Metallocarboxypeptidase D (CPD) is a membrane-bound component of the trans-Golgi network that cycles to the cell surface through exocytic and endocytic pathways. Unlike other members of the metallocarboxypeptidase family, CPD is a multicatalytic enzyme with three carboxypeptidase-like domains, although only the first two domains are predicted to be enzymatically active. To investigate the enzymatic properties of each domain in human CPD, a critical active site Glu in domain I and/or II was mutated to Gln and the protein expressed, purified, and assayed with a wide variety of peptide substrates. CPD with all three domains intact displays >50% activity from pH 5.0 to 7.5 with a maximum at pH 6.5, as does CPD with mutation of domain I. In contrast, the domain II mutant displayed >50% activity from pH 6.5–7.5. CPD with mutations in both domains I and II was completely inactive towards all substrates and at all pH values. A quantitative peptidomics approach was used to compare the activities of CPD domains I and II towards a large number of peptides. CPD cleaved C-terminal Lys or Arg from a subset of the peptides. Most of the identified substrates of domain I contained C-terminal Arg, whereas comparable numbers of Lys- and Arg-containing peptides were substrates of domain II. We also report that some peptides with C-terminal basic residues were not cleaved by either domain I or II, showing the importance of the P1 position for CPD activity. Finally, the preference of domain I for C-terminal Arg was validated through molecular docking experiments. Together with the differences in pH optima, the different substrate specificities of CPD domains I and II allow the enzyme to perform distinct functions in the various locations within the cell. Metallocarboxypeptidases (MCPs) are zinc-dependent enzymes that cleave single amino acids from the C termini of peptides and proteins ,2. The fDrosophila contain three of these carboxypeptidase/transthyretin-like domains  [Carboxypeptidase D (CPD) belongs to the M14B subfamily of MCPs and was originally discovered in duck hepatocytes as a 180-kDa membrane-bound glycoprotein (named gp180) that has the ability to bind duck hepatitis B virus particles ,10. Bovi  ,15,16.Fig 1). CPD is primarily localized in the trans-Golgi network (TGN), and cycles between the TGN and the cell surface [The critical catalytic residues, metal-binding residues, and substrate-binding residues found in all active members of the M14 gene family are conserved in CPD domains I and II but not in domain III. CPD also contains a transmembrane segment of about 20 residues and a C-terminal cytosolic tail of 60 residues  domains of CPD are localized to the lumen of the TGN, exocytic vesicles, and endocytic vesicles, and are exposed to the extracellular environment when the enzyme is transiently present on the cell surface . Based obstrates ,22. Duckl lysine ,22. By cl lysine ,16,22. H3 mutant ,24. All CPD has been implicated in several human diseases. In one study, CPD was found to be downregulated in CD14 positive cells isolated from patients with lupus erythematosus . In a stFig 1). We characterized the enzymatic properties of the full active human CPD and the individual catalytically active domains, using a wide range of substrates and quantitative peptidomics approaches. We found that human CPD domain III is inactive towards all peptides examined, while domains I and II have complementary activities that provide for a wider ability to cleave peptides/proteins with basic C-terminal residues within the secretory pathway, on the cell surface, and within the endocytic pathway. However, not all peptides with C-terminal basic residues were efficiently cleaved by domains I and/or II, indicating the importance of other residues within the peptide. Finally, we modeled the structure of all three domains of hCPD and performed molecular docking experiments with a set of peptide substrates. Our thorough characterization of the substrate specificities of the individual domains of human CPD suggests the functional specialization of each CPD domain to distinct subcellular environments.Despite the numerous studies finding CPD to be associated with human disease, the enzymatic properties of human CPD have not been studied in detail. The objective of the present study was to investigate the properties of human CPD and functionally characterize its individual catalytic domains. For this, we generated catalytically inactive single point mutants for the individual active domains I and II  as well as a double mutant  were grown in FreeStyle 293 Expression Medium  in flasks on a rotary shaker (120 rpm) at 37°C, 8% CO2 and 70% humidity.HEK293T cells (ATCC CRL-3216) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with GlutaMAX and 10% (v/v) fetal bovine serum  at 37°C, 10% COTM-7 expression vector (Merck Millipore) encoding for mouse IgM secretion signal sequence and an N-terminal Strep-Tag II fusion protein. CPD E270Q mutants (according to bovine CPA1 numbering) named here as E350Q, E762Q (for single mutants) and E350Q/E762Q (for the double mutant) were generated by PCR-driven overlap extension [6 cells/ml, grown in FreeStyle 293 Expression Medium for 24 h and then transfected with 1 μg DNA per ml of culture for 7 days. For protein purification, 500 ml of the culture supernatant were equilibrated with 30% ammonium sulphate, centrifuged, filtered through 0.2 μm filters and bound to a 15 ml hydrophobic chromatography column . Protein elution was carried out with a decreasing gradient of ammonium sulphate (30 to 0%) in a 100 mM Tris-HCl, pH 7.5 buffer. The eluted fractions containing CPD  were pooled and loaded onto a 5 ml Strep-Tactin affinity column (IBA GmbH) equilibrated with 5 column volumes (cv) binding buffer , washed with 2 cv of binding buffer and eluted with 8 cv of the elution buffer . Eluted fractions were analyzed by SDS-PAGE, and the purest fractions were pooled and loaded onto a HiLoad Superdex 75 26/60 column  previously equilibrated with a 50 mM Tris-HCl, pH 7.5, 150 mM NaCl buffer. The purified proteins were flash frozen at a concentration of approximately 0.5 mg/ml and stored at -80°C.Human carboxypeptidase D (residues 32–1298) was cloned into the pTriExxtension . BrieflyHEK293T cells were seeded in 24 150-mm cell culture plates and after growing up to 70% confluence, cells were treated with fresh media containing 0.5 μM bortezomib for 1 h. Following incubation, cells were washed three times with cold Dulbecco’s phosphate-buffered saline  supplemented with 0.5 μM bortezomib, immediately scraped and centrifuged at 8000xg for 5 min. For peptide extraction, the cell pellet was resuspended in 1 ml of 80°C water and the mixture was incubated for 20 min in an 80°C water bath. Samples were cooled, transferred to 2 ml low retention microfuge tubes and centrifuged at 13,000xg for 20 min. Soluble fractions containing HEK293T cell peptides were stored at -80°C overnight. Samples were centrifuged again and the supernatant of each tube was collected and concentrated in a vacuum centrifuge to a volume of 1.5 ml. Finally, samples were cooled and acidified with 0.1 M HCl to get a final concentration of 10 mM HCl. After 15 minutes of incubation, samples were centrifuged at 13000xg for 40 min at 4°C and the supernatants stored at -80°C until labelling.Tryptic peptide libraries were generated by digesting five different proteins with trypsin. Trypsin is an endoprotease that cleaves C-terminal to arginine or lysine residues, producing peptides containing basic residues on their C-termini . The proS1 Fig) for 60 min at 37°C in 100 μl of 100 mM Tris-acetate (pH 6.4) containing 100 mM NaCl. Reactions were stopped by adding 50 μl of 500 mM HCl and 1 ml of chloroform. Tubes were mixed and centrifuged for 2 min at 300xg. After centrifugation, 0.5 ml of the chloroform phase was transferred to new tubes and dried overnight at 25°C in a fume hood. Dried samples were resuspended with 200 μl of PBS containing 0.1% of Triton X-100. The amount of product generated was determined by measuring the fluorescence emission of samples at 495 nm upon excitation at 350 nm using a 96-well plate spectrofluorometer. For kinetic analysis, purified enzymes . In all cases, a maximum of 20% of the substrate was hydrolyzed. Kinetic parameters were determined by fitting the obtained data for each enzyme to the Michaelis-Menten equation (y = (Vmax x X) /(Km + X)) using GraphPad Prism software [Vmax is the maximum enzyme velocity and Km is the Michaelis-Menten constant. The pH optimum of purified enzymes was determined with 0.2 mM dansyl-Phe-Ala-Arg in 100 mM Tris-acetate, 150 mM NaCl buffer at the indicated pH.Carboxypeptidase activity was assayed with the fluorescent substrates dansyl-Phe-Ala-Arg, dansyl-Phe-Gly-Arg or dansyl-Phe-Pro-Arg synthesized as described . The asssoftware , where X13C (D12). The labels were dissolved in DMSO to a concentration of 0.4 mg/μl and 5 mg of label was used per sample. At the start of the experiment, the pH of the sample was adjusted to 9.5 with 1M NaOH. Labeling was performed over 8 rounds; 1.6 μl of the label was added to the extract every 20 min. The pH was measured between each round and if necessary, brought back to 9.5, only for the first five rounds. After labeling, 30 μl of 2.5 M glycine was added to quench any unreacted label. Labeled samples for a single experiment were pooled, filtered through a10-kDa centrifugal filter device and 30 μl of 2M hydroxylamine was added to hydrolyze any labeled tyrosines. This was done to ensure that only N-terminal amines and lysine side-chain amines of peptides are TMAB-labeled and not tyrosines. Samples were desalted through C-18 spin columns (Thermo-Scientific) by following the manufacturer’s instructions. Peptides were eluted using 160 μl of 0.5% TFA and 70% acetonitrile, freeze-dried in a vacuum centrifuge, and subjected to liquid chromatography and mass spectrometry as described [Quantitative peptidomics experiments were performed as described previously ,35, withescribed ,35. IdenThe amino acid sequences of human CPD, as well as human CPM and bovine CPA were obtained from the UniProt database . A structure-based sequence alignment between CPD domains I, II and III was generated using PROMALS3D multiple sequence and structure alignment server .i.e., 38% SI with hCPD domain I; 79% SI with domain II and 31% SI with domain III) . Final models contained the catalytic zinc ion, which was transferred from the 1H8L structure. Electrostatic potential calculations for the three domains, including the zinc ion, were carried out using APBS (Adaptive Poisson-Boltzmann Solver) and the corresponding molecular surface represented with PyMOL [Structural models of the different human CPD domains were constructed using the Prime Homology Modeling Workflow implemented in Schrödinger and further subjected to an energy-based refinement to improve their quality ,38. The th PyMOL .The substrate binding sites of the domains were further characterized with SiteMap  by usingThe peptide docking was performed using the standard-precision docking protocol for peptides from Glide . Ten milPichia pastoris was previously used to produce CPD domain I and CPD domain II, as well as other members of the M14 MCP family [Fig 1). Residues 32–1298 of CPD encode the three carboxypeptidase-like domains and lack the C-terminal transmembrane domain and cytosolic tail. The CPD-pTrieX-7 construct was transfected into mammalian HEK293F cells for transient expression, using polyethylenimine as transfection reagent. The secreted recombinant protein showed a major band of 170 kDa by western blot analysis using an anti-Strep-tag II antibody, consistent with the predicted molecular weight for rhCPD (data not shown). Maximum levels of soluble rhCPD in the medium were detected 7 days post-transfection.Several expression systems were explored to produce active recombinant CPD. P family ,42–44. HP family ,22. HoweP family ,46. For Fig 2A) to obtain a high-purity protein product . To characterize the substrate specificity of the individual catalytic domains we generated constructs with single point mutations in domains I or II as well as a double mutant, named here as E350Q, E762Q and E350Q/E762Q, respectively . All mutants were expressed and purified following the protocol optimized for rhCPD . In gel filtration chromatography, all mutants eluted in a single peak with comparable apparent masses (data not shown).A variety of conditions were tested to purify rhCPD from the medium, and optimal results were obtained following a three-step chromatography protocol. The first chromatographic step was a hydrophobic interaction chromatography. The eluates containing the rhCPD protein from the first purification step were pooled and purified over an anti-Strep-tag affinity resin and, further fractionated on a size exclusion column . The construct E350Q shows optimal activity at pH 6.5, with activity >50% of the maximum over the range 5.0–7.5, while the construct E762Q has optimal activity at pH 7.0 and with >50% maximal activity over the range 6.5–7.5 . The double mutant E350Q/E762Q had no detectable enzyme activity at any of the pH values examined .The influence of pH on rhCPD and rhCPD single point mutants were examined using the fluorescent substrate dansyl-Phe-Ala-Arg. The pH optimum of the human protein with both domains I and II active is 6.5, with >50% maximal activity from 5.0–7.5 ; at enzyme concentrations where 70% of dansyl-Phe-Ala-Arg was cleaved, less than 5% of dansyl-Phe-Gly-Arg was hydrolyzed by the enzyme. No activity was detected towards the substrate with a Pro residue in penultimate position . We determined the kinetic parameters for dansyl-Phe-Ala-Arg for the full active rhCPD and the single point mutants E350Q and E762Q  is generally comparable to the kcat of the E762Q mutant with domain I active (8.5 ± 0.5 s-1) and both are smaller than the kcat for rhCPD with both active domains (12.5 ± 0.5 s-1). The lowest Km value was obtained for the full active rhCPD, which showed a Km of 153 ± 16 μM, while single point mutants E762Q and E350Q showed higher Km values of 319 ± 37 μM and 844 ± 139 μM, respectively. The kcat/Km value for rhCPD was approximately 3 times and 10 times higher in comparison with values obtained for the E762Q and E350Q single point mutants, respectively (Table 1).The enzymatic activities of rhCPD were tested with three fluorescent synthetic substrates. To compare substrates, different amounts of the purified enzyme were incubated with 200 μM of each dansylated peptide and the relative amount of product determined. Among the substrates evaluated, dansyl-Phe-Ala-Arg was the peptide most rapidly cleaved by rhCPD .The results obtained using synthetic substrates are useful, but is limited by the availability of substrates. To gain more information about the substrate specificity with a wide range of peptides, we used a quantitative peptidomic approach to address the substrate specificity of human CPD and unravel the contributions of each individual active domain. Quantitative peptidomics allows the study of proteolytic activity by analysing a large number of peptide substrates in a single experiment, thereby providing a more complete understanding of MCP substrate specificities ,35,47,48Fig 4B and S1 Table). Some peptides were extensively cleaved, showing a complete or almost complete decrease in the peak intensity (≥60%) in the sample incubated with the highest concentration of enzyme  and a partial decrease in the peak intensity of the sample incubated with a lower concentration of rhCPD ; these are considered as good substrates of rhCPD . In addition, some peptides were only partially cleaved (decrease in the peak intensity ≥20% and <60%), exhibiting a small decrease in intensity with the highest concentration of enzyme assayed, and no major decrease in the peak intensity with the concentration of 10 nM; these are considered as weak substrates of rhCPD . Some peptides showed an increase in peak intensities that correlated with the amount of rhCPD. In all cases, the peptides that increased upon incubation with CPD lacked C-terminal basic amino acids and are products of rhCPD cleavage . Based on the location of the peptide within the protein and the downstream sequence, we deduced the residue that was cleaved (see cleaved aa in S3 Table).Over 55 peptides were identified by tandem mass spectrometry (MS/MS) and/or close matches with the theoretical mass of the peptides generated with trypsin. Some of these peptides exhibited a peak set with roughly equal peak heights, revealing that these peptides were not substrates or products of rhCPD under the experimental conditions used . Further, the influence of the penultimate (P1) residue on rhCPD substrates and non-substrates was examined . For this analysis, only peptides with permissive C-terminal residues (Lys or Arg) were considered. Good substrates of rhCPD contained Ala, Ser, Phe, Leu, Lys, Thr, or Glu in the P1 position . From these analyses, three products of rhCPD were detected; these peptides were generated by cleavage of peptides containing Arg or Lys at the C-termini and Ala, Phe or Thr in the P1 position (S3 Table). Finally, it is noteworthy to mention that no peptide with Pro, Asp, or Ile in the P1 position ws identified as a rhCPD substrate. Some of these peptides contained C-terminal basic residues (including Arg and Lys), which suggest an important negative influence of these residues on the CPD activity when they are found in P1 position.The majority of peptides with C-terminal (P1’) Lys were either good or weak substrates of rhCPD, while fewer of the peptides with C-terminal Arg were good substrates and the majority of these peptides were non-substrates  of purified rhCPD, of single point mutants E350Q and E762Q, and the double mutant E350Q/E762Q were incubated with the same tryptic peptide library used for the characterization of rhCPD described above. After incubation with the enzyme, the individual reactions were differentially labeled with isotopic TMAB tags, combined and subjected to LC-MS analysis .To gain insight into the substrate specificity of the domains I and II of human CPD, we repeated the quantitative peptidomics and compared rhCPD with the various mutant forms in a single LC/MS run. A schematic of one experiment and labeling strategy is shown in S4 Table). Some peptides were cleaved by the enzyme with both domains I and II active, showing a complete or almost complete decrease in the peak intensity upon incubation with rhCPD; these are considered as substrates (which include good and weak substrates) of rhCPD . Among them, some peptides showed a complete or almost complete decrease in the peak intensity after incubation with either single point mutant E350Q or E762Q ; these are considered as substrates of both domains. However, some peptides substrates of rhCPD revealed a complete or almost complete decrease in the peak intensity upon incubation with the single point mutant E350Q and a slight or no decrease in the peak intensity upon incubation with E762Q; these are considered as preferential substrates of domain II . Another group of peptides revealed a moderate decrease in the peak intensity upon incubation with the single point mutant E762Q and a slight or no decrease in the peak intensity upon incubation with the E350Q mutant; these are considered as preferential substrates of domain I .Over 48 peptides were identified through tandem mass spectrometry (MS/MS) and/or by close matching with the theoretical mass of the peptides generated with trypsin. After the analysis, the results revealed some peptides which showed roughly equal peak heights; these peptides were considered to be neither substrates nor products under the assay conditions used . In contrast, substrates of domain II were more evenly divided between C-terminal Lys versus Arg . Peptides with C-terminal Arg and Lys were also identified as non-substrates of the mutant CPD with single catalytic domains. For these peptides, the identity of the penultimate (P1) residue was further analyzed . Good substrates of the CPD domain I contained only Lys or Phe amino acids in this position. Weak substrates of CPD domain I contained Ala, Thr, Asp, Val, Met, Asn, Gln or Gly in the P1 position . Good substrates of the CPD domain II contained Phe, Lys, Ala or Thr in the P1 position. Weak substrates of the CPD domain II contained Asp, Thr, Ala, Leu, Ser, Met, Val, Asn, Gln or Gly in this position . Again, no peptides with Pro, Ile, or Glu in the P1 position were identified as substrates of either domain under the experimental conditions performed.Analysis of the P1’ residue of substrates of domain I showed a predominance of Arg, with 11 substrates containing C-terminal Arg and only 2 substrates containing C-terminal Lys (S5 Table). Three of these peptides required removal of C-terminal Arg, and these were also products of domain I. The other three peptides required removal of C-terminal Lys, and two of these peptides were only products of domain II and not domain I. The peptide LRVDPVNF was greatly elevated upon incubation with CPD with active domain II, and slightly elevated by incubation with CPD with active domain I (S5 Table). Because the corresponding tryptic peptide contains a Lys on the C-terminus, this finding shows that CPD domain I is capable of cleaving C-terminal Lys, although not as efficiently as CPD domain II cleaves C-terminal Lys from this peptide.Six peptides were identified as products of rhCPD and the domain II only active mutant; these resulted from cleavage of Arg or Lys from tryptic peptides, with Ser, Phe or Val at the P1 position of the cleavage site .The tryptic peptide library provided a large number of peptides with C-terminal basic residues, but few peptides without C-terminal basic residues. To determine if rhCPD was able to cleave any peptide lacking C-terminal basic residues, we performed a third quantitative peptidomics study testing peptides extracted from HEK293T cells treated with bortezomib, a proteasome inhibitor that causes an increase in the levels of many intracellular peptides . After iS6 Table). Of the peptides that contained C-terminal basic residues, some were extensively cleaved, showing a complete or almost complete decrease in the peak intensity upon incubation with the highest concentration of enzyme of 100 nM and a partial decrease in the peak intensity with a lower concentration of 10 nM; these are considered as good substrates of rhCPD . Some peptides were only partially cleaved, exhibiting a small decrease in intensity with the highest concentration of enzyme assayed and no or slight decrease in the peak intensity with the concentration of 10 nM; these are considered as weak substrates of rhCPD . A small number of peptides showed an increase in the peak intensities related with increasing amounts of rhCPD; these are considered as products resultant after the rhCPD cleavage . As expected, analysis of the P1’ residue showed again an exclusive preference for basic residues at this position .More than 60 peptides were identified through tandem mass spectrometry (MS/MS). Unlike the tryptic peptide library, in the HEK293T derived library the majority of peptides identified did not contain C-terminal basic residues. Nevertheless, none of these non-basic ending peptides was affected by the treatment with rhCPD, and thus exhibited a peak set with roughly equal peak heights  shows the electrostatic potential molecular surfaces of the catalytic sites of CPD domains I, II and III, highlighting the differences in electrostatic properties for the three domains. While domain I and II are more similar in terms of electrostatic potential, the active site of domain III completely differs in the distribution of its electrostatic surface potential. In addition, a characterization of the acceptor maps  and donor maps  for these three domains shows that they differ mainly in the properties of the S1’ and S1 region. Domain I has a more clear preference for donor groups in S1 region, due to the acidic properties of some of the residues involved in this site , as compared with domain II . This observation is also reflected in the electrostatic potential maps.The derived models of human CPD domains display active site clefts that are located at the edge of the active-sites grooves within the β-sheet of the catalytic moiety, in agreement with other MCP structures. Inside the catalytic cleft is found the typical catalytic zinc metal ion, which is coordinated with one Glu and to two His residues. The basis of the active-site groove is formed by strands β5, β6, β8 and the N-terminal part of helix α2. The wall of the groove funnel is essentially defined by two projections of the α4–α5 multiple turn and by the α7–β7 hairpin loop, as described previously ,44,52–54S3 Fig, both domains I and II conserve the three protein ligands of the catalytic zinc ion  and the residues directly involved in catalysis . Further, CPD domains I and II display the same residues involved in the S1’ substrate binding subsite , determining a substrate specificity for basic residues. Nonetheless, human CPD domains I and II differ in some residues within the S1’ specificity pocket. One of the major differences is that human CPD domain I contains Ser264 in a position comparable to Leu203 of bovine CPA1 and Ser198 of CPM , whereas CPD domain II contains Asn678 in an equivalent position. Relevant residues at the S1 subsite, such as Tyr248 and Ser199 (following the bovine CPA1 numeration) are essentially conserved between both CPD domains I and II, whereas notable residue variations are observed in one of the loops (α7–β7 hairpin loop) that shapes the border of the funnel and S1 subsite . The equivalent loop in CPM structure has been proposed to play an important role to accommodate the long hydrophobic P1 side chain of bradykinin [As shown in the multiple sequence alignment in adykinin .S3 Fig). In this domain, only the first His residue involved in zinc binding  is present; in contrast, the other two residues involved in metal binding  are replaced by Ala and Asp residues, a change conserved in duck CPD domain III [i.e., Glu270 and Arg127, according to bovine CPA1 numeration) are occupied by Tyr and Gln, respectively. Residues Asn144, Arg145 and Asn146 in bovine CPA1, responsible for the anchoring of the carboxylate group of the substrate, are replaced by a triad of Asp, Thr and Asp in human CPD domain III. Interestingly, the enzyme γ-D-glutamyl-(L)-meso-diaminopimelate peptidase I, which is a member of the M14B subfamily of MCP and a distant relative of CPD, has a triad of Asn, Asp and Gln in homologous positions to the triad Asn144/Arg145/Asn146. Such residue replacement confers to this bacterial enzyme a particular substrate specificity for C-terminal meso-diaminopimelic acid [Unlike domains I and II, human CPD domain III lacks the majority of key residues for CP catalysis . For the sake of simplicity, we used a truncated version of the peptide; i.e., the tetrapeptide GQKR. The best docking pose for GQKR in domain I shows that the C-terminal residue, arginine, interacts with the S1’ region formed by Asp268, Gln335 and Ser264 . As expected from homologous systems, the terminal carboxylate group interacts with the substrate-binding residues Arg218 and Asn217, while the peptide bond subjected to cleavage is located near Arg208. The Zn ion and the main catalytic residue, Glu350, are also surrounding this peptide bond. On the other hand, the lysine at P1 position, places the side chain in the previously described S1 region, interacting with the acidic residues Asp312, Asp314 and Glu315 located at the α7–β7 hairpin loop . In fact, the S1 region displays a marked negative surface electrostatic potential that may explain its preference for basic residues in P1 position. In the case of domain II, GQKR binding mode shows similar interactions for the S1’ region, establishing contacts with Asp682 and Gln747 . Residues Arg635 and Asn634 are stabilizing the C-terminal group, while Arg625, Zn and Glu762 interact with the peptide bond. The lysine at the P1 position in the peptide also interacts with the S1 region, but the type of interactions differ from those of domain I. The positive charge of the lysine makes a π-cation interaction with Tyr724 as well as hydrogen bond contacts with Glu727. In summary, although the binding mode of the tetrapeptide is well reproduced in the two domains, differences in regions S1 and S1’ may explain the differences in substrate specificity.In order to understand the detailed interactions between the peptides substrates within the catalytic sites of the different CPD domains, we performed a preliminary docking experiment with the GQRK predict that this domain is able to bind the peptide with a similar binding mode . The arginine in the C-terminal end interacts strongly with Asp1107, which corresponds to Asp268 and Asp682 in domains I and II, respectively. There is an additional interaction with Asp1064. Lysine in position P1 interacts with Asp1145 and Glu1146, the latter being conserved in the three domains. However, there are some differences in the rest of the residues that form the S1 region. In particular, Asp312 from domain I is aligned to Tyr724 in domain II and Lys1143 in domain III. The properties of these three residues are different enough and may explain the different affinity of the domains for substrates. Accordingly, these results support the hypothesis that domain III might be able to bind peptides and may perform a non-enzymatic function, such as peptide binding and/or transport.As previously explained, the active site of domain III lacks the main catalytic residues, thus being unable to cleave conventional CP substrates. As the function of this domain remains unknown, we wanted to assess its ability to accommodate peptides in its active site. Docking studies with the truncated peptide Table 3 and S9 Table. The longest peptides were truncated to 7 C-terminal residues in order to simplify the docking calculations. The final set of peptides included TITGQKR, LVTDLTK, GAAAGQR, FAATSFR, VDPVNFK and SALGEPKK. The analysis of the best docking poses for these peptides did not explain the different substrate specificity of the two domains, in terms of docking score or particular peptide/receptor interactions. For instance, the best docking poses obtained for VDPVNFK and FAATSFR peptides show very similar interactions, which does not explain why these two peptides have different preference towards the CPD domain I. Therefore, we performed an additional analysis of the docking results, based on the number of expectedly correct docking solutions found within all resulting docking poses for each peptide. The criteria to consider a docking peptide pose as correct are based on the following features: the center of mass of the P1’ and P1 residues should be within 10 Å distance from the center of mass of CPD domain I Asp268 (S1’ pocket) and Glu315 (S1 pocket) residues, respectively, or from CPD domain II Asp682 (S1’ pocket) and Glu727 (S1 pocket) residues, respectively. These criteria are likely to describe the expected general interactions in peptide binding as discussed in the previous section. From this analysis of the docking results, we found that the percentage of correct docking solutions  for each peptide is a good indicator of the observed cleavage of that peptide by each domain; i.e., the higher the percentage of correct poses, the better performance as substrate in the experimental data . For instance, a high percentage of correct docking poses for domain I was found in the case of TITGQKR and FAATSFR peptides. On the other hand, domain II showed preference to bind correctly TITGQKR, LVTDLTK, FAATSFR and VDPVNFK.In order to fully explain the different substrate specificities for the two active CPD domains, we decided to perform an extensive molecular docking study. For this purpose, we selected six peptides, based on the different experimental ratio values observed  shown in The primary goal of the present study was the detailed comparison of the substrate specificity of the first and second catalytic domains of CPD, as well as testing the third carboxypeptidase-like domain for catalytic activity using a broad array of peptides. CPD is the only member of the MCP family that contains multiple carboxypeptidase domains ,15,16. PDrosophila CPD [One important contribution of the present study is that human CPD domain I and II have distinct pH optima, with domain I working best at neutral pH while domain II is optimal at mildly acidic pH values. This observation is consistent with the properties previously reported for duck and hila CPD ,22. indihila CPD . The pH Drosophila CPD which tested a small number of substrates [Drosophila CPD with a single pair of peptides that differed only in the C-terminal residue [Another important finding of the present study is that human CPD domain I and II have differences in their substrate specificities. Both are specific for C-terminal basic residues, with no detectable cleavage of peptides with non-basic residues on the C-terminus. The present study tested dozens of peptides in a peptidomic assay, and extended the previous studies performed with duck and bstrates ,22. The  residue ,22. The 5-Arg6-enkephalin with a kcat of 934 min-1 and Km of 46 μM, whereas Met5-Lys6-enkephalin is cleaved with kcat of 663 min-1 and Km of 375 μM [5-Lys6-enkephalin with a ki of 174 μM and by Leu5-Arg6-enkephalin with a ki of 83 μM [Fig 10). Similarly, CPM and CPZ contain a Ser in this position, and CPE and CPN contain Asn. Perhaps the smaller side chain of the Ser (versus Asn) within the binding site has a superior capability to fit and facilitate the establishment of contacts between the guanidine group of the C-terminal scissile Arg residue, leading to major preference for Arg- versus Lys-containing substrates. The analysis of the docking landscape suggests that there might be some kinetic effects involved in the processes of approaching and entering of the peptides into the active site, which are not fully explained by the structure and energetics of the best docking pose alone. These docking results are consistent with the experimental finding that peptides with a C-terminal lysine have less preference for the active site of domain I, whereas peptides with a C-terminal arginine bind both domains, with a slight preference for domain I.Previous studies using small numbers of substrates have found that other members of the M14B subfamily of MCPs are more efficient at cleaving C-terminal Arg than Lys. For example, CPM cleaves Metf 375 μM . Other sof 83 μM . CPZ is Drosophila CPD, which tested a small number of substrates [Another finding of the present study is that the third carboxypeptidase-like domain of human CPD is inactive as a carboxypeptidase when tested with a fluorescent substrate at a wide range of pH values, or with a wide array of peptides at pH 6.5. In no case was carboxypeptidase activity detected for the form of CPD with mutations in a key catalytic residue in domain I and II. This observation fits with previous studies on duck and bstrates ,22. ThisThree other members of the M14B subfamily of MCPs are also inactive towards standard carboxypeptidase substrates. Two of these, CPX2  and AEBPTaken together, our results establish that both catalytically active domains of CPD play important physiological roles by cleaving a broad range of peptide and protein substrates with C-terminal basic residues or Arg and Lys in the trans-Golgi network, within the secretory and endocytic pathways and in the cell membrane. Although both active domains display a partial overlap of their substrate preferences, our results demonstrate that each active CPD domain has evolved to divergent substrate specificities conserved through evolution. Moreover, the third domain is catalytically inactive against a wide spectrum of peptides, and the biological function of this domain remains unknown.S1 FigReactions containing 200 μM dansyl-Phe-Ala-Arg (filled circles), dansyl-Phe-Gly-Arg (filled squares) or dansyl-Phe-Pro-Arg (triangles/solid line) were incubated with different amounts of enzyme in a 100 mM Tris-acetate, pH 6.5, 150 mM NaCl buffer for 60 min at 37°C.(TIF)Click here for additional data file.S2 FigAcceptor and donor maps for domain I (A and D), II (B and E) and III (C and F). Hydrogen bond acceptors and donors are indicated with a red or blue contour mesh, respectively.(TIF)Click here for additional data file.S3 FigMetal binding residues, catalytic residues, and important substrate binding residues are shown in bold and boxed. Stars above the sequence indicate residues involved in zinc binding. Residues in bold and non-boxed correspond to those residues within the loop α7–β important for substrate binding. Aligned sequences from human CPD domains I, II and III correspond to residues 50–462, 496–873 and 925–1299, respectively. Sequence of human CPM corresponds to residues 18–423. The position numbers below the sequence indicate equivalent positions according to the standard numbering system for bovine CPA.(TIF)Click here for additional data file.S4 FigDetailed view of the pose of the arginine from GQKR peptide at the S1’ regions of CPD domains I (A) and II (B), which mainly differ in the residue that corresponds to Leu203 of bovine CPA1 and Ser198 of CPM, Ser264 in domain I and Ans678 in domain II.(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.


Here, using the α-globin gene cluster as an illustrative model, we provide an overview of the most recent insights into the layers of genome organization across different scales and how this relates to gene activity. It has been known for over a century that chromatin is not randomly distributed within the nucleus. However, the question of how DNA is folded and the influence of such folding on nuclear processes remain topics of intensive current research. A longstanding, unanswered question is whether nuclear organization is simply a reflection of nuclear processes such as transcription and replication, or whether chromatin is folded by independent mechanisms and this  Nuclear organization is currently described at vastly different scales. Many nuclear structures, such as euchromatin, heterochromatin, the nuclear lamina, and non-membrane bound nuclear sub-compartments, can be identified by conventional microscopy. Technological advances have revealed finer detail of nuclear structure, including chromosome territories and the organization of telomeres and centromeres. More recently, sub-microscopic features of nuclear structure such as chromatin folding, chromatin structure, and distribution of chromatin-associated proteins have been determined, and many of these features can now be correlated with specific DNA sequences ,2. An imA key question in this field, discussed here, is whether sub-chromosomal organization is predominantly an emergent property of a wide variety of nuclear processes, or established independently, representing ‘building blocks’ of the genome.It has previously been shown that individual eukaryotic chromosomes fold in such a way that they more frequently interact with themselves rather than with other chromosomes and occupy distinct chromosome territories in the nucleus . AnalyseOne of the first classes of self-interacting domains described relatively large regions of chromatin 100–5 Mb) observed in Hi-C heat maps, which were named topologically associated domains (TADs) ,7. TADs 0–5 Mb obSimilar Hi-C data have also been interpreted from a different perspective that describes larger structures called compartments. These are composed of transcriptionally active or inactive TAD-like structures which cluster together to form functionally distinct nuclear compartments ,12. The More recent analyses of nuclear compartments and TADs using a variety of 3C techniques have revealed previously unrecognized, smaller, self-interacting domains that have been referred to as contact domains , sub-TADA common feature of all self-interacting domains, is that their borders are enriched for boundary elements that among many other proteins commonly bind CTCF, a highly conserved DNA-binding zinc finger protein. CTCF is strongly implicated in the formation of self-interacting domains, which often depends on the presence of pairs of CTCF-binding motifs in convergent orientations at the domain borders ,17–21.The definitions of the structures discussed above are rather vague and their identification somewhat arbitrary . The desIt has been proposed that self-interacting domains are formed by a process of loop extrusion. This is thought to be an active process in which a loop-extruding factor, containing two DNA-binding units, associates with chromatin and travels along the fiber in opposite directions, creating a progressively larger intervening loop, until the factor is stalled at boundary elements, such as convergent CTCF-binding motifs. Hetero-dimerization between CTCF proteins or other boundary proteins then tethers the extrusion factor, defining the limits of the self-interacting domain ,22. It cIn support of the loop extrusion model, it has been shown in various studies that mutation or deletion of specific CTCF-binding sites can disrupt the border of the associated self-interacting domain, and cause a change in genome structure that allows enhancers within the domain to interact with genes in neighboring domains, resulting in mis-regulated gene expression ,17,29–35A possible explanation could be that cohesin is recruited to chromatin in a tissue-specific manner. It has been shown that cohesin is not only associated with CTCF-binding sites, but also with active promoters and enhancers and that dynamic cohesin occupancy of enhancers correlates with tissue-specific interactions between promoters and enhancers . FurtherIt is important to keep in mind that the self-interacting domains observed in 3C analyses represent a population average of a dynamic structure and that the spatial separation between TADs is not absolute. Insulation between domains does not completely prevent the formation of contacts across boundaries  and suchSox9 locus have been shown to be less disruptive to gene expression and cause less extensive changes in phenotypes than those which partially disrupt a self-interacting domain  independent mechanisms underlying self-interacting domains. One might involve a process of loop extrusion that depends on CTCF, cohesin and/or other boundary associated proteins. This sets up constitutive architectural domains that are largely tissue-invariant and separate some regions of the genome into domains that restrict interactions between regulatory elements.cis-elements, increased enhancer-promoter interactions and active transcription may further shape the structure of these domains. Cohesin may also play a role in these interactions (In addition, nuclear processes such as changes in chromatin accessibility, the binding of key transcription factors to ractions . The domractions  and clusAlthough some of the processes driving constitutive and cell type-specific structures may be independent, they may nevertheless influence each other. For example, the constitutive structures thought to be set up by cohesin/CTCF may constrain interactions between regulatory elements and therefore contribute to their specificity . Furthercis-elements, broad, self-interacting domains may appear over relatively large genomic regions upon activation of such elements in specific cell types. Within this region, increased interactions over certain elements can be identified (cis-elements suggest that when such elements, bound by multi-protein complexes, are brought together, they form more stable or frequent interactions than the rest of the self-interacting domain.The structures related to nuclear processes were initially described as chromatin ‘loops’ formed specifically between active promoters, enhancers and boundaries . Howeverentified ,60 (Fig.entified . This isper se in establishing self-interacting domains: a recent study of chromatin conformation during Drosophila embryogenesis showed that the appearance of TADs occurs independently of transcription (Though nuclear processes related to gene expression likely influence genome structure, current evidence argues against a role for transcription cription . HoweverOverall, these studies suggest that the structures observed in contact matrices result from a mixture of different mechanisms and reflect an intricate interplay between form and function .The globin loci have been used extensively to establish general principles of mammalian genome organization and gene regulation and relate perturbations in these processes to human genetic disease. Principles first established at these loci have usually been found to be true of many other loci ,63.To examine the concepts discussed here, we analyzed 3C interaction data for the well-characterized murine α-globin cluster. Some self-interacting domains in this gene-dense 2.5 Mb region appear as relatively discrete structures in mES cells (cis-acting regulatory elements causes the formation of a strong self-interacting domain, in which contacts between the α-globin promoters and enhancers are significantly increased. The boundaries of this domain are formed by predominantly convergent CTCF-binding sites. These become closely apposed specifically in erythroid cells, while no interactions between convergent CTCF sites are observed in mES cells. This shows that this self-interacting domain is not preformed, but becomes established in a cell type-specific manner. It has recently been shown that these CTCF/cohesin boundaries constrain the activity of the α-globin enhancers to the self-interacting domain (Comparison between primary erythroid and mES tissue at higher resolution  shows thg domain .Early studies of nuclear organization of the genome at relatively low resolution showed the existence of self-interacting domains, such as TADs, which were initially interpreted to be evolutionarily conserved and invariant between cell types. More recent analysis at higher resolution suggests that these domains may represent a fractal structure, with many domains contained within previously defined TADs and compartments. In addition, some of these domains appear to be tissue-specific.Current data are consistent with at least two processes contributing to the observed nuclear organization. In some areas of the genome, preformed self-interacting domains may exist, which are formed by processes that are not fully understood, but likely depend on CTCF and cohesin. Processes related to gene expression within these domains impose further organization on the genome in a cell type-specific manner .cis- and trans-acting factors responsible for establishing these domains have been identified, it should be possible to mutate these precisely and rigorously to demonstrate their role in nuclear organization and gene regulation.If the current model is correct, there should be more examples of human genetic diseases that result from perturbations of the formation of self-interacting domains rather than from mutations of genes and their promoters or enhancers. Once the To further understand the origins and effects of self-interacting domains, we require more high-resolution analyses, of individual examples, such as the α-globin cluster discussed here, where interactions between the constituent elements can be related to processes leading to gene expression.


RNase P, the essential activity that performs the 5′ maturation of tRNA precursors, can be achieved either by ribonucleoproteins containing a ribozyme present in the three domains of life or by protein-only enzymes called protein-only RNase P (PRORP) that occur in eukaryote nuclei and organelles. A fast growing list of studies has investigated three-dimensional structures and mode of action of PRORP proteins. Results suggest that similar to ribozymes, PRORP proteins have two main domains. A clear functional analogy can be drawn between the specificity domain of the RNase P ribozyme and PRORP pentatricopeptide repeat domain, and between the ribozyme catalytic domain and PRORP N4BP1, YacP-like Nuclease domain. Moreover, both types of enzymes appear to dock with the acceptor arm of tRNA precursors and make specific contacts with the corner of pre-tRNAs. While some clear differences can still be delineated between PRORP and ribonucleoprotein (RNP) RNase P, the two types of enzymes seem to use, fundamentally, the same catalytic mechanism involving two metal ions. The occurrence of PRORP and RNP RNase P represents a remarkable example of convergent evolution. It might be the unique witness of an ongoing replacement of catalytic RNAs by proteins for enzymatic activities. Escherichia coli [Similar to Janus, the two-faced Roman god of transitions and passages that concomitantly looks to the future and to the past, RNase P has two faces. This essential enzymatic activity that removes the 5′ leader sequences of tRNA precursors occurs either as ribonucleoproteins (RNP) involving a ribozyme  or as prhia coli ,7,8 is uhia coli . RNP RNahia coli . Neverthhia coli .RNP RNases P are present in all three domains of life, their catalytic RNA (P RNA) structure is relatively well conserved in prokaryotes and eukaryote nuclei . HoweverPROtein-only RNase P or PROteinaceous RNase P), have two main domains; a C-terminal catalytic domain belonging to the N4BP1, YacP-like Nuclease (NYN) family [Arabidopsis thaliana [Trypanosoma brucei nucleus and mitochondria [Ostreococcus tauri [Physcomitrella patens [Chlamydomonas reinhardtii [Chlamydomonas utilizes a single PRORP protein for RNase P activity in the nucleus, mitochondria and the chloroplast, thus making the most compact and versatile RNase P machinery described to date in both prokaryotes and eukaryotes [Escherichia coli [Saccharomyces cerevisiae [In contrast, protein-only RNase P enzymes are entirely specific to eukaryotes. These enzymes, termed PRORP (for ) family  and an N) family . The two) family ,20. PRORthaliana ,22,23. Tchondria , and in us tauri , the mosa patens , and thenhardtii . Intereskaryotes . Likewiskaryotes . For inskaryotes , PRORP akaryotes ,29. Remarevisiae , thus shrevisiae . NonetheHere, we review the biochemical, structural, and mechanistic data obtained for protein-only RNase P and discuss how PRORP structure, pre-tRNA recognition, and cleavage differ, but also resemble that of RNP RNase P.Functionally characterized, as well as putative, PRORP sequences are all typified by an α-superhelical domain containing tandem arrays of PPR motifs and a specific NYN metallonuclease domain, bridged by a bipartite zinc-binding domain. Sequences of the N-terminal PPR domains are highly degenerate, but recognizable, by the 3D fold specific for PPR motifs ,18. ReceYacP genes are ubiquitous in prokaryotes and eukaryotes, with the notable exception of fungi [3D(G/A)xN and a second motif DDx15–39(S/T)xDx3DH separated by 50 to 80 amino acids. This NYN signature of PRORP differs significantly from the basic NYN signature [The C-terminal regions of PRORP enzymes contain a metallonuclease domain that was shown to be responsible for the endonuclease activity . This doof fungi . While Nignature , and alsignature .Chlamydomonas CxxC is replaced by CxxA [2-3(R/K) signatures [The connecting zinc-binding domain is split into two subparts upstream and downstream of the NYN domain. Its first part involves a highly conserved CxxC motif, where both cysteines were shown to be required for zinc coordination in Arabidopsis and human ,20,36. H by CxxA . The secgnatures . Here, aBeyond the PPR, the NYN and the connecting domain, other signatures are present in PRORP sequences of specific phyla. For example, a glycine-rich insertion is characteristic of land plant PRORPs. In some phyla, a PPP(Y/F/C)(S/T) motif is found between the NYN domain and the second zinc-binding subdomain. The occurrence of such specific insertions might indicate additional phylum-specific functions, e.g., through interactions with yet unidentified proteins.First analyses of PRORP sequences along with biochemical and biophysical studies, including synchrotron radiation circular dichroism and small angle X-ray scattering, showed that PRORP enzymes are organized in two main domains and contain a high proportion of α-helices ,22. ThisAs expected, the N-terminal PPR domain forms a superhelical structure closely resembling that of TPR (tetratricopeptide repeat) motifs, a domain evolutionary related to PPR and involved in protein-protein interactions ,41. DespThen, the catalytic domain of PRORP implements an α/β/α sandwich fold typical of all structurally-characterized PIN-like and flap nucleases ,46,47. AThe two main domains are bridged by the zinc-binding domain, which appears to have a structural role, i.e., to connect the two functional domains and, thus, to give PRORP its overall structural design . The zinThe comparison of Arabidopsis organellar PRORP1 with nuclear PRORP2 structures revealed that PPR domains in both enzymes are very similar and composed of five and a half PPR and PPR-like motifs corresponding to 11 consecutive α-helices. However, differences are found: for instance, motif PPR2 of PRORP1 has an extension between its two α-helices . The funSimilarly, the comparison of human PRORP structure with plant PRORP structures revealed some discrepancies. Plant PRORP proteins feature a 30-residue-long loop-helix-loop linker inserted between the PPR and the zinc-binding domains which is replaced by a short loop in the human enzyme. In turn the latter has a longer lariat loop in its zinc-binding connecting domain as compared to Arabidopsis PRORPs. The human structure is stabilized by an extended hydrogen bond network and makes a finger between the PPR and NYN domain B, whereaE. coli, the 3′ terminal CCA of tRNAs binds a complementary sequence in a P RNA loop of RNP RNase P. The initial analysis of PRORP bipartite organization suggested that the PPR domain of PRORP might play a role similar to that of the P RNA S domain, while the NYN domain would have a function similar to that of the C domain in RNP RNase P [The capacity of PRORP enzymes to functionally replace RNP enzymes in vivo suggested that both types of RNase P might share a similar mode of action. For its activity, bacterial RNP RNase P docks onto the acceptor arm of the pre-tRNA substrate. In particular, the P RNA specificity (S) domain interacts with the tRNA corner formed by the stacked T and D loops and the catalytic (C) domain contacts the cleavage site between nucleotides −1 and +1 of pre-tRNAs ,53,54. Tobs vary from 1 to 7 min−1, KD values determined with a same protein but different substrates range from 60 to 2300 nM, while KM values determined in multiple turnover conditions vary from 140 to 2000 nM  for PRORP cleavage suggest that protein-only enzymes also use a two-metal-ion mechanism to produce the attacking hydroxide ion and stabilize the transition state [RP-phosphorothioate modification at the canonical cleavage site using Mg2+ as a cofactor, which is not the case of ribozyme RNase P [pro-RP-oxygen of the scissile phosphate is not directly coordinated by a metal in PRORP active site, and suggests that the whole active site may adopt a different configuration. PRORP are proposed to use one hydrated divalent cation to provide the attacking hydroxide and a second hydrated metal ion to protonate the leaving group like the RNase P ribozyme [Beyond the common architectural features and substrate specificities of PRORP and RNP RNase P for pre-tRNA binding, the two types of biocatalyst appear to share a similar catalytic mechanism ,19,60. T RNase P . This suribozyme ,64.2+ ion in the catalytic site increases the affinity of the second metal ion resulting in the cooperativity observed at low concentrations of pre-tRNAs. A comparison of the proposed mechanisms for PRORP and ribozyme RNase P activities shows that both enzymes require catalytic metal ions, although the metal ligands are different. Conserved aspartate residues coordinate the metal ions in PRORP, whereas ribozyme RNase P mainly uses non-bridging phosphodiester oxygens for metal ion coordination.In the latter study, Howard et al. also propose a model where the binding of one MgAltogether, biochemical studies published since the first descriptions of PRORP enzymes reveal that they share common features with RNP RNase P, but also diverge in many aspects. Alongside the discovery of protein-only RNase P, initial appreciations anticipated that the single subunit protein-only RNase P might be a much more efficient enzyme than RNP RNase P and that higher catalytic efficiency might have been the driving force for the replacement of ribozyme RNase P by PRORP during evolution. Pre-tRNA binding and cleavage studies of PRORP now suggest that it is not the case. For example, binding affinities of PRORP proteins for their pre-tRNA substrate are in the micromolar range ,60. ThesRNA binding assays revealed that the two types of enzymes are clearly distinct at the level of substrate recognition in the vicinity of the cleavage point. Hence, PRORP binding to pre-tRNA does not seem to involve specific interactions with pre-tRNA leader and trailer sequences contrary to RNP RNase P. In contrast, the two types of enzyme have a similar organization with the specificity and catalytic domains of P RNAs being analogous to the PPR and NYN domains of PRORP proteins. Moreover, the two types of enzymes appear to dock on the acceptor arm of pre-tRNAs and to make contact with specific residues at the corner of tRNAs formed by the stacking of T and D loops of tRNAs. However, despite these important similarities, PRORP cannot be considered as a true structural mimic of the RNase P ribozyme, at least not in the sense where the exact three-dimensional shape of a nucleic acid is mimicked by a protein for functional reasons, e.g., pathogen proteins shaped as DNA to avoid host defenses or ribosome recycling factors shaped as a tRNA to dissociate ribosomal subunits . Similarnad6 and orf291 RNAs at the level of predicted tRNA-like structures [While major progress has been achieved to understand the biological functions  and evolructures ,67 and tructures . Other cAnswering the open questions regarding PRORP mode of action will rely on the determination of three-dimensional structures of PRORP either in complex with pre-tRNA substrates and/or with 5′ mature tRNA products. Such structures obtained either in solution or at atomic resolution will reveal the exact RNA binding and cleavage properties of PRORP and uncover the plasticity and dynamics of PRORP maturation of tRNA precursors.


The catalytic subunits of SWI/SNF-family and INO80-family chromatin remodelers bind actin and actin-related proteins (Arps) through an N-terminal helicase/SANT-associated (HSA) domain. Between the HSA and ATPase domains lies a conserved post-HSA (pHSA) domain. The HSA domain of Sth1, the catalytic subunit of the yeast SWI/SNF-family remodeler RSC, recruits the Rtt102-Arp7/9 heterotrimer. Rtt102-Arp7/9 regulates RSC function, but the mechanism is unclear. We show that the pHSA domain interacts directly with another conserved region of the catalytic subunit, protrusion-1. Rtt102-Arp7/9 binding to the HSA domain weakens this interaction and promotes the formation of stable, monodisperse complexes with DNA and nucleosomes. A crystal structure of Rtt102-Arp7/9 shows that ATP binds to Arp7 but not Arp9. However, Arp7 does not hydrolyze ATP. Together, the results suggest that Rtt102 and ATP stabilize a conformation of Arp7/9 that potentiates binding to the HSA domain, which releases intramolecular interactions within Sth1 and controls DNA and nucleosome binding. et al. report an interaction of the highly-conserved pHSA and P1 domains of Sth1, the catalytic subunit of the SWI/SNF-family chromatin remodeler RSC. This interaction is released when ATP-bound Rtt102-Arp7/9 binds to the HSA domain, modulating DNA and nucleosome binding by Sth.Bengi Turegun  Remodeler malfunction is often linked to diseases, including cancer2 and cardiovascular disorders3. Remodelers belong to four families: CHD, ISWI, SWI/SNF, and INO80. While the subunit composition of these families varies, they all assemble around a catalytic subunit defined by the presence of a conserved helicase superfamily 2 (SF2) ATPase domain5. The ATPase domain consists of two recA-like subdomains, with the nucleotide-binding and DNA-binding sites located at the interface between subdomains8. DNA-dependent ATP hydrolysis by the catalytic subunit propels changes in chromatin structure, such as histone octamer sliding, ejection, and histone subunit exchange14. These activities are regulated by auxiliary subunits recruited through family-specific domains N and C terminal to the central ATPase domain of the catalytic subunit.Chromatin-remodeling complexes (remodelers) are large, multisubunit complexes that regulate gene expression and genome maintenance15. Actin and Arps have been proposed to regulate the catalytic and nucleosome targeting and sliding activities of their host remodelers22. The HSA domain of Sth1, the catalytic subunit of the budding yeast SWI/SNF-family remodeler RSC (Remodeling the Structure of Chromatin), recruits Arp7 and Arp915. In turn, the Arp7/9 heterodimer forms a tight complex with another auxiliary subunit, Rtt102. Biochemical and structural studies have shown that Rtt102 stabilizes a compact conformation of Arp7/9 that favors binding to the HSA domain, while also promoting high-affinity binding of ATP to one of the Arps24. Arp7/9 regulates the nucleosome sliding, ejection, and ATPase activities of RSC in an Rtt102-dependent manner23. However, the molecular mechanism through which binding of Rtt102-Arp7/9 to the HSA domain of Sth1 regulates its activity is unknown.Two families of remodelers, SWI/SNF and INO80, recruit as auxiliary subunits actin and actin-related proteins (Arps) through a region N terminal to the ATPase domain called the helicase/SANT-associated (HSA) domain29. Interestingly, in Sth1, mutations that restore viability of ΔArp7/9 yeast strains localize to two highly conserved regions: protrusion-1 (P1) within the ATPase domain and a ~60-aa sequence in between the HSA and ATPase domains known as the post-HSA (pHSA) domain15. It is therefore possible that the pHSA domain of Sth1 interacts with the ATPase domain, and this interaction could in principle be regulated by Rtt102-Arp7/9 binding to the neighboring HSA domain. In this study, we test this hypothesis; we show that the pHSA domain interacts with the ATPase domain, directly contacting P1. We further show that binding of Arp7/9 to the HSA domain weakens this interaction in an Rtt102-dependent manner. In addition, the binding of Rtt102-Arp7/9 to the HSA domain reduces the affinity of the ATPase for DNA, while promoting the formation of stable, monodisperse complexes with both DNA and nucleosomes. A nucleotide-bound structure of Rtt102-Arp7/9 reveals that ATP binds only to Arp7, helping stabilize a closed conformation of Arp7 that potentiates binding of Rtt102-Arp7/9 to the HSA domain. Together, the results suggest a molecular mechanism for how actin and Arps might regulate SWI/SNF-family and INO80-family remodelers.Several studies have identified regulatory interactions between the ATPase domain and flanking regions in various catalytic subunits425–1097, lacking both the HSA and pHSA domains, was insoluble and accumulated in bacterial inclusion bodies, whereas a construct lacking only the HSA domain, Sth1365–1097, was soluble. Since Sth1 cannot be expressed without the pHSA domain to test the existence of a hypothetical intramolecular interaction involving the pHSA domain, we designed an internally cleavable construct, whereby the HSA–pHSA region could be severed from the ATPase domain after purification. For this, a canonical TEV protease cleavage site was engineered within the poorly conserved linker between the pHSA and ATPase domains and with minimal changes to the endogenous sequence  contains a non-cleavable N-terminal His-tag for affinity purification and pull-down assays , Sth1 residues P646-P692 were replaced with Rad54 residues P610-P644 (which is 12-aa shorter), such that the swapped region is flanked by conserved sequences that can be unequivocally aligned  deplete a fixed concentration of free DNA through complex formation both in the AMPPNP-bound and ADP-bound states. This assay revealed a similar trend to that observed by fluorescence anisotropy; higher concentrations of Rtt102-Arp7/9-Sth1301–1097 than Sth1301–1097 were required to deplete the DNA band, indicative of a lower DNA-binding affinity of the quaternary complex  complex with DNA than Sth1301–1097 alone, as indicated by a single, slowly migrating band in the native gel , with a slight albeit reproducible advantage for the quaternary complex both in the AMPPNP (apparent KD of 0.46 vs. 0.67 μM) and ADP (apparent KD of 0.51 vs. 0.67 μM) states. Therefore, we conclude that, while the isolated Sth1 catalytic subunit and the Rtt102-Arp7/9-Sth1 quaternary complex have similar affinities for nucleosomes, Rtt102-Arp7/9 appears to confer specificity upon Sth1 by promoting the formation of monodisperse complexes with nucleosomes. These results are consistent with previous findings that the quaternary complex has lower DNA-dependent ATPase activity but increased translocation efficiency on nucleosomes16.In light of the negative effect of Rtt102-Arp7/9 on the affinity of Sth1 for DNA, we asked whether nucleosome binding was similarly affected. Nucleosome binding was measured for Sth1097 Fig. , which i097 Fig. . On the 097 Fig. . Importa301–1097 studied here contains three potential ATP-binding sites, on Sth1, Arp7, and Arp9, such that nucleotide-dependent effects could in principle be due to ATP binding and/or hydrolysis at any of these sites or their combinations. While ATP binding and hydrolysis by actin has been extensively studied32, little is known about the role of ATP in nuclear Arps33. In a previous study, we observed using isothermal titration calorimetry that ATP binds to a single, high-affinity site within Rtt102-Arp7/924, whose location we could not precisely identify. Now that we have found that Rtt102-Arp7/9 affects the interaction of Sth1 with nucleosomes, it is imperative to better understand this regulatory complex, including the location and role of ATP in Rtt102-Arp7/9.The quaternary complex of Rtt102-Arp7/9-Sth134 and Rtt102-Arp7/9-HSA (PDB accession code: 4I6M)23 do not contain ATP bound, presumably because these complexes were crystallized in the presence of high concentrations of phosphate and sulfate ions, which occupy the nucleotide-binding pockets of Arp7 and Arp9. Here, we pre-incubated Rtt102-Arp7/9 with ATP prior to crystallization, and obtained crystals using polyethylene glycol 3350 as the main precipitant. The crystals, which diffracted to a relatively low resolution (3.25 Å), contain two copies of the Rtt102-Arp7/9 ternary complex in the P1 unit cell ell Fig. . The str35. Not surprisingly, we found here that Arp7 lacks ATPase activity, both alone and within the ternary complex Rtt102-Arp7/9, whereas the quaternary complex Rtt102-Arp7/9-Sth1301–1097 displays strong DNA-dependent ATPase activity catalyzed by subunit Sth1 . In contrast, there is no cation associated with the nucleotide in the structure of Arp7. Indeed, Arp7 appears to lack a cation-binding site, as two of the residues that coordinate water molecules that hold the cation in place in actin, Asp-11 and Gln-137, have changed in Arp7 to His-11 and Glu-141, respectively. Specifically, Gln-137 and the cation coordinate a water molecule thought to serve as the nucleophile in the ATP hydrolysis reaction of actin. Strikingly, mutating this residue to glutamic acid in yeast actin (the amino acid found in Arp7) results in lack of viability32. The same study found that residue His-161 in the actin cleft is also absolutely required for yeast viability. Actin His-161 becomes Asn-165 in Arp7. Therefore, the lack of ATPase activity in Arp7 is supported by the structure and previous genetic studies on actin and the Arps36.Monomeric actin is an extremely slow ATPase, and nucleotide hydrolysis is only activated upon polymerizationth1 Fig. . This isrp7 Fig. . In part26. The AutoN and NegC regions of ISWI, N and C terminal to the ATPase domain, negatively regulate ATP hydrolysis and the link between ATP hydrolysis and productive DNA translocation, respectively25. Similarly, the N-terminal region of CSB negatively regulates its ATPase activity27. In SWI/SNF remodelers, the SnAc (Snf2 ATP coupling) domain positively regulates the catalytic activity of the ATPase domain and may also interact with histones37.We have shown here that the pHSA domain of Sth1 interacts with the P1 region of the ATPase domain, and that regulation of this interaction might be an important mechanism to fine-tune the activity of actin/Arp-containing remodelers. This finding adds to a growing list of evidence showing that domains adjacent to the ATPase domain regulate the activity of the catalytic subunits of several remodelers. Thus, N-terminal chromodomains in Chd1 interact with and lock the ATPase domain in an inactive conformationMyceliophthora thermophila was reported38. The structure comprises Snf2 residues Ala-458 to Gly-1128, corresponding to Sth1 residues Ser-383 to Gly-1049 . Although several elements of the structure were not visualized, including parts of P1 and the linker between the pHSA and ATPase domains, the structure offers important clues; it reveals most of the pHSA domain  and a portion of the C-terminal SnAc domain . Both the SnAc and pHSA domains interact extensively with the ATPase domain and, consistent with our findings here, the pHSA domain lies adjacent to P1. The concurrence of our findings and the high conservation of the interacting sequences in the individual SWI/SNF and INO80 families of remodelers ; it participates in inter-recA domain stabilizing interactions39. Thus, two helices of the P1 region within the N-terminal recA domain fold upon two helices of the so-called “Brace” region within the C-terminal recA domain in a crossed-arms manner to stabilize the recA domains around the nucleosome. As a result, the inter-recA conformation observed in this structure is very different from that observed in the absence of nucleosome38. Yet, the pHSA helix lies adjacent to the P1 region in both structures, suggesting that they move as a single entity. If, as our work suggests, the binding of Rtt102-Arp7/9 to the neighboring HSA domain weakens the pHSA–P1 interaction, this could impact the orientation of the recA domains with respect to one another and thus their interaction with the nucleosome, which could possibly explain the formation of the monodisperse Rtt102-Arp7/9-Sth1/nucleosome complex observed here.Given the intrinsic structural flexibility of the recA domains relative to one another24 confirm the presence of a nucleotide-binding site in Arp7. However, we also found that Rtt102-Arp7/9 lacks ATPase activity . There are >100 actin entries in the Protein Data Bank, and to our knowledge the actin/Arp4-SWR1 structure is the first in which actin has no nucleotide bound. In contrast, Arp7, which in isolation has low affinity for ATP (KD = 12 μM), binds nucleotide tightly (KD = 94 nM) as part of the quaternary complex Rtt102-Arp7/9-HSA24. Thus, there appears to be a conserved structural requirement for a closed, nucleotide-bound cleft for the subunit associated with the N-terminal half of the HSA domain and an open, nucleotide-free cleft for the subunit bound closer to the ATPase domain. In other words, we propose that the role of nucleotide binding to actin/Arp-containing remodelers is not regulatory, but structural. If this prediction is correct, it could be expected that the fission yeast remodelers RSC and SWI/SNF, which contain Arp9 and the Arp4 homolog Arp42 (instead of Arp7)42, will have nucleotide-bound Arp42 and nucleotide-free Arp9 at the N-terminal and C-terminal ends of the HSA domain, respectively.The structure of ATP-bound Rtt102-Arp7/9 described here and previous biochemical dataity Fig. , suggestIn summary, the biochemical and structural data presented here suggest that Rtt102 and ATP stabilize a conformation of Arp7/9 that potentiates binding to the HSA domain, which in turn releases intramolecular interactions between the pHSA domain and the P1 region of the ATPase domain. These interactions might control the affinity and specificity of the ATPase for DNA and nucleosomes, ultimately regulating the translocation efficiency of the remodeler. Because both the pHSA domain and P1 are highly conserved separately within the SWI/SNF and INO80 remodeler families, this could represent a general mechanism for how actin and Arps regulate the activities of their host remodelers.24. For Sth1301-1097P1Swap, the Sth1 fragments 301–645 and 693-1097 were primer-extended to add S. solfataricus Rad54 residues 610–644. A silent EcoRI site was introduced during extension to ligate the two Sth1 fragments. For protein purification, cells were resuspended in lysis buffer , lysed using a Microfluidizer apparatus (Microfluidics), and clarified by centrifugation. Lysates were purified on a Ni-NTA affinity column (Qiagen) and washed extensively with lysis buffer. Proteins were eluted with 250 mM imidazole and bound to a HiTrap Heparin HP column  equilibrated in sample buffer , and 4 mM benzamidine). Proteins were eluted using a 200–800 mM NaCl gradient, and further purified on a SD-200 gel filtration column  equilibrated with sample buffer. To reconstitute the Rtt102-Arp7/9 ternary complex, Rtt102 and Arp7/924 were mixed at a 2:1 molar ratio and purified through a SD-200 gel filtration column equilibrated in sample buffer (without benzamidine).Sth1 constructs and complexes were cloned and expressed as described2, and 0.1 mM AMPPNP). A 1-mL volume of the reaction was then incubated for 4 h with 250 μL of Ni-NTA resin (Qiagen) equilibrated in co-purification buffer. Unbound protein was flowed through a PolyPrep column (Bio-Rad), and the resin was washed with 5 mL of co-purification buffer, followed by elution with 250 mM imidazole. The samples were then analyzed by SDS-PAGE, and the Coomassie-stained bands were quantified densitometrically using the program ImageJ43. For experiments with Sth1 alone, the ATPase level after cleavage was set to 1. To account for loading differences (before and after co-purification), the loss of ATPase after co-purification was calculated by normalizing the intensities of the His-tagged N-terminal fragments and adjusting the intensities of the ATPase bands accordingly. For experiments with Arp7/9, the ATPase levels were normalized using the intensities of Arp7 and Arp9 as an internal reference. The normalized ATPase band intensities for the TEV-digested samples were divided by 0.91, a factor accounting for the loss of intensity of the ATPase band upon TEV cleavage.A TEV site was engineered between Sth1 residues 424 and 432 by site-directed mutagenesis . Purified Sth1 constructs at 6 μM (±Arp7/9 and Rtt102) were incubated with 20 μg of TEV protease overnight in co-purification buffer  with 100 μg TEV protease. A volume of 500 μL of the digested sample was injected onto a Bio-Sil SEC 250 HPLC column (Bio-Rad) at a flow rate of 0.5 mL/min, and 0.25 mL fractions were collected starting at 15 min post injection. As a control, 500 μL of undigested Sth1, supplemented with an equivalent amount of TEV purification buffer, was also analyzed.Sth12 and 50 mM NaCl). Equimolar (100 μM) amounts of each strand were mixed at 95 °C and annealed by slow cooling to room temperature. For anisotropy experiments, the oligos contained a fluorescein label at the 5′-end.Oligos (5′-TCCATGTCCATGGATACGTGG-3′ and 5′-TCCACGTATCCATGGACATGGA-3′) were injected onto a Symmetry300 C18 column (Waters) equilibrated with 0.1 M TEAA pH 7.0 and purified on a 0–20% acetonitrile gradient. Following cycles of lyophilization and resuspension in 50% ethanol to fully remove the TEAA and acetonitrile, the oligos were resuspended in hybridization buffer  was equilibrated in hybridization buffer ) for 30 min at 20 °C. The anisotropy of free DNA was recorded with a Cary Eclipse fluorescence spectrophotometer (Varian) by excitation at 485 nm and recording the average anisotropy between 515 and 520 nm. Measurements were performed in triplicate. Sth1 (±Rtt102-Arp7/9) at ~20–40 μM was titrated, and recordings were taken after 4 min of stirring. The changes in anisotropy were obtained by subtracting the anisotropy of free DNA from that of DNA–protein complexes. Data were fitted to a single-site, hyperbolic binding isotherm with the program Igor (WaveMetrics) to obtain dissociation constants.388–1097 at 100 μM was incubated with a 1.5-fold molar excess of the double-stranded oligo (described above) for 2 h at 20 °C. The complex was loaded onto a TSKgel SuperSW2000 column (Tosoh Bioscience) connected inline with a DAWN HELEOS MALS detector and an Optilab rEX refractive index detector for mass analysis. Molecular masses were calculated with the program Astra (Wyatt Technology).Sth144. Briefly, Xenopus laevis core histones were expressed in BL21 Rosetta cells and purified from inclusion bodies. Histone octamers were reconstituted and purified by gel filtration chromatography. To determine the optimal ratio for nucleosome assembly, histone octamers were mixed with varying concentrations of 601 nucleosomal DNA for test assemblies. Nucleosomes were assembled according to the optimal DNA:histone ratio and purified by gel filtration chromatography.Histones were assembled and purified using the salt gradient dialysis method as previously described2, and 1 mM ADP (or AMPPNP). The concentration of nucleosomes or free DNA was held constant at 50 μM, and Sth1 constructs were added at a range of concentrations to a maximum of 10 μM. The reactions were incubated for 30 min at room temperature, and 10 μL aliquots were loaded onto a 4–20% TBE gel (Bio-Rad) in 0.5 × TBE. The gels were run at 4 °C and 150 V for 3 h (nucleosome) or 45 min (free DNA), and then stained with SYBR Gold  and imaged on a Bio-Rad Gel Doc Imager. The bands corresponding to the nucleosome core particle or the 21-bp DNA duplex were quantified densitometrically using the program ImageJ. For each lane, the nucleosome band was quantified and normalized to the no-Sth1 control, and these values were subtracted from 1 to obtain the fraction of nucleosomes bound.Native gel-shift assays were performed in 20 mM HEPES pH 7.5, 50 mM NaCl, 5% glycerol, 1 mM DTT, 0.1 mg/mL BSA, 1 mM MgClRtt102-Arp7/9 at 10 mg/mL was incubated with 1 mM ATP for 30 min, and then crystallized at 16 °C using the sitting drop method. Needle-like crystals were obtained in drops consisting of a 1:1 (v/v) mixture of protein and well solution (20% (v/v) PEG 3350 and 200 mM ammonium sulfate). These initial crystals were used to streak-seed hanging drops consisting of a 1:1.2 (v/v) mixture of protein and well solution (20% (v/v) PEG 3350, 170 mM ammonium sulfate, and 17 mM EDTA). The resulting larger crystals were flash-frozen in liquid nitrogen, using as cryo-protectant 25% glycerol added to the crystallization solution.45, using as a search model PDB entry 4I6M (the HSA domain was not included in the search). Model building and refinement were performed with the programs Coot46 and Phenix. The use of the program Rosetta47 in combination with Phenix significantly accelerated the convergence of the refinement during the initial steps. The Ramachandran plot of the final model revealed 97.0 and 0.12 percent of resides in favored and outlier regions, respectively. Composite omit maps . A molecular replacement solution was obtained with the program Phenixaps Fig.  were cal2, 0.1 mg/mL BSA, 200 μM MESG, and 1 unit of purine nucleoside phosphorylase. The reactions were stopped by addition of 250 mM EDTA, and the phosphate release was determined by measuring the absorbance at 360 nm48. For Sth1 constructs and complexes, the experiment was performed ±30 μM (nucleotide concentration) of the plasmid pRSF-Duet-1 (Novagen).Proteins at 5 nM were incubated with 1 mM ATP for 20 min at 22 °C in 20 mM HEPES pH 7.0, 50 mM potassium acetate, 2.5 mM MgClThe atomic coordinates and structure factors for the ATP-bound Rtt102-Arp7/9 complex described in this study have been deposited in the Protein Data Bank (accession code: 5TGC). The data that support the findings of this study are available from the corresponding author upon request.Supplementary Information


Pachyrhizus erosus (yam bean), which is a neglected and underutilized legume species. The chemical properties of flour produced from the yam bean include 5.8% moisture content, 5.7% crude fat, 6.2% crude fiber, and 85% available carbohydrate, indicating appropriate shelf‐stable flour, low fat, and abundant energy. The results also showed a reducing sugar content of 2.0% and 21.0% starch. Pasting temperature was 70.6°C with peak viscosity of 14.5 BU, which supports ease of cooking of the flour. The swelling power obtained was 752.9 g/100 g at 85°C with a solubility index of 54%. Water holding capacity (WHC) obtained for the flour was 363.88%, whereas swelling volume was 14.0 ml and makes the flour appropriate for the production of infant foods. The P. erosus flour therefore exhibits good functional and chemical properties that would make the flour quite suitable as a substitute for other flours in food systems.Many plant species that are suitable for food across the world are neglected and underutilized. In order to increase their diversified food uses and thus help enhance food and nutrition security, we studied the chemical and functional properties of  The genus comprises five species , commonly prepared in West Africa. This is buttressed by studies by Zanklan, Ahouangonou, Becker, Pawelzik, and Grüneberg  flour was a modification of the method described by Dorporto, Mugridge, Garcia, and Vina  and weighed. The weighed samples were spread thinly on separate drying trays and dried in a mechanical dryer  maintained at 55°C for 5.5 hr. The dried slices were weighed again and milled using a hammer mill  to an average particle size of 250 μm. The resulting P. erosus flour sample was sealed in double laminated sealable polyethylene bags and stored in a freezer for further analysis  equipped with a 10 cm‐g sensitivity cartridge. Ten percent slurry of the flour sample was prepared with distilled water and the slurry was heated uniformly (1.5°C/min) from 25°C to 95°C, held at 95°C for 15 min, and cooled at the same rate to 50°C  method was used for the post hoc multi comparison test. All the analyses were done with Statistical Package for Social Scientists (16.0) software.All determinations from the laboratory experiments were done in replicates. The statistical analyses were conducted using ANOVA. Significant statistical differences in samples were tested at 33.1P. erosus flour is shown in Table P. erosus flour was about 5.8%, which is within the acceptable range for commercial flours. Comparing the root (83.7%) and flour (5.8%) moisture values, it is obvious that the oven drying of the samples was effective in causing a reduction in the moisture content of the flour to ensure long shelf‐life. The moisture content recorded for P. erosus flour is lower than that reported by Ukpabi  root flour as 9.0%, which is similar to the value obtained for the dried P. erosus tubers used in this study. This value is more than that of other root crops like cassava, sweet potato, and yam. Sabanis and Tzia  but similar to corn (7.5%) and rice (7.0%). Similarly, P. erosus flour, due to its relatively high protein content and possibly its leguminous nature (with relatively high lysine and tryptophan content), can be composited with cereal flours and used as weaning foods for children to resolve nutritional deficiency problems. Probably, this can serve as a cost‐saving exercise since the current over‐reliance of soybean is becoming financially unbearable for most malnourished care givers.Crude protein obtained for the 8% Table . On dry‐n et al. , the autand Tzia  also repP. erosus flour  and only a small fraction of the value for taro flour (267.9 BU). Oguntunde  reportedP. erosus flour during prolonged cooking at 95°C was 7.5 BU. This is the difference between the peak viscosity and the viscosity after holding for 15 min at 95°C, an indication of the paste stability. Adebowale, Sanni, and Awonorin (P. erosus flour was able to withstand more heating and shear stress than taro flour (15.3 BU) and sweet potato flour (23.40 BU). The final viscosity was 16.50 BU, producing a setback viscosity of 2.00 BU from peak (and 9.50 BU from holding at 95°C). Final viscosity indicates the ability of starch to form various paste or gel after cooling  is an important parameter to be considered in the preparation of mashed snack foods and extruded foods as well as baked products. Water holding capacity (WHC) obtained for the flour sample was 363.88% Table . In theio et al.  reportedo et al.  for red Oledinma . High waOledinma . The higP. erosus flour was 14.0 ml (Table The swelling volume recorded for ml Table . Swellin4P. erosus flour is appropriate to guarantee a stable shelf‐life of the product. Its low fat content also reduces the risk of oxidation and prevents off‐flavor development resulting from rancidity. The low fat content of this flour is advantageous as a substitute for health food particularly for over‐weight individuals. The high protein content of P. erosus flour makes it quite suitable for weaning foods for children, and the high sugar content and lower pasting as well as the high water holding capacity makes it better flour in the production of infant supplement foods. These desirable attributes of the P. erosus flour make it suitable for partial or whole substitution of traditional flours for the production of different flour‐based foods.The low moisture content of the None declared.


In nearly all diploid eukaryotes, meiosis is the key phase in sexual reproduction. The typical process is completed by a round of DNA replication followed by two consecutive rounds of chromosome cell division . SCs are fully assembled during the pachytene, one of the five MI sub-stages   interacts with SYCP3 , a series of constructs were screened. One construct of the mSYCP2 NTR 1–390 aa) was successfully crystallized, and the corresponding structure was determined with 2.1 Å resolution using the single anomalous dispersion method of the Selenium atom  and an SLD (Spt16M-like domain). The ARLD is composed of fourteen helices  that may provide a platform for interactions with other partners. The SLD consists of a twisted ten-stranded β-sheet (β1 to β10) flanked by two helices (α15 to α16). Computational and structural homolog search analysis revealed that the ARLD structure is highly similar to that of the “required for cell differentiation (RCD-1)” protein . We also performed a DALI search for the SLD. Overall, the SLD structure is highly similar to the middle domain of the histone chaperone FACT . The Spt16M domain structure adopts a canonical Pleckstrin Homology (PH) domain architecture, which comprises a seven-stranded antiparallel-barrel that buries a hydrophobic core and has one end capped by a C-terminal helix. We speculate that the SLD may participate in a chromatin related process.To further address the functional role of mSYCP2 NTR, yeast two-hybrid screening assays were performed using the mSYCP2 NTR as bait, and a matched cDNA library prepared from adult mouse testis was used as the prey. Following the standard procedure, all clones were selected using nutrition-restricted plates. Fifteen mmol/L 3-amino-1,2,4-triazole (3-AT) was added to the growth medium each time to inhibit low levels of His expression in a leaky manner when a potential candidate was validated.http://www.ncbi.nlm.nih.gov/tools/vecscreen/), and then database searches were performed using the BLAST Service  (http://www.ncbi.nlm.nih.gov/BLAST). The identified proteins included two centromere components, CENP J and CENP F, meiosis specific function unknown protein Fam183b, transcription factor Pou5f2 and spermatogenesis associated 24 (Spata24)  of CENP J is required and sufficient for mSYCP2 NTR binding Fig. . To valiin vitro. It is possible that in addition to the SLD, another part of SYCP2 may be required for this association.Our results, for the first time, provide insight into the possible role of SYCP2 in SC assembly/disassembly. Our structural analysis revealed that mSYCP2 NTR contains two separate subdomains (an ARLD and a SLD) and that the overall conformation appears as a “twisted arm”. The ARLD domain belongs to the armadillo-repeat protein family. Previously published results showed that typical armadillo-repeat units often form a superhelix, which typically provides a platform for many protein partners that transduce Wnt signaling, such as β-catenin. We found that the ARLD of mSYCP2 can also associate with different protein partners, including CENP J and CENP F. We also tried to further address more specific residues of the ARLD that interact with CENP J and CENP F. Three SYCP2-NTR mutants  were built. These mutants were then transfected into COS7 cells and the co-IP experiment was conducted. However, we did not observe any differences between the wild type and mutants in terms of interacting with CENP J and CENP F . The complex structure of SYCP2 with CENP J or CENP F maybe required to map out the detailed interactions. Interestingly, the SLD domain structurally resembles a Spt16M molecule, which is known as the well-recognized histone protein H2A-H2B; thus, the SLD may be involved in chromatin binding. It was reported that SYCP2 is tightly bound to chromatin in wild type mice Below is the link to the electronic supplementary material.