a Gram-positive multidrug-resistant (MDR) bacterium of human skin. It is a health concern as it is involved in nosocomial infections. Genome-based screening approach of the complete genome of Staphylococcus virus PH15 allowed the identification of an endolysin gene . Bioinformatics analysis of the Ph28 protein predicted that it is a two-domain enzyme composed by a CHAP (22-112) and MurNAc-LAA (171-349) domain. Phylogenetic analysis and molecular modelling studies revealed the structural and evolutionary features of both domains. The MurNAc-LAA domain was cloned, and expressed in E. coli BL21 (DE3). In turbidity reduction assays, the recombinant enzyme can lyse more efficiently untreated S. epidermidis cells, compared to other Staphylococcus strains, suggesting enhanced specificity for S. epidermidis. These results suggest that the MurNAc-LAA domain from Ph28 endolysin may represent a promising new enzybiotic. Given the worldwide increase in antibiotic resistant bacteria, bacteriophage derived endolysins represent a very promising new alternative class of antibacterials in the fight against infectious diseases. Endolysins are able to degrade the prokaryotic cell wall, and therefore have potential to be exploited for biotechnological and medical purposes.  The emergence of multidrug-resistant (MDR) bacteria is an extremely important threat to public health worldwide. The diversity of resistance mechanisms that contributes to the development of MDR may lead to pandrug resistance (PDR) ,2. ConsiPeptidoglycan hydrolases  attract considerable interest as potential antibacterial tools . These eEndolysins are large group of enzymes with diverse specificity towards the various bonds within the peptidoglycan (PG) ,13,14. FS. epidermidis is a Gram-positive commensal bacterium of the human skin microbiome . Th. ThStaph strains  revealed strains B. For thS. epidermidis, display a modular architecture. They are composed by an enzymatic catalytic domain (ECD), located at the N-terminal part of the protein, and at least one cell wall binding domain (CBD), which is located at the C-terminal part. The ECD and CBD domains are linked by a flexible interdomain polypeptide  and under different culture conditions . On the other hand, the MurNac-LAA domain was expressed in E. coli BL21(DE3) and its catalytic activity and specificity was evaluated. The most common assay for determining the endolysins activity is based on the drop in optical density of the substrate after addition of the enzyme [S. epidermidis cells, as shown in To demonstrate that ph28 gene codes for an active endolysin, the full-length protein as well as the MurNac-LAA domain were cloned and expressed in e enzyme ,35. ThusS. epidermidis strains  as well as different Staphylococcus species and the results are listed in S. epidermidis strains. On the other hand, it displays higher lytic activity towards S. epidermidis strains, compared to other Staphylococcus species , suggesting a higher preference and presumably enhanced specificity for S. epidermidis.The specificity of the catalytic MurNac-LAA domain was further assessed using a range of different vice-versa. The C-score for the MurNAc-LAA and CHAP domain were 0.35 and 0.38, respectively, suggesting the validity of both models [+2 was predicted based on PDB structure 3QAYA. The molecular model of the CHAP domain in complex with Ca+2 was predicted based on PDB structure 4OLKA. The program PyMOL (www.pymol.org) was used for structures visualization. Aminoacid sequence alignments were carried out using Clustal O . Retrievh models ,29. The h models  and COACh models  based onThe nucleotide sequence encoding the MurNAc-LAA domain (aa171-349) was synthesized and amplified by PCR. The PCR reaction was carried out in a total volume of 50 μL, containing 8 pmoles of each primer: 5’- ATGACAAACAAAACGAGAAGTC-3’ and 5’- TTAATTAATAGCGCTTGCTATTGACTTTGT-3’ 10 ng template DNA, 0.5 mM dNTPs, 25 μL 2× HF reaction buffer and 1U Pfu DNA polymerase. The PCR-primers were designed according to the Ph28 gene sequence. The PCR protocol was composed by an initial denaturation at 94 °C for 30 sec, then 25 cycles of 15 sec at 94 °C, 15 sec at 65 °C and 1 min at 72 °C. A final extension of time at 72 °C for 10 min was performed after the 25th cycle. The PCR product was cloned to the pEXP5-CT/TOPO-TA vector (Invitrogen).E. coli BL21(DE3) cells with the recombinant plasmid were grown at 37 °C in 1 L LB medium containing ampicillin (100 μg/mL). When the absorbance of the culture at 600 nm was 0.4 AU, the expression of MurNAc-LAA domain was induced by the addition of 1 mM isopropyl 1-thio-β-galactopyranoside (IPTG). Following five hours incubation, the culture was centrifuged at 10,000 rpm for 10 min. The cell pellet was resuspended in potassium phosphate buffer  and sonicated. Cell debris were removed by centrifugation at 13,000 rpm for 5 min.Transformed −1) was determined by fitting the single exponential decay equation to the experimental data (absorbance). E. coli BL21(DE3) cells were used as negative control. The in vitro assessment of peptidoglycan-degrading activity of MurNAc-LAA domain was performed using the turbidimetry assay as described previously ,31,32. ES. epidermidis bacteriophage PH15 endolysin is a two domain protein (CHAP and MurNAc-LAA). The the MurNAc-LAA domain was cloned, expressed in E. coli BL21 (DE3), and its ability to catalyze the lysis of S. epidermidis was demonstrated. These results suggest that the MurNAc-LAA from Staphylococcus phage PH15 may represent a potential new enzybiotic. In conclusion, phages have evolved several classes of endolysins aiming at degrading the cell wall of their bacterial hosts. Although endolysins have a common enzymatic activity , several distinct classes exist with different catalytic mechanisms, structures, and domain architectures. The


Many studies report age as a risk factor for BoHV-1 infection or seropositivity. However, it is unclear whether this pattern reflects true epidemiological causation or is a consequence of study design and other issues. Here, we seek to understand the age-related dynamics of BoHV-1 seroprevalence in seasonal calving Irish dairy herds and provide decision support for the design and implementation of effective BoHV-1 testing strategies. We analysed seroprevalence data from dairy herds taken during two Irish seroprevalence surveys conducted between 2010 and 2017. Age-dependent seroprevalence profiles were constructed for herds that were seropositive and unvaccinated. Some of these profiles revealed a sudden increase in seroprevalence between adjacent age-cohorts, from absent or low to close to 100% of seropositive animals. By coupling the outcome of our data analysis with simulation output of an individual-based model at the herd scale, we have shown that these sudden increases are related to extensive virus circulation within a herd for a limited time, which may then subsequently remain latent over the following years. BoHV-1 outbreaks in dairy cattle herds affect animals independent of age and lead to almost 100% seroconversion in all age groups, or at least in all animals within a single epidemiological unit. In the absence of circulating infection, there is a year-on-year increase in the age-cohort at which seroprevalence changes from low to high. The findings of this study inform recommendations regarding testing regimes in the context of contingency planning or an eradication programme in seasonal calving dairy herds. Bovine Herpes Virus Type 1 (BoHV-1) is a highly contagious virus of cattle and is known to be the causative agent of the acute respiratory disease infectious bovine rhinotracheitis (IBR). The virus occurs worldwide and is endemic in a number of food-producing countries . Infecti50/mL nasal discharge; [When a naïve animal is exposed to BoHV-1, a so called primary infection can occur  and viruscharge; , 7) and scharge; –11. Follscharge; . BoHV-1 scharge; .A number of alternative pathways for BoHV-1 transmission have been reported in the literature. Direct contact between animals (e.g. nose to nose) is considered the most important route of transmission within farms . AerosolInfections with BoHV-1 occur worldwide, although there are differences in prevalence and incidence , includiwww.animalhealthireland.ie). The current study was undertaken to inform the consideration of options for a national eradication programme in Ireland.In the Republic of Ireland, information on BoHV-1 prevalence is available from recent cross-sectional studies –19. ThesAn understanding of the dynamics of BoHV-1 seroprevalence within herds is essential to guide control decisions and inform programme design. The published literature contains a number of retrospective studies where age is repeatedly identified as a risk factor for BoHV-1 seropositivity e.g. , 19–22. The objectives of this study were (1) to understand the age-related dynamics of BoHV-1 seroprevalence in unvaccinated dairy herds with seasonal calving, and (2) to determine how vaccine usage affects these dynamic patterns. The findings are discussed in the context of IBR control and surveillance.The data used for this study originated from two Irish seroprevalence studies undertaken by the Moorepark Animal and Grassland Research and Innovation Centre, managed by Teagasc (The Agriculture and Food Development Authority), between the years 2010 and 2017. The data have already been used in previous studies and we refer to  for detaIn brief, the first seroprevalence study was carried out on 24 dairy herds from which blood samples were taken at least once between 2010 and 2013. Herds selected for the study were Teagasc research farms or participants in the Dairy Information System . These dhttps://www.icbf.com/wp/?page_id=149), provided by the Irish Cattle Breeding Federation (ICBF). Blood samples were collected from the entire herd, tested for BoHV-1 antibodies and the herd´s vaccination status recorded. Information on the frequency of vaccination and the type of vaccine used were not recorded. Antibodies were determined using IDVet gE and Qiagen gB ELISA in vaccinated and non-vaccinated herds, respectively. Again, the sample-to-positive ratio was used for classifying antibody titres as positive, negative or inconclusive by referring to the manufacturer’s instructions.The second seroprevalence study was conducted in 2015 on 57 dairy herds. Herds under investigation were members of a breeding information service  to ensure that potential increases/decreases in prevalence are not dominated by moves into or out of each herd.Based on the date of birth of each tested animal, we calculated the age in days at the time of sampling. Animals were then grouped into age cohorts by year from 0 to 9. Animals older than 9 years were assigned to cohort > 9.BoHV-1 seroprevalence was estimated at herd and age-cohort level for each round of sampling, calculated as the number of seropositive cattle divided by the total number of cattle tested. For each age cohort, a Gaussian confidence interval was calculated on the basis of the apparent prevalence of seropositive animals at the 95% significance level.In a final step, we created two overlapping subsets of the data (A & B). Dataset A included serological results from herds that at the time of sampling were not vaccinated against BoHV-1. All herds that were seronegative or not completely sampled (e.g. missing young-stock data) were excluded. For herds that were tested multiple times the data of the first sampling was used. In dataset A, each herd was included only once. Thus 3994 test results from 15 unvaccinated seropositive dairy herds were used for dataset A. Dataset B included all herds that were tested at least twice, irrespective of their vaccination status. All test results from these herds were included in dataset B, with the exception of results from herds that were not completely sampled for a given round of testing. 8972 test results from 13 multiple-tested herds were retained in dataset B. These multiple tested herds were investigated to study the influence of vaccination on the within-herd prevalence. Six herds were already vaccinating before sampling was conducted or had started to vaccinate within the sampling period. All herds in dataset B showed a decline in herd-level/age-related seroprevalence over time . The documentation of the model follows the ODD protocol ; –28 and ihttps://www.scala-lang.org/).The model simulates the spread of BoHV-1 in a seasonal calving dairy herd, where young stock and lactating cows are not epidemiologically separated and replacement stock are home-bred. The simulated herds are similar in structure  and management practices. The herds are simulated without in-moves and hence represent epidemiologically closed herds. The model simulations are not intended to replicate the herds in the data but rather to highlight general BoHV-1 serology dynamics. The model design comprises two components . The demWe simulated 15 separate dairy herds, each for 20 years. BoHV-1 infection was seeded in all herds at the same point in time by infecting a random animal (seed infection).In Scenario A, we simulated the course of infection in the herds while excluding reactivation of latently infected cattle. The serological status of each animal in the simulated herd was recorded during the simulation period. The seroprevalence profiles across age-cohorts of the simulated herd were evaluated at three intervals: immediately after infection ; at a time point selected at random between years two and five, and at 12 years after the initial seeding of infection.Scenario B was similar to A but this time a reactivation event was simulated 4.5 years after the initial seeding of infection. The reactivated individual was forced to infect one naïve animal, thereby triggering a primary infection during its infectious period. Again, three seroprevalence profiles were reported: at 6 months, at 5 years (i.e. 6 months after the reactivation event), and at a time point selected at random between nine and 12 years after the initial seeding of infection (i.e. 4.5 to 7.5 years after the reactivation event).Among the 17 unvaccinated dairy herds, two herds returned 100% negative test results. 15 dairy herds were non-negative (Dataset A) and within-herd seroprevalence varied between 0.8 and 96.2%. The overall seroprevalence in these 15 positive herds was 31.7% (SD 8%).The overall seroprevalence of antibodies against BoHV-1 in these herds increased with age  with movements both into and out of the herd over several age-cohorts. As depicted in Figure Our understanding of the dynamics of BoHV-1 seroprevalence has been greatly improved by comparing aggregated serological results (across herds) with an interpretation of outcome patterns . Indeed, the benefit of visualising data in this more complex but complete manner has been recognised previously as a more appropriate starting point prior to technical aggregation . In the In Irish dairy farming, different management groups  often form independent epidemiological units, due to, for example, separate housing or grazing on separate blocks of land. There would be a need for an independent evaluation of seroprevalence profiles within each management group, if these were found to be epidemiologically distinct on a single farm. Since we were not able to extract these management groups from the available data, we considered a whole herd as a single epidemiological unit in our modelling work.By coupling the outcome of our data analysis with simulation output of an individual-based model at the herd scale, we have shown that the youngest age-cohort with a high seroprevalence profile in a naïve herd is related to the time interval since infection ceased to circulate . It is intrinsic to the interpretation of age-dependent seroprevalence profiles that only the most recent outbreak can be deduced from the profile. Indeed, a successful reactivation event (i.e. one that initiates further primary infections) within the herd or the introduction of the virus by other pathways (that results in active virus circulation) will each result in a further outbreak that will mask the previous serological profile  in each herd. This age-threshold cohort provides useful information for surveillance and monitoring. Population-based screening of BoHV-1 herd-level prevalence should target the older age-cohorts, whereas detection of recent virus circulation (i.e. major outbreaks) should address the youngest age-cohorts in a herd without maternal immunity but with epidemiological contact with older latently infected animals. Efficacy of vaccination or other intervention strategies (e.g. biosecurity) should be monitored by observing the longitudinal shift in the age of the age-threshold cohort and through surveillance of the younger cohorts to identify potential new exposure. Further research is needed to explore data for seasonal calving beef herds to see if patterns there are as clear as in dairy herds.Additional file 1: ODD protocol of the simulation model.Additional file 2: Age-related prevalence profiles without seasonal calving.Additional file 3: Effect of vaccination.


The multistage compression of axisymmetric samples constituting a simulation of the real technological process and hot-rolling performed on a semi-industrial line were carried out using three cooling variants: in water, in air, and after isothermal heating and cooling in water. The temperature at the end of the thermomechanical treatment for all variants was 850 °C. On the basis of detailed structural studies, it was found that the main mechanism for removing the effects of the strain hardening that occurred during the four-stage compression involved the dynamic recrystallization occurring in the first and second stages, the hot formability and dynamic recovery in successive stages of deformation, and the static and/or metadynamic recrystallization that occurred at intervals between individual deformations, as well as after the last deformation during isothermal heating. Analysis of the phase composition and structure allowed us to conclude that the tested steels have an austenitic-ferritic structure with carbide precipitates. Research using scanning and transmission electron microscopy identified κ-3AlC and M7C3 carbides in both the analyzed steels. In addition, complex carbides based on Nb and Ti were identified in X98 steel; C carbides occurred in the entire volume of the material. Slow cooling after thermomechanical treatment influenced the formation of larger κ-carbides at the border of the austenite and ferrite grains than in the case of rapid cooling. The size and morphology of the carbides found in the examined steels was varied. Back-scattered electron diffraction studies showed that wide-angle boundaries dominated in these steels.In this paper, analytical results are compared for the newly developed steels, Fe-Mn-Al-C (X105) and Fe-Mn-Al-Nb-Ti-C (X98), after being hot-rolled and also after undergoing thermomechanical treatment in a Gleeble simulator. These steels have a relatively low density (~6.68 g/cm Due to their excellent mechanical properties, relatively high plasticity, and forecasted relatively low production costs , Fe-Mn-Al-C steels, which are the subject of this analysis, can be potentially used for elements of transport infrastructure and vehicles such as cars, buses, or trains, as well as in all kinds of constructions where the weight of the structure is one of the most important criteria for the selection of materials ,14,15,163AlC carbides and others. They found that κ-carbides are formed from areas enriched in carbon through spinodal decomposition and are key determinants of the properties of these steels [3AlCx carbides in Fe-Al-Mn-C steels can occur in both austenite and ferrite, depending on the content of the alloying elements. In Fe-Al-Mn-C alloys with a high manganese content (above 10%), Mn atoms take the place of Fe atoms in the above carbide, and the stoichiometry of the Fe3AlCx carbide is transformed into 3AlCx [Rana et al. described multiphase TRIPLEX Fe-Mn-Al-C steels with a structure consisting of three phases: austenite, ferrite, and κ-e steels ,26. The Mn)3AlCx ,25,26. EMn)3AlCx .3AlC carbides and α-ferrite were found along the austenite grain boundaries and inside the austenite. The κ-carbides were then three to six times larger than in the case of fast-cooled (water) steels, while reducing the plasticity and strength of these steels. Therefore, with the current state of knowledge about the structural mechanisms of this steel group, it is recommended to quickly cool Fe-Mn-Al-C steel to avoid the formation of large κ-carbides [Chen et al. showed that, in Fe-Mn-Al-C steels, recrystallization occurred during a traditional hot-rolling process, and the microstructure after hot-rolling showed elongated austenite grains with annealing twins ,28. Aftecarbides ,28,29,30carbides ,7,8,10. In describing the structure of Fe-Mn-Al-C steel after hot-rolling, Bausch, Frommeyer et al. noted the formation of ferrite bands ,4,31. ThThe mechanical properties of high-manganese steels can be shaped by suitably-selected thermomechanical treatment, which is the preferred method of producing mass products in economic terms. The use of appropriate heat and plastic deformation treatment allows the best relationship between the strength properties and plasticity of these steels to be obtained ,37,38,39The tests were performed on experimental high-manganese X98MnAlNbTi24-11 (X98) and X105MnAlSi24-11 (X105) TRIPLEX steels, whose chemical composition is given in −1. Linear variable displacement transducers and strain gauges for measuring pressure allowed feedback to be obtained, which enabled accurate performance and high repeatability of the set mechanical quantities of the planned process. To reduce friction, graphite and tantalum foils were used between the sample surface and the anvil surfaces, while both surfaces were coated with high-temperature nickel grease  given by Ekelunda .(1)ε˙=2w—equal peripheral speed of rollers 0.74 m/s;V1, h2—thickness of before and after passes;hΔh—absolute crease;R—roll radius, amounting to 0.275 m.−1. Similar plastic deformation rates were used for plastically deformed samples in the Gleeble simulator. It should be taken into account that under industrial conditions the plastic deformation rates in the final culverts reach values up to 50 s−1 and even up to 100 s−1. The parameters of the hot-rolling process were developed assuming that the size of the value of plastic deformation was determined by the allowable force of the band pressure on the roll. The basis for determining the pressure force during rolling was determining the average unit pressure. Proper description of this value for specific conditions always consists in choosing a method appropriate for a given process; therefore, after performing the appropriate analyses, it was considered that it was best to use the Zjuzin method to determine the average unit pressure exerted by the sheet on the rolling mill [The plastic deformation rates for subsequent culverts were, respectively, 9.5, 10, 10.3, and 10.1 sing mill ,42. The ing mill ,43. The Variant 1—supersaturation of steel after plastic deformation in the last culvert with a deformation value of 20%, under deformation strengthening conditions controlled by dynamic healing;Variant 2—cooling of steel in the air after plastic deformation in the last culvert with a deformation value of 20%, under deformation strengthening conditions controlled by static and metadynamic recrystallization;Variant 3—supersaturation of steel after plastic deformation in the last culvert with a 20% deformation value and isothermal annealing at its deformation temperature for 30 s, in conditions ensuring the assumed proportion of statically recrystallized austenite grains.The purpose of each cooling variant was:3COOH);Total of 950 mL 99% acetic acid (CH4).Total of 50 mL 60% perchloric acid , as well as on a SUPRA 35 scanning electron microscope  at an accelerating voltage of 20 kV using secondary electron detection (SE). In order to examine the chemical and phase composition of the samples’ micro-areas, as well as precipitates and particles, an EDS detector and a diffraction camera coupled to the abovementioned microscope in an Edrid Trident XM4 system were used. Tests using backscattered electron diffraction (EBSD) were performed at an accelerating voltage of 15 kV, a working distance of 17 mm, and a step size of 0.20 μm. Samples for structural examination using a transmission electron microscope (TEM) were cut and then prepared by mechanically grinding them to a thickness of 60 µm, followed by ion beam polishing. Transmission electron microscope studies were conducted at an accelerating voltage of 300 kV. TEM investigations were performed using a probe Cs-corrected FEI Titan 80-300 S/TEM microscope  equipped with EDAX EDS. Selected area electron diffraction patters were obtained with a camera length of 215–330 mm and a C2-50 condenser aperture. In STEM mode, HAADF (high-angle annular dark field) was used with a convergence angle of 24 mrad.The main purpose of the research was to analyze the structural changes of X98 and X105 steels after simulation of several hot compression stages and the actual hot-rolling of trial sheet sections, together with the determination of the structural processes occurring in the intervals between individual hot forming cycles. max, from 0.13 to 0.28. An effect of deformation temperature on εmax was also noted . However, in the case of X105 steel, the deformation εmax had a value of 0.16 at 1150 °C and increased with the reduction in the compression temperature to 850 °C to about 0.28, so the plasticizing deformation values increased by about 75%. The initiation of dynamic recrystallization occurred before reaching the deformation εmax, corresponding to the maximum value of the plasticizing stress. Knowledge of these processes is important in order to precisely design the thermomechanical treatment process under industrial conditions. The initiation of dynamic recrystallization at 850 °C took place after a true strain of about 0.2 for both steels. The production of a fine-grained structure as a result of dynamic recrystallization, under industrial conditions, is therefore achievable provided that these thermomechanical treatment parameters are mapped onto industrial mills [A detailed analysis of the σ–ε curves for the tested high-manganese TRIPLEX steels obtained on the Gleeble 3800 simulator indicated that the strain rate had a large impact on the value of the flow stress, which for the applied deformation conditions ranged from 110 to 485 MPa . It was so noted . For exaal mills ,37,44,45−1, the value of the maximum plasticizing stress at which the dynamic recrystallization process was initiated was achieved at a deformation value of 0.183 for X98 steel and 0.163 for X105 steel. The plastic deformation applied for both the first (at 1100 °C) and the second (at 1050 °C) deformation stages was 0.23 . Additionally, the temperature of the first stage of deformation (1100 °C) was 50 °C higher than in the case of tests performed in the single-stage compression of the tested steels, while on the basis of these test results a clear trend could be seen that, along with the increase in deformation temperature at a constant rate of plastic deformation, in this case 10 s−1, the critical crush value decreased from 0.253 to 0.183 for X98 steel and from 0.279 to 0.163 for X105 steel. These changes were necessary to achieve maximum plasticizing stress and thus to initiate the process controlling the deformation strengthening, which was dynamic recrystallization. The change in the value of the plastic deformation rate from 7 s−1 in the first stage of deformation at 1100 °C, and its increase to a value of 10 s−1 in the last (fourth) stage of deformation, was an attempt to map the actual plastic deformation rates used on the rolling line. Based on the results of one-stage compression tests, it was found that the reduction in the deformation rate at a constant test temperature caused a decrease in the value of the deformation value. For example, for X98 steel, hot plastic deformation at 1050 °C, together with the reduction of the deformation rate from 10 to 1 s−1, the deformation value decreased from 0.183 to 0.166, this being necessary to achieve maximum plasticizing stress and thus to initiate the process of dynamic recrystallization that controlled the deformation strengthening. This trend was maintained for both the tested X98 and X105 steels in the range of the tested plastic deformation rate from 10 to 0.1 s−1. Based on this analysis, it was found that a plastic deformation rate of 7 s−1 in the first stage of deformation at 1100 °C and its increase to a value of 8 s−1 in the second stage of deformation were sufficient to initiate the process of dynamic recrystallization that controlled the deformation strengthening. In the next stage of deformation at 950 °C, the used value of 0.23 was on the border of the critical crush necessary to initiate dynamic recrystallization, which was determined in one-stage deformation tests of 0.233 and 0.214  the main processes controlling deformation strengthening to be dynamic recrystallization and static recrystallization, occurring between subsequent deformations . The avee to 65% b. In bote to 65% b.7C3 carbide at grain boundaries in both steels was also noted. Based on EBSD research, it was found that the four-stage heat and plastic deformation treatment completed with cooling variants 1–3 in both steels was characterized by obtaining a structure with the dominance of wide-angle borders, whose percentage was generally smaller than after deformation using the Gleeble simulator and ranged from 60% to 71% for X98 steel and 47% to 71% for X105 steel  additions was on average around 11%, and in the case of the reference X105 steel its share was definitely higher and amounted to about 27%;After hot plastic deformation using the Gleeble simulator and a semi-industrial rolling line, the structure of both tested steels was similar in terms of phase composition. X98 steel, due to its Nb and Ti content, was characterized by a significantly smaller size of austenite grain and a share in the structure of, among others compound carbides, C. However, both the tested steels differed significantly, as in the initial state after forging, with regard to the participation and arrangement of ferrite. In X98 steel, after simulated plastic deformation (Gleeble) with different cooling variants, ferrite was quite evenly distributed in its structure, with the largest areas of ferrite revealed in option 2. In X105 steel, ferrite occurred in the form of elongated areas in the direction of rolling in variants 1 and 2, and fine grains in option 3. These clear differences in the distribution and form of ferrite in the simulated deformations no longer found their analogy in the structure of the steel after rolling, where the ferrite in all cooling variants was arranged in the form of highly-elongated strands in the direction of rolling, which resulted from a low tendency to undergo recrystallization. In addition, the formation of ferrite bands parallel to the rolling direction was affected by the high concentration of aluminum in the steels tested, which was also noted by Bausch et al. [It was found that the process controlling deformation strengthening at all stages of the hot plastic deformation was dynamic recrystallization, together with static recrystallization in the intervals between subsequent stages, especially between the last stages. Cooling of the analyzed steels after thermomechanical treatment in air promoted metadynamic recrystallization and an increase in the average grain size. The applied isothermal annealing after plastic formation caused fragmentation of the structure, because the main processes removing the effects of deformation strengthening were metadynamic and static recrystallization;On the basis of EBSD tests in each of the presented states , it was found that the studied steels were dominated by wide-angle boundaries (misorientation angle Ѳ ≥ 15°), with the exception of X105 steel from the second cooling variant after rolling. Deformation twins with a misorientation angle of 58°–62° were also disclosed, while for most variants with X98 steel their share was definitely greater. The share of low-angle boundaries of about 20%–30% (misorientation angle Ѳ < 15°) in both the tested steels may indicate that the recrystallization process was not completed with the adopted thermomechanical treatment plan;7C3-type carbides and nanometric κ-3AlC carbides located inside the austenite and ferrite grains, as well as at the grain boundaries. Only in variant 2, cooling after heat treatment, were the carbides in question definitely larger and located mainly on grain boundaries, which may significantly reduce the mechanical properties of the steels tested after this type of treatment. M7C3-type carbides with orthorhombic crystal lattices were revealed in both the steels examined in the austenite and ferrite grains;Research on the structure of the X98 and X105 steels using a transmission electron microscope allowed the identification of M3AlC carbides were also found. In X98 steel, the size of the κ-carbide was smaller and ranged from a few to 160 nm, while in X105 steel both carbides had a size ranging from several nanometers up to nearly 1 µm.It was also found, on the basis of tests using SEM coupled with an EDS spectrometer, that the X98 steel had dispersive carbides based on Nb and Ti, which were released in austenite, ferrite, and the grain boundaries. The size of these carbides ranged from several nanometers to about 15 µm. At the grain boundaries in both the analyzed steels, AlN precipitations of up to 3 µm were also noted. At the boundaries of austenite and ferrite grains in both the investigated steels, κ-Based on the results of our research and analysis, the following conclusions can be drawn: