Acinetobacter baumannii was characterized and its dimeric crystal structure was unveiled, demonstrating the extra-domain-mediated dimerization of MltA for the first time. Based on the current structural and biochemical studies, the working mechanism of MltA with various functionally related proteins on the bacterial outer membrane was modeled.MltA from the human pathogen  N-acetylmuramic acid and N-acetylglucosamine in peptidoglycans. Although substrate recognition and cleavage by MltA have been examined by previous structural and mutagenesis studies, the overall mechanism of MltA in conjunction with other functionally related molecules on the outer membrane of bacterial cells for peptidoglycan degradation has remained elusive. In this study, the crystal structure of MltA from the virulent human pathogen Acinetobacter baumannii is characterized and presented. The study indicated that MltA from A. baumannii forms homodimers via an extra domain which is specific to this species. Furthermore, the working mechanism of MltA with various functionally related proteins on the bacterial outer membrane was modeled based on the structural and biochemical analysis.Peptidoglycan digestion by murein-degrading enzymes is a critical process in bacterial cell growth and/or cell division. The membrane-bound lytic murein transglycosylase A (MltA) is a murein-degrading enzyme; it catalyzes the cleavage of the β-1,4-glycosidic linkage between  Biochemical and structural studies revealed that abMltA forms homodimers in solution; these dimers may serve as the functional unit of this enzyme. The structure of abMltA showed a distinct extra domain in domain 2, which was similar to that in MltA from N. gonorrhoeae, and dimerization was mediated by this extra domain. Further, we propose the working mechanism of MltA with various functionally related proteins on the bacterial outer membrane based on the current structural and biochemical analysis.Although previous studies examining MltA from 2.2.1.A. baumannii corresponding to amino acids Gly23–Asn388 was constructed by inserting the synthesized gene product into a pET-28a vector. The gene sequence was derived from GenBank (ID SSM89718) and synthesized by BIONICS . The expression plasmid was delivered into E. coli strain BL21 (DE3) using heat shock at 42°C. The transformed bacteria were spread on a lysogeny broth (LB) agar plate containing kanamycin and incubated for 16 h at 37°C. A single colony was picked and cultured overnight at 37°C in 5 ml LB containing 50 µg ml−1 kanamycin. Subsequently, the cells were transferred and cultured in 1 l LB. When the optical density, measured at 600 nm, reached approximately 0.6–0.7, the culture was cooled on ice and 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to the medium to induce gene expression. After adding IPTG, the cells were further cultured for 15 h at 20°C on a shaking incubator. Bacterial cells expressing the target gene product were harvested by centrifugation and the cell pellet was resuspended in 10 ml lysis buffer . The cells were disrupted in lysis buffer by sonication on ice with six bursts of 30 s each and a 60 s interval between each burst. To remove the cell debris and collect the supernatant, the cell lysate was centrifuged at 10 000g for 20 min at 4°C. The collected supernatant was mixed with nickel–nitrilotriacetic acid (Ni–NTA) resin solution  by gentle agitation for 2 h at 4°C. The resulting mixture was loaded onto a gravity-flow column. The Ni–NTA resin was washed with 30 ml washing buffer  to remove unbound proteins. 2.6 ml elution buffer  was then loaded into the column to elute the bound protein. The resulting eluate was concentrated to 50 mg ml−1 and sequentially subjected to size-exclusion chromatography (SEC). SEC purification was performed using an ÄKTAexplorer system  equipped with a Superdex 200 Increase 10/300 GL 24 ml column  pre-equilibrated with SEC buffer . The peak fractions from SEC were pooled, concentrated to 33.8 mg ml−1, flash-frozen in liquid nitrogen and stored at −80°C until further use. Protein purity was assessed by SDS–PAGE.The expression plasmid for N-terminally truncated MltA from 2.2.−1 protein solution in 20 mM Tris–HCl pH 8.0, 150 mM NaCl was mixed with an equal volume of reservoir solution and the droplet was allowed to equilibrate against 300 µl reservoir solution. The crystals were produced from a buffer comprising 0.1 M CHES pH 9.5, 20%(w/v) PEG 8000. They appeared in 60 days and grew to maximum dimensions of 0.2 × 0.2 × 0.4 mm. For data collection, the crystals were soaked in mother liquor supplemented with 30%(v/v) glycerol as a cryoprotectant solution, mounted and flash-cooled in a nitrogen stream at −178°C. The diffraction data were collected on the 5C beamline at Pohang Accelerator Laboratory (PAL), Pohang, Republic of Korea at a wavelength of 1.000 Å. The diffraction data were indexed, integrated and scaled using HKL-2000 . The mobile phase buffer flowed at a rate of 0.3 ml min−1 at 25°C. A DAWN TREOS MALS detector , which was interconnected with an ÄKTAexplorer system , was used for the MALS experiment. The molecular mass of bovine serum albumin was used as a reference value. The absolute molecular mass was assessed using ASTRA (Wyatt Technology).The absolute molar mass of abMltA in solution was determined by MALS. The target protein, purified by affinity chromatography using Ni–NTA, was filtered with a 0.2 µm syringe filter and loaded onto a Superdex 200 10/300 gel-filtration column  that had been pre-equilibrated in SEC buffer  according to the manufacturer’s protocols. Mutagenesis was then confirmed by sequencing. Mutant proteins were prepared using the method used for the purification of wild-type abMltA.2.6.Clustal Omega .The amino-acid sequences of abMltA from various species were analyzed with 2.7.7esj.Coordinates and structure factors have been deposited in the RCSB Protein Data Bank with PDB code 3.3.1.b)]. During the SEC purification process, two homogeneous protein samples corresponding to oligomer and dimer fractions were produced, and only the dimer factions could successfully be crystallized [Fig. 1b)]. The 2.06 Å resolution crystal structure of abMltA was finally resolved and refined to Rwork = 21.77% and Rfree = 26.40%. The crystallographic and refinement statistics are summarized in Table 1The limited knowledge of MltA, with limited structural information, is often attributed to its insolubility due to its membrane-association. However, we found that a codon-optimized, signal peptide-removed version of abMltA (corresponding to amino acids Gly23–Asn388) was soluble. A rapid two-step chromatography, involving affinity chromatography followed by size-exclusion chromatography (SEC), generated a large amount of soluble abMltA protein (20 mg per litre of LB culture) with high purity (∼90% purity) . The final model of each molecule was constructed from Ala44 to Asn388. 20 residues from the N-terminus could not be represented in the final model due to poor electron density that was not traceable. The crystal structure of abMltA showed the presence of ten α-helices and 16 β-sheets. It exhibited a typical domain composition, with two distinct domains: domain 1 and domain 2 [Fig. 1d)]. Interestingly, however, abMltA contained an extra domain composed of residues Asp151–Asp184, which was distinctly separated from domain 2 [Fig. 1e)]. Domain 1 consisted of four α-helices (H1–H3 and H10) and eight β-sheets (S1–S3 and S12–S16), while domain 2 consisted of five α-helices (H5–H9) and six β-sheets (S4–S5 and S8–S11) [Fig. 1d)]. For the construction of domain 1, three α-helices (H1–H3) and three β-sheets (S1–S3) at the N-terminus formed a double-ψ β-barrel structure with one α-helix (H10) and five β-sheets (S12–S16) at the C-terminus, which is a common topology for MltA proteins [Figs. 1d) and 1e)]. Domain 2 was composed of ∼130 residues in the middle of MltA [Fig. 1e)]. The extra domain, comprising one α-helix (H4) and two β-sheets (S6 and S7), was composed of ∼30 residues located between the N-terminal region of domain 1 and domain 2 [Fig. 1e)]. B-factor analysis showed that three loops, the H7–H8 connecting loop in domain 2 and the S12–S13 connecting loop and the S14–S15 connecting loop in domain 1, demonstrated the highest B-factor values, indicating that these loops might be flexible [Fig. 1f)]. All three loops with high B-factor values were located near the substrate-binding pocket. As demonstrated in a previous study performed with ecMltA, these loops were not directly involved in substrate interaction. These flexible loops around the active site might help to access the substrate in the active site of MltA ]. A deep tentative substrate-binding pocket was distinctly formed between domain 1 and domain 2, including the extra domain [Fig. 1g)].Two molecules, chain h)]. Superimposition of chain A on chain B indicated that domain 2 and the extra domain were tilted 20° away from domain 1 when domain 1 was superimposed [Fig. 1h)]. This domain rotation in chain B resulted in a wider substrate-binding pocket. The lengths of the entrance to the substrate-binding pocket in chain A and chain B were 12.4 and 20.7 Å, respectively. The interdomain movement induced upon binding of the substrate is one of the main characteristics of MltA  of 3.2 Å . As we observed dimerization of abMltA in solution, we analyzed the crystallographic packing to search for symmetric molecules and to further understand the tentative dimeric structure of abMltA. An alternative dimeric structure was formed by chain A and chain B′, as demonstrated by crystallographic packing [Fig. 2b)]. Since two types of interactions between chains A and B and between chains A and B′ can be formed for crystallographic packing, we analyzed the protein–protein interaction (PPI) in both the AB dimer and the AB′ dimer using the PDBePISA PPI-calculating server  of 1.000, where the score ranges from 0 to 1 as the relevance of the interface for complex formation increases, whereas the interface of the AB dimer has a score of 0.00 [Fig. 2c)]. In the AB′ dimer the total dimer surface buried 2296 Å2 (a monomer surface area of 1148 Å2), which represents 6.7% of the total surface area. Hydrogen bonds  and salt bridges  were the main forces responsible for the formation of this dimeric interface [Fig. 2c)]. Asp151, Glu164, Lys168 and Lys177 from one molecule formed salt bridges with the same residues from the other molecule. Tyr162, Arg169 and Gly235 from each molecule were involved in the formation of hydrogen bonds at the dimeric interface [Fig. 2d)]. In the case of the AB dimer, the dimer buried a total surface area of 918 Å2 (a monomer surface area of 474 Å2), which represents 2.8% of the total surface area as calculated by PDBePISA. This interface analysis indicated that the AB′ dimer might be formed in solution [Fig. 2c)]. To confirm this result, we performed a mutagenesis study. Since Asp151, Tyr162 and Glu164 are the primary interface residues involved in the formation of the AB′ dimer, they were mutated to lysine, producing a D151K, Y162K and E164K triple mutant. We analyzed the effect of the mutations on the formation of dimers using SEC. Although the single mutants did not affect dimer formation (data not shown), as indicated in Fig. 2e) the triple mutant  showed a definite disruptive effect, demonstrating a new peak corresponding to the size of a monomer in the SEC profile. The newly formed peak at the void volume might be attributed to aggregated triple mutant of abMltA [Fig. 2e)]. This aggregated particle formed by mutation might be explained by (i) the direct effect of the triple mutation on the solubility of abMltA or (ii) the disruptive effect of the triple mutation on the formation of the dimer and the production of insoluble monomer. The molecular mass of the newly generated tentative monomer peak following triple mutagenesis was further calculated by MALS to confirm the mutagenesis effect. The MALS results showed that the molecular weight of the peak corresponding to the size of a monomer produced by the triple mutant was 44.8 kDa, indicating that this peak indeed represented the monomeric form of abMltA that was produced by disruption of the AB′ dimer interface. These results strongly indicated that abMltA forms a dimer in solution. The dimer formation was mediated by the extra domain of abMltA.Although its molecular architecture has not been determined by structural studies, a higher ordered murein-synthesizing machinery composed of PBPs, MipA and MltA has consistently been demonstrated by biochemical, biophysical and cellular studies  and E. coli (ecMltA), has been studied. Hence, these ortholog structures were selected as structural homologs from the DALI server. Considering that most bacteria contain an MltA enzyme, it is interesting that only two ortholog structures have been examined to date. This might be because MltA is localized on the membrane, making its structural study difficult to perform because of solubility issues. ngMltA (PDB entry 2g6g), the MltA domain of LtgG (PDB entry 6qk4) and two different states of ecMltA , in that order, were the top four matches obtained using the DALI server ]. In particular, ngMltA, the protein with the highest structural similarity according to the DALI search, showed an additional loop and β-sheet in domain 1 [Fig. 3b)]. Although the overall fold of domain 1 of ngMltA was quite different from that of abMltA, interestingly the extra domain of abMltA was located in the same position as that of ngMltA [Fig. 3b)]. However, the structure of the extra domain of ngMltA differed from that of abMltA [Fig. 3b)]. A pairwise structural comparison showed that the locations of domain 1 and domain 2 were not identical . The structural dynamic movement of each domain of MltA observed upon accommodating the substrate in ecMltA is one of the main features of MltA ]. The structure of chain A of abMltA was more similar to that of the closed conformation of ecMltA. However, abMltA did not include the substrate, indicating that substrate binding might not be the only reason for domain movement. Although domain 1 and the extra domain of chain B were shifted outwards a little compared with those of chain A, the structure of chain B was also more closely related to the closed conformation when compared with that of the open conformation of ecMltA [Fig. 3f)].The structural comparison performed by superposition of monomeric abMltA with MltA from different species showed that the domain compositions and locations were different between each species, exhibiting an r.m.s.d. of 3.2 Å with ngMltA, 5.6 Å with LtgG, 2.7 Å with an ecMltA–substrate complex and 8.8 Å with ecMltA, although the overall folds of domain 1 and domain 2 were similar [Table 2g) and 3h)]. To speculate on the function of this extra domain by comparing the structural homologs, we again performed a DALI search ]. About 35 additional residues in ngMltA formed a structurally different domain compared with that of abMltA. According to a previous structural study, the structure of ngMltA was solved in a monomeric form and the function of this extra domain has been suggested to be a protein–protein interaction module that might be used for binding to other proteins ]. Sequence alignment also showed that the previously identified amino-acid residues involved in substrate binding in the ecMltA system  were completely conserved throughout different species, indicating that the strategies for recognition of the substrate in the active site might be very similar among MltA orthologs [Fig. 4b)]. To analyze the substrate binding of abMltA, the structure of abMltA was superimposed with that of ecMltA complexed with a substrate analog. This structural comparison study showed that the locations of seven residues, Thr119, Tyr121, Gln182, Ser184, Tyr200, Asp317 and Asp328, involved in substrate binding in the ecMltA system were structurally conserved as Thr125, Tyr127, Gln213, Ser215, Tyr230, Asp342 and Asp354, respectively, in abMltA [Fig. 4c)]. This confirmed that the seven conserved residues in abMltA might be used for the recognition of substrate via a strategy similar to that of the substrate recognition performed by the ecMltA system. Since the cysteine residue in the N-terminal region, which is a well known lipid-modification residue involved in membrane anchoring, was completely conserved across all species, the site of action at the membrane of MltA will be common [Fig. 4b)]. The presence and the sequence of the extra domain vary among different species. ecMltA does not exhibit the extra domain, while the sequence and the structure of the extra domain of ngMltA were different from those of abMltA [Figs. 3i) and 4b)]. This indicated that the extra domain-mediated dimerization of MltA might be specific to abMltA.Analysis of the evolutionarily conserved amino-acid positions in abMltA based on the phylogenetic relations between homologous sequences using the d)]. Although MltA functions in a monomeric form, as determined in a previous study performed using the representative Gram-negative bacterium E. coli ].Based on the results of previous studies and those observed in the current structural study, we conclude that MltA anchors on the outer membrane of the bacterial cell via lipid-modified cysteine residues at the N-terminus. Membrane-bound MltA recognizes the peptidoglycan using a deep substrate-binding groove, which is formed by seven highly conserved amino-acid residues and is involved in the peptidoglycan-recycling process with MipA and PBPs [Fig. 4MltA, 7esjPDB reference:


The wire drawing process is commonly perceived as one of the best studied metal forming processes in almost every aspect; however, when considering elastic deformation, researchers usually focus on the uniaxial tensile forces after the material exits the drawing die and not the elastic deformation region before entering the drawing die, even though it may have a significant impact on the strength parameters and the nature of metal flow inside the drawing die. The aim of this research is to theoretically and experimentally identify the deformation in the elastic region and to further link the shape of this region and the values of stress occurring in it with the geometrical parameters of the drawing process and assess its impact on its strength parameters. In order to achieve the assumed goals, numerical analyses using the finite element method and experimental research on the drawing process in laboratory conditions were carried out using Vickers hardness tests and resistance strain gauges measuring deformation in stationary and non-stationary conditions. The obtained results indicate that the shape and the extent of the region of elastic deformations generated in the material before the plastic deformation region during the drawing process depends on the applied deformation coefficient and stationarity of the process. It is commonly accepted that the drawing process of round wires is one of the most well-known metal forming processes, both theoretically and technologically, not only due to its circular symmetricity and stationarity but also because many formulas allow for fairly accurate calculations of drawing forces , the coefficient of friction, residual stresses in the material, unit pressure values, etc. ,2,3,4,5.It is assumed that when an elastic–plastic body is subjected to a sufficient stress field, the plastic deformation region is generally accompanied by an elastic deformation region. When considering the wire drawing process, the location of the elastic–plastic region is usually situated inside of the die reduction angle, and to meet the criteria required for material transition to plastic state, it is necessary for it to be entirely proceeded by the elastic state. For the purposes of this analysis, four regions of strains throughout the length of the drawn material were distinguished and are presented in Two parallel hypotheses concerning theoretical analysis of the wire drawing process may be considered regarding the classical literature on the subject, i.e., the flat cross-section hypothesis, which assumes that a selected set of points constituting a plane perpendicular to the drawing axis before entering the drawing die will remain at this plane throughout the entire process and after its completion B; and thRegarding more recent scholarly sources, however, attempts have been made to determine the elastic–plastic deformation boundary inside of the die reduction angle, e.g., using the method of characteristics , using tA wide range of scientific papers concerning the generally understood process of drawing of round wires were written in the 20th and 21st centuries, both in terms of the conventional drawing process and innovative methods, such as accumulated angular drawing (AAD), which tests the influence of the linearity of dies throughout the process; drawing with the use of ultrasound; drawing with an elevated temperature of the die ; or drawMost of the available wire drawing process scientific analyses are based on the rigid-plastic body model. Such simplification does not fully take into account the phenomena occurring in the material before entering the elastic–plastic deformation region inside of the drawing die, and the assumption that the material before entering the die reduction angle is stress-free is not true, as it is obvious that elastic–plastic deformations must be preceded by elastic deformations . Among rThere are many scientific works concerning various metal forming processes and the elastic and elastic–plastic deformations occurring during the process, such as electroplasticity-assisted bending process , where tThe focus of the article in on the fundamental research of a cognitive nature and not on application aspects. The conditions of the conducted experiment (velocity of the process and exogenous factors) vary from those in the drawing operations carried out on an industrial scale. Their selection was the key element necessary to isolate the tested parameters, such as the changes in the drawing force and elastic deformation of the material before entering the drawing die, which are normally concealed by changes in the temperature of the material.Nevertheless, on the basis of the analysis of the conducted research, it is possible to propose a number of significant postulates important from a practical point of view and transfer them (after appropriate verification) to the industrial drawing process.The observed increase in the drawing force in the non-stationary phase of the drawing process may be associated with the evolution (disappearance) of the elastic deformation region situated in front of the plastic deformation region. Therefore, it may be hypothesized that other process parameters (the presented study proved the influence of the size of the deformation coefficient λ) also have an impact on the shape of the elastic deformation region, and thus on the value of the drawing force in the stationary phase. After empirical verification of this hypothesis, it is possible to search for a new concept of minimizing the force parameters of the drawing process.As is commonly known, in industrial drawing processes, the elastic back tension of a controlled value is often used in order to reduce the unit pressure of the material on the surface of the drawing die. At the same time, should the critical value be surpassed, it causes a significant increase in the drawing force and lowers the material’s deformability. This back tension is in synergy with the natural region of plastic deformation analyzed in this study. There is a prospective possibility that not only the value of the back tension, but also the elastic deformation region tested in the current work, affects the value and distribution of metal unit pressure on the drawing die’s surface, and thus the wear and tear of the tool or lubricants used. Moreover, the unit pressure and the coefficient of friction determine the temperature gradient across the material cross-section , which to some extent determines the state of residual stress in the wire after the drawing process.The studied phenomenon can also be analyzed from the point of view of defects occurring in drawn materials. The classical model of Avitzur’s central burst defects is based on a rigid-plastic body model, while in fact in the observed area of fractures, the material is probably in an elastic state. The elastic deformation region at the axis of the drawn material analyzed in this paper transforms into a new form and cracks appear, which depend not only on the kinematic incompatibility of the velocity field of the material particles, but also on the stress state. Recognition of this phenomenon gives hope for the development of a new mechanism for the emergence of central burst defects, and maybe even for the development of new technological recommendations, making it possible to eliminate the defects in question.In order to empirically determine the length of the elastic deformation region generated behind the die in the axi-symmetric wire drawing process of copper, the authors of this paper conducted laboratory tests with as low as possible drawing velocity. Copper wire rod of ETP grade and chemical composition, as specified in The starting point of the conducted research were finite element method (FEM) simulations of the wire drawing process. The strain rates and stresses present at the wire cross-section were analyzed in order to identify elastic–plastic and elastic deformation regions, which functioned as a reference for empirical research conducted in real conditions.The most important part of the research was the experimental determination of the length of the elastic deformation region and the values of strains occurring in it in order to ensure that the authors carried out wire drawing tests at a very low drawing velocity  on a testing machine  with a maximum tensile force possible to be applied equal to 50 kN registered by an independent measuring system fixed with the jaws. The assembly was rigged with mechanical gears. The strain was measured using resistance strain gauges  placed on the samples, as shown in Traditionally, the wire drawing process is considered to be stationary, and the macroscopic effect of this stationarity is the constant drawing force necessary to conduct this process. The authors made an assumption that if an elastic deformation region is being generated in the material before entering the die reduction angle, which in any way affects the strength parameters of the process, then from the moment when the material dimensions before the drawing die are smaller than the dimensions of the elastic deformation region, it is expected to have an effect on the drawing force. In order to verify this hypothesis, an analysis of the drawing process was carried out using a drawing die, with a diameter of 10 mm, using two various process parameters, i.e., with the deformation coefficient λ = 1.33  and λ = 1.1 after an additional draw of the input material to diameter of 10.5 mm prior to strain gauge measurements, which would allow us to determine the influence of the applied cross-section reduction on the length of the elastic deformations region generated before entering the die reduction angle. An initial estimation of the length of the elastic deformation region was based on the observed increases in the recorded drawing force at the end of the process. More complex analysis of the length of the elastic deformation region was carried out using precise strain gauge measurements. In each case, measurements were made in stationary conditions, i.e., when the strain gauge was placed far from the end of the material, and in non-stationary conditions, i.e., when the strain gauge was placed close enough to the end of the drawn material that the initial estimation based on the drawing force suggested that the length of the material was lower than the expected length of the elastic deformation region. Strain gauges used during analysis measured strains in longitudinal and radial directions.The last part of the analysis was the Vickers hardness tests performed at the cross-section along the axis of the samples after the drawing process using a TUKON 2500 hardness tester  with a test load accuracy of +/−1% and an accuracy of the indent’s diameter measurement of 0.02 mm. Starting 0.5 mm from the edge and with the same intervals between measurements, 19 indentations were made on the samples taken from stationary and non-stationary conditions, which allowed for their thorough analysis in comparison with the hardness of the input sample.Numerical analysis of wire drawing process with the use of the FEM method was performed in commercial SFTC DEFORM-3D software . The Newton–Raphson method was selected for the analysis with Sparse Solver, which were chosen as an optimal set resulting from previously conducted and verified numerical simulations of drawing processes. A model of the die was set as elastic, and the material properties of tungsten carbide (TC) with 19% of Co were assigned for this object. For the drawn wire, an elastic–plastic model was selected, and material properties of Cu-ETP grade copper were chosen, with a work-hardening curve representing the plastic behavior of the metal. The most important material boundary conditions of FEM simulations are presented in The initial diameter of the wire before its deformation was set to 11.55 mm, and a 10 mm diameter was chosen for the die bearing, which gave a total amount of deformation at the level of λ = 1.33 during the process. −1. When the process reaches non-stationary conditions, strain rates values increase at the axis of the wire and slightly decrease in the areas close to its surface. As the process proceeds, continuous strain rate distribution changes, and higher values can be observed outside the direct deformation region, close to the end of the wire. The shape of the elastic–plastic deformation region is similar than the aforementioned determined using the method of characteristics described by Pater and Samołyk [Results of FEM analysis in the form of effective strain rate characteristics are shown in  Samołyk  or obtai Samołyk . Compari Samołyk . In orde Samołyk .Fundamental mechanical properties of the input material were identified in the uniaxial static tensile tests and Vickers hardness tests. The stress–strain curve obtained in the tensile test with the use of resistance strain gauges enabled us to measure deformations with much higher resolution in comparison with the classic extensometers, and as is easily noticeable, there is a very small range of uniform deformations, which may be estimated at 0.2% or 2‰, as presented in After defining the basic mechanical properties of the input material, the elastic constants of the material, i.e., the Poisson’s ratio and the Young’s modulus, were determined. In order to attain these values, ultrasonic, using a supersonic flaw detector OmniScan MX2  and strain gauge using Zwick 100kN ProLine  methods were used for comparison, and in both cases similar results were obtained. σ—Accuracy of measurement; E—Young’s modulusThe key research challenge of the current paper was the empirical determination of the length of the elastic deformation region and the values of strains occurring in this region, which would make it possible to verify the aforementioned numerical simulations. In order to perform the experiment, the strain gauge method was used; however, due to the fact that strain gauges show high sensitivity to temperature variations and that the designed test stand excluded the possibility of using compensating sensors into the system, it was necessary to assess the temperature changes of the material throughout the representative drawing process. Such an assessment was made using a flat thermocouple recording the surface temperature of the rod just before entering the die and a second thermocouple placed at the end of the drawn material in a 30 mm deep hole drilled beforehand. The recorded values along with the drawing force are shown in The main part of the conducted research was the analysis of the shape and length of the elastic deformation region, which was carried out with two initial diameters, the first being 11.55 mm (deformation coefficient λ = 1.33), and the second one after the additional draw being 10.5 mm in diameter (deformation coefficient λ = 1.1). The measured velocity of the material after exiting the drawing die was approx. 0.1 mm/s, which along with the known geometry of the die and the deformation coefficients allowed us to calculate the velocity of the material before entering the die and the mean velocity of the material inside the deformation region. All the above data are presented collectively in In order to verify the hypothesis assumed in the Materials and Methods section, drawing forces for both considered deformation coefficients were measured using five separate samples obtained under the same conditions and of the same length. A detailed analysis of the drawing force values at the end of the process was performed, and in each of the tested cases, an instantaneous increase in the recorded values was observed just before the end of the drawn material. This allowed us to preliminarily estimate the length of the elastic deformation region based on the calculated drawing velocity and the recorded duration of the increase in accordance with Formula (3). Representative examples are presented in Based on the recorded data, calculated velocities of the process for each of the samples and Formula (3), the average values of the length of elastic deformation region for each deformation coefficient were estimated for a given die geometry and are presented in More complex and thorough analysis of the shape and length of the elastic deformation region generated in the material behind the elastic–plastic region was conducted using resistance strain gauges measuring longitudinal and radial strains in stationary and non-stationary conditions, as described in the Materials and Methods section. The graph presented in From data presented in The recorded data shown in The final stage of the carried-out research included Vickers hardness tests conducted on the cross-section of the samples with a 10 mm diameter after the drawing process. Samples of the cross-section were taken from stationary and non-stationary phases of the process for both applied deformation coefficients. The average values calculated from all 5 drawn samples are presented in (1)The results of the FEM simulation indicate the differentiation of the shape of the elastic deformation region in stationary conditions, where a much greater range of deformations was recorded at the wire surface in comparison with non-stationary conditions, i.e., at the end of the process, where the values of elastic deformations are greater along the material axis.(2)The presence of the elastic deformation region was indirectly verified by drawing force measurements with different drawing parameters (various deformation coefficients λ). The increase in the recorded drawing force at the end of the process was related to the potential length of the elastic deformation region. It was proven that with the increase of the deformation coefficient, the length of this region increases even several times and can reach even 28.6 mm in length.(3)It was proven that the range of the elastic deformation region in non-stationary conditions is greater along the wire axis than at the surface using strain gauge method. In addition, it was stated that the length of the elastic deformation region at the surface of the drawn wire in stationary conditions is definitely greater than at the material surface in non-stationary conditions. The tests confirmed that the applied deformation coefficient affects the lengths and shape of the considered strain regions.(4)The hardness tests of samples taken from the cross-section in non-stationary phase of the process show that the recorded values at the axis were the highest with slightly lower values at the surface of the material and definitely the lowest in between. However, considering the samples taken from stationary conditions the measured values were higher at the edge of the material than at the axis, which confirms the differences in the amount of strain depending on the process phase. The greatest differences between the stationary and non-stationary conditions are visible at the axis of the drawn material. The measured values are again influenced by the applied deformation coefficient throughout the drawing process.(5)The shape of the elastic deformation region before entering the die reduction angle was confirmed to be in compliance with the FEM simulations conducted throughout this research paper among the tested areas.Taking into consideration the obtained FEM simulations, empirical research results and conducted calculations, the following may be stated:


According to the microscopic structures of the dough samples, a decrease of the starch area may be clearly seen for the samples with high levels of GBF addition in wheat flour. The bread evaluation showed that the specific volume, porosity and elasticity increased, whereas the firmness, gumminess and chewiness decreased up to a level of 15% GBF addition in wheat flour. The color parameters L*, a* and b* of the bread samples indicated a darkening effect of GBF on the crumb and crust. From the sensory point of view, the bread up to a 15% GBF addition was well-appreciated by the panelists. According to the data obtained, GBF could be recommended for use as an improver, especially up to a level of 15% addition in the bread-making industry.Germinated bean flour (GBF) was obtained and incorporated in different levels  into dough and bread made from refined wheat flour. The incorporation of GBF into wheat flour led to a decrease of the water absorption value, dough consistency, baking strength, extensibility and improved tolerance for mixing, total gas production and α-amylase activity. Tan  Bread is one of the most consumed foods in the world. Nowadays, we are trying to diversify the bread products in order to comply with consumer demands . AdditioHowever, the incorporation of germinated legumes grains must be carefully chosen by taking into account the sensory preferences of consumers, the nutritional profile of legumes grains and their impact on bread from a technological point of view. In this sense, beans, a legume that is highlighted by a nutritional profile appreciated by nutritionists, are of particular interest for use in bread making.Fabaceae, genus Phaseolus, species vulgaris. Beans are of particular nutritional interest, because they are an important source of protein and carbohydrates [Borlotti beans belong to the family hydrates , phenolihydrates  and havehydrates , diabetehydrates , cardiovhydrates  and metahydrates . Beans ahydrates . Studieshydrates , which ihydrates . In the hydrates . Beans ahydrates ,32,35. Thydrates . The nuthydrates . Throughhydrates ; mineralhydrates , decreashydrates , trypsinhydrates , and acthydrates .However, there are few studies made on the possibility of using germinated beans in a bread-making recipe. This study presents in detail the effect of the addition of germinated bean flour on the rheological properties of dough, its microstructure and on the quality of the bread. All these aspects need to be highlighted in order to determine exactly the optimal amount of germinated bean flour in a refined wheat bread production recipe.Phaseolus vulgaris) cultivated in Suceava County, Romania. The beans were germinated, lyophilized and milled before they were used in the wheat flour. The germination was made in dark conditions at a temperature of 25 °C and a constant humidity of 80%, according to the methods reported in our previous studies [Commercial refined wheat flour was used. Wheat flour type 650 was bought from the S.C. Dizing S.R.L. company . Germinated bean flour was obtained from beans . The wheat flour was analyzed for the gluten deformation index and wet gluten content according to Romanian standard SR 90/2007. The falling number value of the wheat flour was analyzed according to the ICC 107/1 method.The raw materials and bread samples were analyzed according to ICC standard methods: moisture content (ICC 110/1), ash content (ICC 104/1), protein content (ICC 105/2) and fat content (ICC 136). To be certain that the bean flour after germination and lyophilization can be incorporated into the purchased wheat flour, this was analyzed through a microbiological point of view as follows: yeast and molds according to SR ISO 7954:2001, An Alveo Consistograph  was used to determine the dough rheological properties during mixing (Consistograph test) and extension (Alveograph test). The Consistograph test was made according to ICC 171 and AACC 54–50 approval. The Alveograph test was done according to ICC 121, AACC 54–30A and ISO 5530/4 approval at a 14% moisture basis at constant hydration. The Consistograph test at a 14% moisture basis determined the water absorption capacity (WA), maximum pressure (PrMax), tolerance to kneading (Tol), consistency of the dough after 250 s (D250) and consistency of the dough after 450 s (D450). The Alveograph test determined the maximum pressure (P), dough extensibility (L), index of swelling (G), baking strength (W) and configuration ratio of the Alveograph curve (P/L).2 volume production , maximum height of gaseous production , volume of the gas retained in the dough at the end of the test  and retention coefficient . For this purpose, dough samples were obtained by kneading 250-g mixed flours, 5-g salt and 7-g compressed yeast of the Saccharomyces cerevisiae type, according to the Consistograph water absorption value. The falling number test determined the falling number value .The dough rheological properties during fermentation used a Rheofermentometer device  according to AACC89–01.01 approval. The falling number values were made using a falling number device  according to ICC 107/1 approval. The Rheofermentometer test determined the total COG’), loss modulus (G”) and loss tangent (tan δ) were carried out at a constant stress of 15 Pa for the frequency sweep tests and during heating from 25 to 100 °C at a heating rate of 4 °C per min at a frequency of 1 Hz and a fixed strain of 0.001.The dough fundamental rheological properties were made using a HAAKE MARS 40 rheometer device  with a plate and plate system of 40 mm in diameter and a gap of 2 mm. The dough samples were mixing at the optimum Consistograph water absorption by using the Alveo Consistograph and then placed between rheometer plates and rested before analysis 5 min for relaxation. Frequency sweep tests from 1 to 20 Hz were performed at 25 °C in a range of linear viscoelasticity previously established. The storage modulus (The epifluorescence light microscopy (EFLM) images of dough with different levels of germinated bean flour addition were observed at room temperature with a Motic AE 31  equipped with catadioptric objectives LWD PH 203 (N.A. 0.4). The dough samples were prepared, and the images were obtained and analyzed according to methods reported in our previous studies ,46.Saccharomyces cerevisiae type (3%); sodium chloride (1.5%) and distilled water, according to the water absorption capacity value of wheat-germinated bean flours.The bread samples were obtained through the following steps: dosing the ingredients ; mixing them for 15 min in in a heavy duty mixer , divided in three pieces of 400 g each; leavening for fermentation for 60 min at 30 °C in a fermentation chamber  and baking the dough samples for 30 min to 220 °C in an electrical bakery convection oven with steam production, ventilation and humidification . The ingredients used in the sample production were: white wheat flour type 650; germinated bean flour ; compressed yeast of the The specific volume (seed displacement method), porosity and elasticity were determined in accordance with the SR 90: 2007 standard method.L* (darkness/brightness), a* (shade of red/green) and b* (shade of blue/yellow) were analyzed. The determination was based on the CIE Lab* color system. The field in which the absorption of electromagnetic radiation was achieved was UV-VIS.Crumb and crust colors of the bread samples were made using the Konica Minolta CR-400 colorimeter . The parameters The texture characteristics of the bread sample were determined using the texturometer device TVT-6700 . This texturometer was equipped with a 10-kg load cell. The firmness, gumminess, cohesiveness, resilience and chewiness were determined. For this purpose, the bread samples were cut into 50-mm-high slices and subjected to two compression cycles up to 20% of their initial height. For this, a cylindrical probe of 45 mm was used at a speed of 1.0 mm/s, a trigger force of 5 g and a recovery period between compressions of 15 s.To highlight the microstructure of the crumb, the Motic SMZ-140 stereo microscope  with a 20x objective was used, to a resolution of 2048 × 1536 pixels.For the sensory determinations, a 9-point hedonic scale was used, and the fallowing characteristics were evaluated: appearance, color, aroma, taste, smell, texture and global acceptability. The sensory characteristics of the bread samples were evaluated by a panel of 20 semi-trained judges.All data were expressed as the mean ± standard deviation. Statistical analysis was carried out with Statistical Package for Social Science statistical package . A one-way analysis of variance (ANOVA) with Tukey’s test was used to determine the significant differences at a 5% level.The physical–chemical characteristics of the wheat flour used as the base material were the following: 14.6% moisture, 0.66% ash content, 12.3% protein, 1.12% fat, 30.4% wet gluten and 3-mm gluten deformation index. The falling number of the wheat flour was 356 s. According to the data obtained, the wheat flour was of a very strong quality for bread making and had a low α amylase activity.Bacillus cereus, aflatoxin less than 1.4 ppb, ochratoxin 32.62 ppb and zearalenone 96.38 ppb. According to the data obtained, the germinated bean flour was from the microbiological point of view in the limits range recommended by the European Union and, therefore, may be used as ingredients in food products [The germinated bean flour presented the following physical–chemical characteristics: 10.1% moisture, 3.0% ash, 26% protein and 1.4% fat. According to the data obtained, the germinated bean flour presented a high protein content, the values being in agreement with those reported by Kassegn et al.  and Poblproducts ,50.The Consistograph data are shown in The Alveograph characteristics are shown in The Rheofermentometer characteristics and the falling number values are shown in G’), loss modulus (G”) and loss tangent (tan δ) strongly depend on the frequency. As it may be seen, the G’ was higher than G” in all frequency ranges, indicating that the elastic properties of the dough samples were more prominent than the viscous ones. Tan δ, which is the ratio of viscous and elastic components of the dough, was lower than 1 for all the analyzed samples.The frequency sweep tests are shown in G’, G” and tan δ variations. As it may be seen, the G’ and G” decreased up to a certain temperature due to protein denaturation, fallowed by an increase of their values due to the starch gelatinization process. After gelatinization, the starch hydrolysis reduced the dough consistency, which began to increase only after the α-amylase activity was inactivated.A temperature sweep of the dough mix rheological properties is shown in EFLM was used to investigate the dough samples with different levels of germinated bean flour additions . RhodamiThe bread physical characteristics are shown in p < 0.05) different when low levels of GBF were added to the wheat flour and decreased when high levels of GBF were incorporated in the bread recipe.All the bread physical characteristics  are of particular importance for the quality of the finished products, because they are related to consumer acceptability, so it is necessary to monitor the influence of the addition of GBF on their values. A GBF addition up to 15% had the effect of improving specific volume and porosity values. Improving the specific volume of bread is closely related to increasing the porosity, both of which are desirable and may be an indicator of the freshness of this food product . The valL*, a* and b* changed depending on the GBF addition level in the wheat flour. Evaluating the color parameters is important, because the color of the bread is a feature closely related to its quality [ quality . The col quality .L* (brightness) decreased due to the addition of germinated bean flour. Thus, it can be concluded that the bread samples with the addition of GBF had a lower brightness, both of the crust and of the crumbs. Regarding the parameter a*, it was observed that the control sample had the lowest value, and then, it increased with the increase of the GBF addition level. Thus, it can be concluded that the GBF addition had the effect of increasing the red value of the samples. The color data obtained also showed that the value of yellow increased due to the GBF addition in the wheat flour. The L*, a* and b* value trends were similar for both bread areas analyzed, namely the crust and crumbs.As it may be seen, the value of the parameter p < 0.05) than the control sample. The cohesiveness of the samples decreased with the increased level of GBF additions. The GBF addition also influenced the resilience parameter. The data evolution of this parameter corresponded to that of the elasticity parameter that was previously determined.The textures of the bread samples are shown in The microstructure of the breadcrumbs was characterized by pores that are formed due to the release of gas during the fermentation process of the dough and gas retention during fermentation and bread baking . A breadFrom The sensory analysis highlights consumer preferences. Performing a sensory analysis tests is necessary to remove the risk of market failure, if the products are to be placed on the market . AppearaFrom p < 0.05) in the nutritional profile were for the protein, ash and carbohydrates contents because of the GBF composition, which presented higher protein and ash contents than the wheat flour, which it partially substituted.The bread compositional analysis shown in According to Consistograph data, the GBF addition decreased the water absorption value of the mixes up to 51.4%. This may be due to the fact that, during the germination process, a part of the starch and protein contents of the bean grains were lost. Additionally, these components changed their structures, which may have had a decreasing effect on the water absorption value. During germination, the protein part may be hydrolyzed by proteases into amino acids and lower molecular peptides, and starch may be hydrolyzed by amylases to dextrins . The tolTo Alveograph, the increased of maximum pressure is up to 18.62% for the sample with the highest level of GBF addition in wheat flour. This effect may be due to the increase of viscosity of dough samples with GBF addition, which lead to a denser dough with a higher resistance. The data obtained suggested that the chemical action of GBF on dough system was less than the physical one. Even if GBF is a germinated flour which is an active one from the enzymatic point of view its enzymatic effects on dough rheological properties on Alveograph are not evident ones. The maximum pressure of wheat flour dough is higher for all samples with GBF addition compared to the control one. This may be caused by the interactions between gluten proteins and GBF fibers, which values ranged between 14% and 19% on raw bean . The preDuring the fermentation process, the maximum height of gaseous production (H’m) increased when low levels of GBF were incorporated in dough recipe and decreased when high levels of GBF were added in wheat flour. This may be due to the increase level of α-amylase activity through GBF addition in wheat flour. According to our data, the falling number value decreased up to 25.28% for GBF_25 sample compared to the control one. The FN value expressed α-amylase activity in mixes through their viscosity, which is a much lower one, as the α-amylase enzyme activity from the mixes increased . The H’mδ increased in a frequency-dependent manner. G’ and G” increased with high levels of GBF addition in wheat flour, indicating that an addition GBF improved the dough viscoelasticity. The tan δ lower than 1 for all dough samples showed a solid-like behavior of the dough samples [δ presented higher values for dough samples with GBF addition, which indicated the decreased in the ratio of elastic structure. The effects of temperature on dough rheological properties with different levels of GBF addition in wheat flour were shown on temperature sweep graphs. As it may be seen, the paste temperature of the control sample was lower than the dough samples with a GBF addition in dough recipe. The increased values of temperature led to an increased value of dough viscoelasticity due to the starch gelatinization process. With the increasing degree of starch gelatinization, the dough viscoelasticity decreased. The starch gelatinization depends on α-amylase activity increased [G’ and G” values and higher tan δ values compared to the control sample when dough mixes are in the starch gelatinization phase. In the cooking stability phase, when α-amylase acted on starch due to the fact that it was in the optimum activity range, the dough viscoelasticity decreased for all the dough samples, even more with the increased level of the α-amylase from the mixed flours [G’ and G” values and higher tan δ values compared to the control one in the cooking stability phase.The effect of GBF addition in wheat flour on dynamic dough rheological properties shown that tan  samples . Tan δ pncreased . It is wncreased . Therefod flours . TherefoThe microstructure of the control sample A–F showe2. This could explain the increase in bread volume with the addition of GBF in wheat flour [Usually, the addition of legume flours in the bread-making recipe has the effect of negatively influencing the physical characteristics of the finished products , but gerat flour ,74. Probat flour . A decreat flour . The decat flour . Therefoat flour . The resat flour ,79.A higher porosity of the bread is related to an increased volume , which ia*  can be attributed to the pigment present in the outer shell of the borlotti-type beans. The decrease in the brightness of the samples can be attributed to the increase in the amount of protein [b* indicator (yellow color) is also related to the increase in the amount of protein with the increase level of GBF addition in wheat flour [Regarding the color parameters values, studies have shown that the increase of the value of the parameter  protein . Differe protein ,90. Taki protein , then it protein . Differe protein . Phenoli protein . The incat flour . A higheat flour . Howeverat flour ,96 but, at flour . TherefoThe firmness of the bread was influenced by GBF addition in wheat flour. This may be influenced by the interaction between protein and starch, which changed due to GBF addition in wheat flour, a fact which may lead to an increase in the firmness parameter . The infRegarding the crumb microstructure, data have shown that the GBF addition has a direct influence on the size of the pores of the bread and their density. Thus, particles of different sizes of GBF addition absorb water differently, which has an influence on the crumb microstructure. Thus, it was shown that larger particles lead to a loaf of bread with a more compact structure, with smaller pores and thicker, as it may be seen in the present study . The chaRegarding the results of the sensory analysis obtained, it can be concluded that an addition of 5%, 10% and 15% germinated bean flour, respectively, led to a better assessment by the panelists compared to the sample without any GBF addition due to the nutritional composition of the beans but, also, due to the advantages of the germination process over the sensory profile of the bean grains. Studies shown that, during the germination process, take place the activation of enzymes in the bean grains , enzymesp < 0.05) compared to the control sample. The protein content significant increased (p < 0.05) with the increase level of GBF addition in wheat flour up to 44.2% for the GBF_25 sample. This increased are due to the high protein content of the GBF whose protein content is twice as high as that of wheat flour sample. A significant increased (p < 0.05) of fat and ash content also may be seen for the bread samples with different levels of GBF additions up to 23.45% and 80.39% for the GBF_25 sample. This increase is due to the high level of fat and ash from GBF sample compared to the wheat flour, which presented lower values for these parameters. The carbohydrates content significant (p < 0.05) decreased with the increase level of GBF addition in wheat flour due of the substitution of wheat flour (which is a refined one with high starch content) by GBF with a high protein content compared to wheat flour. The caloric values of the bread samples with GBF addition were lower (p < 0.05) than the control sample. However, the decreases were a small one of only 0.49% for the GBF_20 sample, for which was recorded the lowest caloric value.All the macronutrient content of the bread samples were significant different  in wheat flour led to significant changes on the dough rheological properties, microstructural properties and bread quality. The presence of active enzyme, dietary fiber and non-gluten proteins from GBF were responsible for the decrease of the dough consistency, baking strength and extensibility mainly due to the gluten network modifications shown by EFLM. The dilution effect of the gluten network would be responsible for the less-elastic structure highlighted by a tan δ value increase. A more α-amylase activity in the dough samples due to a GBF addition led to a decrease in the dynamic moduli values when the dough temperature increased, showing a less viscoelasticity fact highlighted also by a falling number values decrease. During fermentation, the maximum height of gaseous production and total CO


Precise chromosome segregation relies on kinetochores. How kinetochores are precisely assembled on centromeres through the cell cycle remains poorly understood. Centromeres in most eukaryotes are epigenetically marked by nucleosomes containing the histone H3 variant, CENP-A. Here, we demonstrated that Ccp1, an anti–CENP-A loading factor, interacts with the N terminus of CENP-T to promote the assembly of the outer kinetochore Ndc80 complex. This work further suggests that competitive exclusion between Ccp1 and Ndc80 at the N terminus of CENP-T via phosphorylation ensures precise kinetochore assembly during mitosis. In addition, CENP-T is critical for Ccp1 centromeric localization, which in turn regulates CENP-A distribution. Our results reveal a previously unrecognized mechanism underlying kinetochore assembly through the cell cycle. ccp1Δ. The CIM domain can be phosphorylated by CDK1 (cyclin-dependent kinase 1). Phosphorylation of CIM weakens its interaction with Ccp1. Consistent with this, Ccp1 dissociates from centromeres through all stages of the cell cycle in the phosphomimetic mutant of the CIM domain, whereas in the phospho-null mutant of the domain, Ccp1 associates with centromeres during mitosis. We further show that the phospho-null mutant disrupts the positioning of the Ndc80 complex during mitosis, resulting in chromosome missegregation. This work suggests that competitive exclusion between Ccp1 and Ndc80 at the N terminus of CENP-T via phosphorylation ensures precise kinetochore assembly during mitosis and uncovers a previously unrecognized mechanism underlying kinetochore assembly through the cell cycle.Kinetochores, a protein complex assembled on centromeres, mediate chromosome segregation. In most eukaryotes, centromeres are epigenetically specified by the histone H3 variant CENP-A. CENP-T, an inner kinetochore protein, serves as a platform for the assembly of the outer kinetochore Ndc80 complex during mitosis. How CENP-T is regulated through the cell cycle remains unclear. Ccp1 (counteracter of CENP-A loading protein 1) associates with centromeres during interphase but delocalizes from centromeres during mitosis. Here, we demonstrated that Ccp1 directly interacts with CENP-T. CENP-T is important for the association of Ccp1 with centromeres, whereas CENP-T centromeric localization depends on Mis16, a homolog of human RbAp48/46. We identified a Ccp1-interaction motif (CIM) at the N terminus of CENP-T, which is adjacent to the Ndc80 receptor motif. The CIM domain is required for Ccp1 centromeric localization, and the CIM domain–deleted mutant phenocopies  The precise inheritance of genetic information relies on the accurate segregation of chromosomes in mitosis and meiosis. Kinetochores are large protein complexes assembled on centromeres and play a crucial role in chromosome segregation. The kinetochore links the chromosome to microtubule polymers, drives the movement of chromosomes, and ensures correct microtubule–kinetochores attachment –3. The kThe kinetochore comprises an outer region and an inner region. The outer kinetochore interacts with microtubules and is assembled on the platform of the inner kinetochore. The inner kinetochore consists of a complex of 14 to 16 subunits known as the constitutive centromere–associated network (CCAN) that is directly built on centromeric chromatin 4–9. Most 1012CENP-T, an integral component of CCAN, is also a histone fold–containing protein. CENP-T provides a platform for the assembly of the Ndc80 complex (Ndc80C), an essential outer kinetochore component, during mitosis , 15–18. 1521CENP-T has been shown to interact with three other histone fold–containing proteins, CENP-W, CENP-S, and CENP-X, to form the heterotetrameric nucleosome-like structure in vitro , 26. TheSchizosaccharomyces pombe contains large regional centromeres and is considered to be a model system for centromere study. The CENP-A homolog, Cnp1, is enriched in centromere cores, which are surrounded by pericentromeric heterochromatin  family protein, has been shown to play an important role in antagonizing the loading of CENP-A in fission yeast . Ccp1 foCnp20 in fission yeast. We further identified a conserved Ccp1-interaction motif (CIM) at the N terminus of CENP-TCnp20, which is adjacent to the Ndc80 receptor motif. We demonstrated that CIM is important for Ccp1 localization. Furthermore, our data suggested that CDK1-mediated phosphorylation of the CIM motif at the onset of mitosis dissociates Ccp1 from CENP-TCnp20, allowing proper positioning of Ndc80C. Ccp1 associates with centromeres during mitosis in the phospho-null mutant of the CIM domain, leading to mislocalization of Ndc80C and severe chromosome segregation defects. Our study uncovers a previously unrecognized mechanism regulating kinetochore organization in regional centromeres.Here using mass spectrometry, we found that Ccp1 interacts directly with CENP-TCnp1 and CENP-TCnp20, associated with Ccp1 . Our previous TAP purification of CENP-ACnp1 also revealed that CENP-ACnp1 interacts with Ccp1 (Cnp20 and Ccp1 was further confirmed by coimmunoprecipitation (co-IP)  residues. A conserved Ndc80 receptor motif from aa 70 to aa 87 was also identified in the N terminus  B. The C terminus .Cnp20 in fission yeast is essential for cell growth  mutant of CENP-TCnp20, cnp20-9, by random mutagenesis. The cnp20-9 mutant grows normally at room temperature, but does not survive at 36 °C . Consistent with the key role of CENP-TCnp20 in the assembly of the Ndc80 complex, we found that the association of Ndc80-GFP with centromeres is lost in cnp20-9 at the restrictive temperature , whereas the localization of GFP-Ccp1 at centromeres only has a mild reduction in the CENP-A ts mutant, cnp1-1 . We next checked the distribution of CENP-TCnp20-GFP in ccp1Δ and found that its centromere localization was unaffected in the mutant . In addition, we found that CENP-ACnp1-GFP partially reduced its centromere localization in cnp20-9 at the restrictive temperature . We have previously shown that Ccp1 dissociates from centromeres in the mis16 mutant . It is thus likely that Mis16 may regulate Ccp1 localization via CENP-TCnp20. It has been shown that Mis16 centromeric localization is not affected by the cnp1-1 mutant at the restrictive temperature , indicating that the dimeric structure is vital for its interaction with CENP-TCnp20.To further confirm that Ccp1 interacts with CENP-TP-TCnp20 A. Our prP-TCnp20 . The yeaCnp20 with Ccp1, we constructed the N-terminal and C-terminal domains of CENP-TCnp20: Cnp20-N (aa 1 to 368) and Cnp20-C (aa 369 to 479), respectively. Our yeast two-hybrid assays indicated that Ccp1 interacts with the N-terminal domain but not with the C terminus . To narrow down the Ccp1-interacting region of CENP-TCnp20, we constructed a series of N-terminal fragments of CENP-TCnp20, including aa 1 to 88, aa 89 to 196, and aa 197 to 368, and conducted yeast two-hybrid assays using Ccp1 as a bait. We found that Ccp1 associates with aa 1 to 88 . Our results indicate that the first 55 amino acids of CENP-TCnp20 are the minimal interaction domain with Ccp1, which we named the Ccp1-interacting motif (CIM). The CIM domain lies close but does not overlap with the Ndc80 receptor motif .In order to identify the interacting domain of CENP-T 1 to 88 B. Furtheor motif D. Our se1-56 and purified full-length Ccp1. We found that Cnp201-56 was able to pull down Ccp1 in vitro, indicating that the CIM domain indeed directly interacts with Ccp1 .To further investigate how the CIM domain physically interacts with Ccp1, we performed in vitro pull-down assays using a peptide derived from Cnp20ith Ccp1 E. We alsCnp20 in Ccp1 localization, a CIM domain–deleted version of cnp20+ (cnp20-Δ1–55) was constructed under the control of its endogenous promoter and inserted at the ade6+ locus in the cnp20-9 mutant background. As a control, the full-length cnp20+ and the first 88 amino acid–deleted cnp20 (cnp20-Δ1–88) were also inserted in ade6+ in the same manner. The full-length cnp20+ can completely rescue the growth defect of cnp20-9 at the restrictive temperature. On the other hand, cells carrying cnp20-Δ1–88 grow well at room temperature, but not at 36 °C, the same as the cnp20-9 mutant alone  by CRISPR-Cas9. As a control, we also created a strain in which the first 30 amino acids in CENP-TCnp20 (cnp20-Δ1–30) were deleted , though the GFP signal is weaker . We concluded that the CIM domain is required for Ccp1 centromeric localization. Our data suggest that CENP-T regulates the recruitments of Ccp1 to centromeres via its CIM domain.We next deleted the CIM domain of the endogenous  deleted B. To dets weaker C and D. ccp1Δ mutant is sensitive to the microtubule destabilizing drug, thiabendazole (TBZ) due to chromosome segregation defects . Reporter genes inserted within the centromeric core region are silenced stochastically. The silencing state correlates with the CENP-A nucleosome occupancy in centromeres , suggesting that the level of CENP-Acnp1 in centromeres substantially increase in these cells. Together, these results indicate that cnp20-ΔCIM phenocopies the ccp1Δ mutant, consistent with the idea that CENP-TCnp20 recruits Ccp1 through its CIM domain to centromeres during interphase.The epigenetic state of tromeres , 43. Delre cores . To invee region . We founntly red G and H. d  at the endogenous sites by CRISPR-Cas9  and examined the distribution pattern of GFP-Ccp1 throughout the cell cycle in the mutant background. As a control, we also created a mutant in which 6 serine and threonine residues within CIM were replaced by alanine (cnp20-6A) . These data indicate that dephosphorylation of the CIM of CENP-TCnp20 results in a tight association of Ccp1 with centromeres. Consistent with this idea, our yeast two-hybrid assays demonstrated that, unlike Cnp20-14D, Cnp20-14A showed a strong interaction with Ccp1  A. GFP-Ccll cycle B and C. ith Ccp1 F. Togethcnp20-14A on chromosome segregation, we analyzed the mutant using TBZ growth assays. We found that cnp20-14A is highly sensitive to TBZ, even more strongly than the cnp20-ΔCIM mutant , indicating that cnp20-14A leads to severe chromosome segregation defects. We further utilized time-lapse microscopy to examine chromosome segregation in cnp20-14A. The mutant cells expressing the GFP-tagged Spc25, a subunit of Ndc80C and the Sad1-mCherry (spindle pole body [SPB] marker), were analyzed. After the onset of mitosis, kinetochores oscillate between the two SPBs and segregate toward the SPBs during anaphase. Wild-type cells complete the segregation within 14 min ; actually unlike the cnp20-Δ1–55 mutant, cnp20-14D is even more resistant to TBZ than WT .These data suggest that dissociation of Ccp1 from centromeres by phosphorylation of CIM at the onset of mitosis promotes the interaction between CENP-T and Ndc80C, leading to the proper assembly of the outer kinetochores. In fact, we found that  than WT D. It is Cnp20 at the onset of mitosis. To test this, we performed the in vitro kinase assays using human recombinant CDK1-cyclin B and a synthesized peptide derived from Cnp201-55. The highly conserved human CDK1 is known to be capable of functionally replacing the CDK1/Cdc2 in fission yeast  to detect changes in phosphorylation status. We observed migrating bands slower than their nonphosphorylated counterpart from the in vitro assay using Cnp201-55. However, no slow migrating bands were identified in the assay with the Cnp201-55-14A peptide . These data support the idea that the CIM domain is phosphorylated by CDK1. Furthermore, consistent with the functional studies described above, our in vitro binding assay showed that phosphorylation of the CIM domain by CDK1 results in reduced interaction between the CIM domain and Ccp1 .CDK1/Cdc2 in fission yeast is essential for viability. The temperature-sensitive alleles of perature , preventCnp20 with centromere depends on Mis16 . It is possible that Mis16 recruits Ccp1 to centromeres through CENP-TCnp20.Assembly of the outer kinetochore during mitosis is tightly regulated. But the molecular mechanism for this regulation, especially in regional centromeres, remains poorly understood. CENP-T, a key inner kinetochore component, is known to promote the recruitment of Ndc80C, a complex essential for kinetochore–microtubule interactions during mitosis , 15–20. 15cnp20-14A cells displayed mislocalized Scp25-GFP signal and the mutant cells are viable, suggesting that alternative mechanisms are also used in recruiting the Ndc80 complex during mitosis.We further identified the first 55 amino acids at the N terminus of CENP-T as the CIM. Notably, the CIM domain is situated next to the Ndc80 receptor motif. We found that the CIM domain can be phosphorylated. Phosphorylation of the CIM domain disrupts the interaction between CENP-T and Ccp1, leading to dissociation of Ccp1 from centromeres. On the other hand, dephosphorylation of the domain results in tight interaction of CENP-T with Ccp1 and consequently the association of Ccp1 with centromeres during mitosis. We further showed that dephosphorylation of CIM disrupts the interaction between CENP-T and Ndc80C. As a result, the phospho-null mutant of CIM exhibits mislocalization of the Ndc80 complex during mitosis and severe chromosome segregation defects. Our data suggest that occupancy of the Ccp1 homodimer at the CIM domain during interphase prevents the interaction of the adjacent Ndc80 receptor motif of CENP-T with Ndc80C. Removing Ccp1 from CENP-T makes the Ndc80 receptor motif accessible during mitosis, which may allow Ndc80C to interact with CENP-T to be properly assembled. Nevertheless, we found that only ∼20% of the S. pombe, M. musculus, and H. sapiens . The Ndc80 receptor motif which is next to the CIM domain is considered as a conserved domain for the interaction between the Ndc80 complex and CENP-T by previous studies . It is likely that both the Ndc80 receptor motif and the Ndc80 complex coevolve during evolution to maintain the interaction. Similar to the Ndc80 receptor motif, we believe that the CIM domain is also functionally conserved.We found that multiple amino acids in the CIM domains are conserved across different species, especially in  studies , 21–24. tudies 21, 21–24. SI Appendix, Fig. S9). We believe that the truncated BD-Cnp20-N is likely self-activating. On the other hand, the self-activation activity of full-length BD-Cnp20 may be inhibited by the configuration of the full-length CENP-TCnp20 protein.We used both the yeast two-hybrid system and in vitro pull-down assays to confirm the interaction between the CIM domain and Ccp1. Nevertheless, we noted that our yeast two-hybrid results are not completely consistent with our in vitro pull-down data. We think it is likely due to the fact that two assays were performed in two very different systems. Unlike the yeast two-hybrid system, the pull-down assays were performed in the nonstringent conditions with much higher protein levels. Also, the truncated BD-Cnp20-N in our yeast two-hybrid assays showed interactions with both AD (empty vector) and AD-Ccp1-4A (the homodimer mutant), but the full-length BD-Cnp20 did not show any interactions with AD or AD-Ccp1-4A (Our in vitro kinase assays further demonstrated that the CIM domain is phosphorylated by CDK1. This was further supported by the fact that constitutive overexpression of Cdc13/Cyclin B results in dissociation of Ccp1 from centromeres. Presumably, overexpression of Cdc13 causes CENP-T to be phosphorylated during interphase, which disrupts the interaction between the CIM domain of CENP-T and Ccp1, a phenomenon that is similarly observed in the Cnp20-14D mutant. Our in vitro kinase assays indicated that the domain contains multiple phosphorylation sites. Consistent with this, our point mutation analysis revealed that phosphorylation of all of the 14 potential phosphorylation sites within CIM is required for the dissociation of Ccp1 from centromeres during mitosis. It has been shown that CDK1 in multiple species specifically phosphorylates the N terminus of CENP-T, especially the Ndc80 receptor motif, during mitosis , 21–24. 21Our study uncovers a previously unrecognized mechanism behind the regulation of kinetochore organization in regional centromeres. This work also provides a molecular explanation for the observed Ccp1 dissociation from centromeres during mitosis. Based on our data, we propose the following model: The CIM domain of CENP-T is phosphorylated at the onset of mitosis by CDK1. The phosphorylation expels Ccp1 from CENP-T, which allows the Ndc80 receptor motif to interact with Ndc80C. At the end of mitosis, the CIM domain is dephosphorylated by an unknown phosphatase. This leads to the reassociation of Ccp1 with the CIM domain F, which 47S. cerevisiae contains a single nucleosome (ccp1Δ mutant, the CIM-deleted mutant is sensitive to TBZ and also exhibited increased silencing in centromeres, indicative of an increased amount of CENP-A nucleosomes in the region. We propose that CENP-T recruits Ccp1 to centromeres at the end of mitosis to remove excessive CENP-A nucleosomes and allow room for the incorporation of sufficient CENP-T–containing particles to centromeres.Whereas the point centromere in cleosome , 54, CENcleosome , 56. Thecleosome , 27. CENcleosome , 26. Howcleosome . In thiscnp20 was created by marker reconstitution mutagenesis (SI Appendix, SI Materials and Methods. Fission yeast strains used in this study are listed in SI Appendix, Table S1.Standard media and genetic analysis for fission yeast were used . The temagenesis . DetailsSupplementary FileSupplementary File