Protein domains are generally thought to correspond to units of evolution. New research raises questions about how such domains are defined with bioinformatics tools and sheds light on how evolution has enabled partial domains to be viable. Genome Biology evaluate the implications of having the query sequence only matching part of a Pfam family, which is an intriguing finding, given that a Pfam family is considered to be an evolutionary unit.With the rapid expansion in the number of determined protein sequences - over 92 million in UniProt in March 2015 - an ever-increasing number of biologists are using bioinformatics tools for annotation of these sequences. One widely used strategy is to identify occurrences of Pfam families within the sequence of interest  in GenomIn discussing these articles, it must be emphasized that the definition of a protein domain is complex. One approach to define a protein domain is from a structural perspective. This was highlighted in Wetlaufer’s seminal article , where hAnother approach, currently used in Pfam , definesIn many proteins, the structural and the sequence-based approaches yield a very similar definition of the domains forming a protein. However, this is not always the case. In Pfam , the families are primarily derived based on sequence alignment, but additionally take into account the concept that a Pfam family is likely to be a structural domain. However, as we do not have the structures for all proteins, a Pfam family subsequently could be split once a structure is determined - but of course this takes time. Moreover, the structural definition of a domain involves substantial subjective input, and algorithms are able only to provide guidance. The extent of the problem of defining domains is highlighted in a recent study to map CATH (for ‘class-architecture-topology-homologous superfamily’)  to SCOP Genome Biology articles investigated partial matches to Pfam domains. Triant and Pearson [Two recent  Pearson  show tha Pearson  domains  Pearson  domains,A related question is considered by Prakash and Bateman . They idPhotobacterium phosphoreum LuxF [A particularly striking case (illustrated schematically in Figure eum LuxF . This doThe difficulties that can occur in Pfam-based protein annotations can also arise when other strategies are applied, such as in prediction of protein structure. For example, a web-based resource for template-based modelling is available for the community - Phyre2 . These t


On a single Bragg reflection, the intensity ratio in resonant diffraction below and above the Ti absorption K edge demonstrates a domain contrast up to a factor of ∼270, thus implementing a non-contact, non-destructive imaging technique with micrometre spatial resolution, applicable to samples of arbitrarily large dimensions.The identification and high-resolution mapping of the absolute crystallographic structure in multi-domain ferroelectric KTiOPO 4. Resonant  X-ray diffraction spectra collected across the absorption K edge of Ti (4.966 keV) on a single Bragg reflection demonstrate a huge intensity ratio above and below the edge, providing a polar domain contrast of ∼270. This allows one to map the spatial domain distribution in a periodically inverted sample, with a resolution of ∼1 µm achieved with a microfocused beam. This non-contact, non-destructive technique is well suited for samples of large dimensions , and its potential is particularly relevant in the context of physical phenomena connected with an absence of inversion symmetry, which require characterization of the underlying absolute atomic structure (such as in the case of magnetoelectric coupling and multiferroics).A novel method is presented for the identification of the absolute crystallographic structure in multi-domain polar materials such as ferroelectric KTiOPO For example, multiferroic materials often exhibit magnetic structures that are either driven by the underlying low symmetry of the crystal structure, or lead to a spontaneous symmetry breaking, often giving rise to strong magnetoelectric coupling. However, in relating the spin structures to the underlying atomic ordering, the latter must be characterized with equal confidence. One type of structure of interest is represented by polar systems and ferroelectrics.via the application of an external electric field using patterned electrodes. Materials such as KTiOPO4 (KTP), LiNbO3 and LiTaO3 can offer very good conversion efficiencies for any desired frequency within their transparency range.Among the crystal classes lacking inversion symmetry, those possessing at least a direction whose two senses are geometrically or physically different (polar direction) are called piezoelectric; among those, pyroelectric crystal classes possess a polar direction which has no symmetrically equivalent directions, and are therefore capable of sustaining a permanent electric dipole moment. Such systems are energetically degenerate  with their spatially inverted counterparts, leading to the possibility of multi-domain states. Ferroelectrics are a subset of pyroelectric crystals whose defining property is the ability to switch the sense of spontaneous polarization upon application of an electric field of magnitude greater than the coercive field. Piezoelectric crystals also exhibit nonlinear optical susceptibility leading to second-harmonic generation: monochromatic light waves passing through the crystal induce new waves of twice the incident frequency. A way to exploit this feature for a variety of applications is the quasi-phase-matching technique, implemented through the modulation of the second-harmonic coefficient along the direction of propagation. This can be obtained by preparing a ferroelectric crystal in a state of periodically spaced domains of alternating polarity, et al., 2011via the electron beam, besides the constraints placed on sample thickness in the case of TEM. Surface-sensitive techniques such as atomic force microscopy and electrostatic force microscopy are also employed, although the results may be difficult to interpret.A variety of experimental techniques to reveal the integrity of the domain periodicity and the quality of the resulting crystal have been traditionally employed  X-ray diffraction (RXD) which, in contrast to those outlined above, combines the features of being non-destructive and non-invasive with the further advantage of directly probing the squared structure factor of the diffraction cross section, and therefore of being related to the atomic positions, without the need for a phase-sensitive measurement apparatus. This technique measures the domain-specific intensity of the resonant structure factor in the material under investigation, rather than the phase shifts and interference between waves diffracted from opposite domains. As such, it is able to determine the absolute orientation in a sample with any arbitrary pattern of either single or multiple domains. Moreover, our approach is different from traditional resonant X-ray techniques in that it only requires diffraction profiles from a single Bragg reflection and is applicable to crystals of arbitrarily large dimensions; therefore, we suggest that this method can be established as a valuable tool in the characterization of those materials whose absolute structure plays an important role in the setting of physical properties such as the magnetoelectric effect and multiferroicity. By mapping the inversion domains in large crystals to establish their monodomain nature or obtain a spatial image of the domains, the information thus acquired can be combined, for example, with chiral spin structure data to obtain insight about the exchange interactions responsible for the magnetic structure and magnetoelectric coupling.E is sufficiently close to an absorption edge of one of the chemical species of the compound  with a grating period of Λ = 9.02 µm. In this standard bulk poling technique  by means of a U27 undulator insertion device and a channel-cut Si(111) monochromator, while the sample was mounted on a Newport six-circle kappa diffractometer at ambient temperature, equipped with translation motors to perform raster scans over the sample surface. A Pilatus 100K photon-counting area detector was used to collect the diffracted radiation. In order to maximize lateral resolution in the high-resolution domain imaging measurement, a micron-sized focal spot is required: this was achieved by means of a Kirkpatrick–Baez (KB) mirror system mounted on the diffractometer. This assembly allowed us to reach a beam spot size of 1.2 µm  × 1.5 µm .The experiment has been carried out at the Materials and Magnetism beamline I16 at Diamond Light Source, making use of its microfocus capability. The energy of the incident radiation was tuned at the Ti 3.K edge, as shown in the fluorescence scan across the absorption edge in the inset of Fig. 1The incident-beam energy was tuned to the Ti j atoms at positions A’ and ‘B’ is A’. Therefore the requirement outlined above implies that E = 5.007 keV above the edge, and E = 4.96 keV below the edge, thus satisfying all conditions.From the simulations, it was apparent that the reflection (417) is particularly sensitive to the domain composition. The reason for this can be explained as follows. The ultimate aim of the measurement is to discriminate between domains from the intensities collected at two fixed energies (above and below the edge). The ideal situation is one in which a significant intensity contrast between domains exists at an energy below the edge, and this contrast remains significant but reverses its sign at an energy above the edge. In this situation, the contrast is strong at both fixed energies and changes dramatically with energy across the edge. The former makes the ratio of intensities below and above the edge highly sensitive to the domain composition, while the latter allows domain orientation to be determined even when the total signal is highly inhomogeneous. The structure factor contributing to the intensity of reflection E = 4.96 and 5.007 keV), where the energy above the edge has been specifically chosen to correspond to an intensity of almost zero, in order to enhance the contrast between domains. At this stage the beam was defocused (with both main mirrors and KB mirrors out) and the beam spot was defined by the sample slits to be 0.015 × 0.05 mm. The results are plotted in Fig. 2A low-resolution map of the spatial distribution of domains was obtained by rastering the sample aligned on said reflection at the two different energies below and above the edge . As a way of confirming the validity of the technique, this measurement has been repeated on the two opposite surfaces of the sample, an operation that, given the symmetry of the crystal, is equivalent to collecting data on the same surface from the two opposite polar domains. Given the prevalence of multiple scattering peaks, the energy spectra have been repeated at three different azimuth angles, spaced by 1°, and averaged together with the exclusion of the outlying data points, in order to give a reliable profile. The results are plotted in Fig. 1E = 4.96 and 5.007 keV). The results are in Fig. 3x) × 1.75 µm . The stability of the microfocused beam has been characterized, resulting in a drift in position which is largely linear and of the order of a few microns per 12 h, roughly corresponding to the time necessary to acquire one map. The drift in position is corrected when extracting the point-by-point ratio between intensities from the two different maps, as part of the overall shift that had to be applied to correct for the centre of rotation. To obtain a quantitative estimate of the purity of the domains induced by the electric poling, we have collected a series of one-dimensional translation scans that cut through the a-poled domains along the direction x. One of these scans is plotted in Fig. 4a). The corresponding domain fraction profile has been extracted by comparison with the simulation of the sum of two incoherent diffraction domains ‘A’ and ‘B’ related by inversion, to which end the ratio of intensities between energies below and above the edge has been employed: F is the domain fraction (from 0 = domain ‘B’ to 1 = domain ‘A’), A’ (‘B’) at the two energies. The results for this specific scan cutting across the domains  indicate that the fraction of crystallographic domain ‘A’ ranges from ∼0.18 to ∼0.4. This same procedure has been extended to the whole high-resolution map, thus obtaining the complete two-dimensional pattern of domains in Fig. 5Since the experimental capability to distinguish between the crystallographic domains had been established, we proceeded to identify and map the surface area artificially poled by the electric field. To this end, we have made use of the microfocusing provided by the KB mirrors. The same procedure used in the case of the low-resolution map has been applied, rastering the sample aligned on the reflection (417) at the same energies below and above the edge . In the experiment by Lyford et al. . Since we obtain a semi-sinusoidal function for the domain modulation, the main features of the satellite distribution appear as one constant term , one main harmonic separated from the main reflection by 1/N reciprocal-space units , plus much weaker contributions from the finer details of the modulation. Comparing this with the analysis in Lyford et al. (2015This information can be compared with the results obtained on the same sample in Lyford  al. 2015 by meansns Fig. 4b. In th al. 2015, the bea al. 2015, it can 4.K edge, when measured on opposite surfaces of the sample to emulate diffraction from opposite monodomains, displayed domain contrast up to a factor of ∼270. The finely tuned sensitivity provided by this contrast has been exploited to obtain a µm-resolution image of the spatial distribution of domains, and to extract the domain fraction by comparison with a simple diffraction model based on the tabulated scattering coefficients of the resonant atom.In the case of periodically domain-inverted KTP, the energy dependence of the resonant X-ray diffraction from a single Bragg reflection has been exploited to determine the absolute crystallographic structure. The intensity ratio between two energy points below and above the Ti The quality of the sample under investigation has been thoroughly characterized in its periodically domain-inverted region, resulting in a domain fraction modulated as a sinusoidal curve varying within a range of about 20%, within our spatial resolution; this allows us to assess the efficacy of the electric poling as quite inferior to the ideal complete domain switching.The method introduced in this paper is of considerable potential, being applicable to samples in a state of either single or multiple domains in an arbitrary pattern and, equally importantly, of arbitrary dimensions, which confers it a significant advantage over other long-established techniques dependent on resonant X-ray scattering. A promising line of work in this respect is the crystallographic characterization necessary to the comprehension of those magnetic phenomena that are closely connected to the non-centrosymmetric nature of the underlying atomic structure, such as displayed in multiferroic materials. The contrast mechanism, exploited here in a scanning microscopy setup, is also well suited for development in full-field imaging techniques such as topography, thus enabling one to perform real-time characterization of domain dynamics, for instance to follow domain evolution under applied external fields.


Staphylococcus aureus (MRSA). Here, we find that the lytic activity of the full-length LysGH15 and its CHAP domain is dependent on calcium ions. To elucidate the molecular mechanism, the structures of three individual domains of LysGH15 were determined. Unexpectedly, the crystal structure of the LysGH15 CHAP domain reveals an “EF-hand-like” calcium-binding site near the Cys-His-Glu-Asn quartet active site groove. To date, the calcium-binding site in the LysGH15 CHAP domain is unique among homologous proteins, and it represents the first reported calcium-binding site in the CHAP family. More importantly, the calcium ion plays an important role as a switch that modulates the CHAP domain between the active and inactive states. Structure-guided mutagenesis of the amidase-2 domain reveals that both the zinc ion and E282 are required in catalysis and enable us to propose a catalytic mechanism. Nuclear magnetic resonance (NMR) spectroscopy and titration-guided mutagenesis identify residues  in the SH3b domain that are involved in the interactions with the substrate. To the best of our knowledge, our results constitute the first structural information on the biochemical features of a staphylococcal phage lysin and represent a pivotal step forward in understanding this type of lysin.The lysin LysGH15, which is derived from the staphylococcal phage GH15, demonstrates a wide lytic spectrum and strong lytic activity against methicillin-resistant  Staphylococcus aureus (MRSA). Here, we report that the lytic activity of LysGH15 and its CHAP domain is dependent on calcium ions. To elucidate the molecular mechanism, we determined the structures of three individual LysGH15 domains using X-ray crystallography or nuclear magnetic resonance (NMR). The crystal structure unexpectedly reveals an “EF-hand-like” calcium-binding site near the Cys-His-Glu-Asn quartet active site groove in the LysGH15 CHAP domain. Furthermore, the calcium ion plays an important role as a switch that modulates the lytic activity of the CHAP domain. Additionally, structure-guided mutagenesis also confirms that both E282 and the zinc ion play an important role in maintaining the lytic activity of the LysGH15 amidase-2 domain. Moreover, the NMR structure and titration-guided mutagenesis identify residues in the LysGH15 SH3b domain that are involved in the interactions with the substrate. The structure of LysGH15 is the first determined lysin structure from a staphylococcal phage, and these results represent a pivotal step forward in understanding this type of lysin.The staphylococcal phage lysin LysGH15 demonstrates great potential against methicillin-resistant  Staphylococcus aureus is a common habitant of the human skin and respiratory tract, several highly pathogenic strains are major causes of hospital-associated infections and can be life threatening, particularly in immunocompromised patients S. aureus (MRSA) infection, particularly as caused by community-associated MRSA (CA-MRSA) isolates, has dramatically increased worldwide, which raises serious concerns within the medical community Although Lysin  is a cell wall hydrolase that is synthesized at the end of the phage lytic life cycle and is involved in cell lysis and the release of progeny particles from host cells in vitro and in vivocysteine, histidine-dependent amidohydrolases/peptidases), a central amidase-2 domain , and a C-terminal SH3b domain  In our previous study, we reported that LysGH15, which is encoded by the staphylococcal phage GH15, demonstrates strong lytic activity against MRSA In this study, we report that the lytic activity of the LysGH15 CHAP domain is critically dependent on calcium ions. To elucidate the molecular mechanism, we determined the structures of three individual LysGH15 domains. The crystal structure of the CHAP domain unexpectedly reveals a calcium-binding site near the Cys-His-Glu-Asn quartet active site  . Sedimentation velocity experiments using analytical ultracentrifugation (AUC) indicated that the three individual domains all exist as monomers in solution .Bioinformatic analysis PSIPRED  and knowThe activity of each individual catalytic domain was determined Table 1.PSI-BLAST analysis using the National Center for Biotechnology Information (NCBI) database reveals that the amidase-2 domain contains a conserved zinc-binding site. To determine the role of the zinc ion in the activity of the amidase-2 domain, the combination of the CHAP and amidase-2 domains was pretreated with EDTA (1 mM), and excess EDTA was removed by dialysis. Surprisingly, the lytic activity of the combination was completely abolished. Additionally, the individual CHAP domain is also sensitive to EDTA Figure 2E. coli, but the protein exhibited degradation. Although the introduction of a C-terminal His-tag improved the stability of LysGH15, the slow degradation of LysGH15 was not amenable to crystallization, which hindered our initial attempts to crystallize full-length LysGH15. However, the crystallization of individual LysGH15 domains was possible, and the respective structures were successfully determined (Table S1). The X-ray crystal structures of the CHAP and amidase-2 domains were determined using the selenium single-wavelength anomalous dispersion (Se-SAD) and iodide single-wavelength anomalous dispersion (I-SAD) methods, respectively, due to the lack of homologous structures. Crystals of the individual CHAP domain encompassing residues 1–165 and of the amidase-2 domain encompassing residues 165–403 were obtained, as shown in Table S2). The NMR spectra of the SH3b domain were acquired using a construct containing residues 368–495 . Iterative rounds of model building followed by refinement result in a model with good statistics and geometry (Table S1). The quality of the electron density permits the unambiguous modeling of residues 1–164. The final model is refined to 2.69 Å resolution . The two molecules form a dimer that is mediated by hydrogen bonding interactions at the central interface by one molecule of Bis-Tris-propane that was present in the crystallization reservoir solution. However, a Bis-Tris-propane molecule does not mediate homodimer formation of the CHAP domain in solution and cannot affect the activity of the CHAP domain. The CHAP domain forms a globular structure that is comprised of three α-helices that are packed against six β-sheets, as shown in The CHAP domain crystallizes in the 2+ ion that forms the classical 12-residue  calcium-binding site. The presence of the calcium was further confirmed using inductively coupled plasma atomic emission spectrometry (ICP-AES) analysis. The side chains of D45, D47, and D56 and the main chains of Y49 and H51 are 2.36, 2.37, 2.13, 2.32, and 2.27 Å, respectively, from the Ca2+ ion, as shown in 2+ is approximately 27 µM . The circular dichroism (CD) spectroscopy showed that the presence/absence of Ca2+ does not affect the secondary structures of the LysGH15 CHAP domain . However, the fluorescence-based thermal shift assays showed ∼2°C shift in Tm of the CHAP domain with/without Ca2+, which indicated that Ca2+ has a slight contribution to the protein thermostability .Notably, as shown in Staphylococcus saprophyticus SSP0609 and several other proteins  . The architecture and size of this groove indicate that it most likely serves as the binding site for a portion of the peptidoglycan. In the center of the groove, C54, H117, E134, and N136 form the Cys-His-Glu-Asn quartet. C54 is positioned at the beginning of helix α1, followed by H117 at the beginning of strand β3, E134 at the end of strand β4, and N136 proximal to the beginning of the loop that links strands β4 and β5 . This potential active site is solvent exposed and lies within a shallow groove on the protein surface, which is consistent with its ability to cleave a highly crosslinked and branched polymer.The structure of the amidase-2 domain (residues 165–403) was determined at 2.27 Å resolution. As shown in Bacillus phage (PDB ID: 1YB0) α5α6 are well conserved because they contain the zinc-binding sites and conserved residues, including N275 and E282.We compared the structure of the LysGH15 amidase-2 domain with other proteins available in the PDB, as shown in N-acetylmuramoyl-L-alanine amidases Mutation of the three zinc-binding residues to alanine results in a complete loss of the activity  of the LysGH15 amidase-2 domain, as shown in β1β2, Lβ2β3, Lβ6β7, and Lβ7β8) exhibit larger RMSD values compared with the β-strand regions in the structure, suggesting that these loops exhibit higher flexibility. The overall structure of the LysGH15 SH3b domain is very similar to the SH3b domain of the ALE-1 protein  , the peptide “AGGGGG” was used to perform NMR titrations of the 15N-labeled LysGH15 SH3b domain, as previously reported for the SH3b of ALE-1 1H-15N HSQC spectra of the LysGH15 SH3b domain, as shown in Figure S5A. The equilibrium dissociation constant KD is approximately 3.0 mM, as obtained by fitting the titration curve of the CSPs , which indicates that the binding is quite weak and may explain the inability to cocrystallize the ALE-1 SH3b domain with the polyglycine peptide in a previous study In light of the sequence and structural similarities with the SH3b domain of ALE-1, in addition to an identical target bacteria . The potential binding site is consistent with the polyglycine binding site of the ALE-1 SH3b domain that was proposed by previous docking and mutagenesis analysis Residues exhibiting large CSP values are clearly clustered in the sequence, and mapping of the CSP results onto the structure of the LysGH15 SH3b domain reveal that these residues may interact with the peptide. The residues with significant CSPs are largely from β1, β2, β5, loop LTo determine the effect of these residues on the binding, these residues were mutated to alanine in the individual SH3b domain of LysGH15. As shown in In this study, we report that the lytic activity of the LysGH15 and its CHAP domain is critically dependent on a calcium ion. To confirm these findings, the structures of the individual CHAP, amidase-2, and SH3b domains of LysGH15 were determined. This study represents the first report of the structure of a staphylococcal phage lysin.Interestingly, the structural studies reveal that the CHAP domain of LysGH15 contains a calcium-binding site that is located near the active site groove. This finding is unexpected because the calcium-binding site is not detectable through sequence-based searches alone 2+-binding motif. The “EF-hand-like” motif differs from the classical EF-hand as follows: (i) the length of the Ca2+-binding loop is shorter or longer than 12 residues and/or (ii) the secondary structure elements of the flanking regions are not two helices Bacillus anthracis (PDB ID: 1ACC) Clostridium thermocellum (PDB ID: 1DAQ) A classical EF-hand protein contains a helix–loop–helix CaE. coli glutathionylspermidine (GSP) synthetase, which operates via a nucleophilic mechanism involving C59 as the catalytic nucleophile As in the thermolysin-like protease, the calcium ion plays an important role as a switch that modulates the protease between active and inactive states according to the biological demand http://pfam.sanger.ac.uk/family/PF05257), which exhibit high sequence identity with the LysGH15 CHAP domain in the calcium-binding site  and in the Cys-His-Glu-Asp proteolytic active site  also exhibit this sequence identity.Some members  in the Pfam sequence database  and were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The coding regions for the CHAP (residues 1–165), amidase-2 (residues 165–403), and SH3b (residues 368–495) domains were cloned into the pMCSG7 vector as previously reported lysGH15 gene was subcloned into the pET-26b vector. Mutations were designed based on these constructs and were generated using the QuikChange Site-Directed Mutagenesis Kit following the manufacturer’s instructions (Stratagene). All of the recombinant plasmids were sequenced to verify the sequence.The genes for the full-length LysGH15 and its three individual domains  were amplified using corresponding primers that were designed based on the full-length E. coli BL21(DE3) (Tiangen Biotechnology). The cells were grown in Luria-Bertani (LB) medium at 37°C until the OD600 reached 0.8. The culture was then induced with 0.2 mM isopropyl-β-D-thiogalactoside (IPTG) for 20 h at 16°C. Cells were harvested by centrifugation at 4,670×g for 30 min and were resuspended in phosphate-buffered saline . After lysis by sonication, the cell debris was removed by centrifugation at 38,900×g for 30 min. The supernatant was applied to a nickel-nitrilotriacetic acid (Ni-NTA) resin gravity column (Qiagen) that had been previously equilibrated with PBS. The column was washed using 100 ml of lysis buffer containing 20 mM imidazole, followed by a 50 mM imidazole wash. Finally, the protein was eluted with PBS containing 500 mM imidazole. After buffer exchange, the 6× His-tag was removed using tobacco etch virus (TEV) proteolysis (except for full-length LysGH15). Uncut protein was removed using a second Ni-affinity chromatography step. The proteins without a His-tag were concentrated and applied to a Superdex G200 size-exclusion chromatography column (Amersham) that was preequilibrated with 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl (500 mM NaCl for full-length LysGH15). For the SH3b domain, 40 mM Na3PO4 and 50 mM NaCl, pH 6.5, were used. Fractions containing the purified target protein were pooled and stored at −80°C until further analysis.The plasmids harboring the target gene, which encoded 6× His-tagged proteins, were transformed into E. coli BL21(DE3) strain that contained the pMCSG7-CHAP vector was grown in M9 medium containing glucose (0.2% M/V), MgSO4 (1 mM), and ampicillin (100 µg/ml) at 37°C until the OD600 reached 0.8. Subsequently, selenomethionine was added to the culture (50 µg/ml). The subsequent purification steps were similar to those used for the native protein.The E. coli BL21(DE3). The cells were grown in M9 medium containing glucose (0.2% M/V), MgSO4 (1 mM), and ampicillin (100 µg/ml). 15N ammonium chloride and/or 13C glucose was used as the sole nitrogen and carbon sources, respectively, for isotope labeling. Labeled SH3b was purified using an identical procedure as that used for the native protein.The plasmid pMCSG7-SH3b was transformed into 2SO4, 30% (wt/vol) PEG 3,000, and 0.1 M xylitol. For phase determination, crystals of amidase-2 were soaked for 5 min in crystallization solution supplemented with 10 mM potassium iodide (KI) prior to cryoprotection and freezing.All of the proteins were initially screened for crystallization using the hanging-drop vapor diffusion method and commercially available sparse matrix screens at 16°C. Crystals were obtained by mixing 1 µl of the protein solution with an equal volume of the reservoir solution and equilibrating the mixed drop against 300 µl of the reservoir solution. Crystals of CHAP and Se-Met-CHAP were grown in a solution containing 0.1 M Bis-Tris-propane, pH 7.5, and 3.8 M sodium formate using 10–15 mg/ml of the proteins. The crystal of the amidase-2 domain was grown using 10–18 mg/ml of the proteins and a reservoir solution containing 0.1 M Tris-HCl, pH 9.0, 0.2 M Li2DF structure determination pipeline Table S1.Diffraction data for native amidase-2 domain crystals and crystals soaked with KI were collected at beamline BL5.0.1 . Otherwise, diffraction data for the native CHAP domain crystal and anomalous diffraction data for the selenomethionine CHAP domain crystal were collected at 100 K using an ADSC Q315 CCD detector at beamline BL17U1 of the Shanghai Synchrotron Radiation Facility (SSRF). The data sets were indexed, integrated, and scaled using the HKL2000 software 15N and/or 13C) contained 0.02% (w/v) sodium 2,2-dimethylsilapentane-5-sulfonate (DSS) and 10% (v/v) 2H2O. All NMR experiments were performed at 298 K on an Agilent DD2 600 MHz NMR spectrometer that was equipped with a Z-gradient triple-resonance cryoprobe, as previously described with some modifications 1H-15N and 1H-13C HSQC and, three-dimensional CBCA(CO)NH, HNCACB, HNCO, HN(CA)CO, HBHA(CO)NH, HCCH-TOCSY, and CCH-TOCSY experiments were performed for SH3b backbone and side chain assignments. Three-dimensional 1H-15N and 1H-13C NOESY-HSQC spectra with mixing times of 150 ms were collected to generate distance restraints. All data were processed using NMRPipe 15N and 13C chemical shifts were referenced indirectly Table S2.NMR samples of the SH3b domain (labeled by ATCC) and was used throughout the study. Staphylococcal lytic assays were performed using an overnight culture of USA300 grown at 37°C in tryptic soy broth (TSB) supplemented with 1% wt/vol yeast extract. Staphylococci were washed in PBS (pH 7.4) and resuspended at an OD600 of approximately 1.0. Bacteria were mixed with the wt or mutant proteins, and the OD600 was kinetically measured in a spectrophotometer for 120 min. All assays were performed in triplicate. Peptidoglycan was isolated from stationary phase cultures of USA300, as previously described with some modifications 578 of approximately 0.6. When the protein was processed using EDTA (1 mM), the excess EDTA was removed by dialysis. The peptidoglycan lytic activity was measured as the decrease in the OD578 for 60 min.The CA-MRSA strain USA300-TCH1516 was obtained from the American Type Culture Collection (AGGGGG” was synthesized by Sangon Biotech (Shanghai) Co., Ltd. A stock solution (100 mM) of the peptide was prepared in a buffer that was identical to that used for the SH3b protein sample. The interaction between SH3b and the peptide was detected by monitoring the two-dimensional 1H-15N HSQC spectra of SH3b during the titration. The observed CSPs were calculated as previously described HN and δN are the changes in the 1HN and 15N chemical shifts, respectively. The equilibrium dissociation constants (KD) were estimated by fitting the CSPs to the following equation:max is the CSP at the theoretical saturated condition, which was also obtained from the fit; r is the molar ratio of the peptide to the protein; Cpro is the concentration of the initial protein solution; and Clig is the stock concentration of the peptide.The peptide “2SO4. The optical density was measured at 450 nm in a spectrophotometer.The protocols of ELISA used here were similar to those previously described with some modifications www.analyticalultracentrifugation.com).Sedimentation velocity experiments were performed using a Beckman XL-I analytical ultracentrifuge at 20°C as previously described Inductively coupled plasma atomic emission spectrometry  was used for the metallic evaluation of protein samples at Tsinghua University, as previously described with modifications 2 was stepwise injected into 50 µM the Ca2+-free CHAP protein sample. The data were analyzed with the MicroCal Origin software.Measurements were conducted at 16°C using an ITC-200 microcalorimeter  as described previously 2+ was adjusted to 0.15 mg/ml in 20 mM Tris, pH 7.5, 200 mM NaCl prior to the measurements. Wavelength spectra were recorded at 20°C using a 0.1-cm path length cuvette. Each scan was obtained by recording every 1 nm with a bandwidth of 1 nm between the wavelength ranges of 200–260 nm.CD spectra were acquired on a Chirascan CD Spectrometer (Applied Photophysics) according to the previous description 2+ in a buffer  supplemented with a 1,000 dilution of SYPRO Orange dye (Invitrogen) as described previously Thermal shift assays were conducted using 0.2 mg/ml of the CHAP protein with or without CaThe atomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB) under accession codes 4OLK for the CHAP domain, 4OLS for the amidase-2 domain, and 2MK5 for the SH3b domain. The BMRB accession ID for the SH3b domain is 19752.t-test at a level of significance of P<0.05.Statistical significance was determined using the unpaired two-tailed Student’s Figure S1Purification of LysGH15 and its three individual domains. (A) Size-exclusion chromatography (SEC) of the full-length LysGH15 and its three individual domains . SDS-PAGE analysis (inset) of the protein collected from each major peak. (B) Analytical ultracentrifugation (AUC) analysis of the three individual LysGH15 domains .(TIF)Click here for additional data file.Figure S2The equilibrium dissociation constant of the CHAP domain in the presence of calcium ions was determined using ITC. (A) 600 µM CaCl2 was stepwise injected into 50 µM the CHAP protein samples. (B) 600 µM CaCl2 was injected into the buffer as control.(TIF)Click here for additional data file.Figure S32+ binding to the LysGH15 CHAP domain.The biophysical effect of Ca (A) Circular dichroism (CD) spectroscopy of the CHAP domain with/without Ca2+. (B) The thermal shift assays of the CHAP domain with/without Ca2+.(TIF)Click here for additional data file.Figure S4Alignment of sequences and surface rendering of the groove. (A) The CHAP domain. (Left), alignment of the 35 unique protein sequences indicating the conserved residues; (right), surface rendering of the groove in the LysGH15 CHAP domain. (B) The amidase-2 domain. (Left), alignment of the 44 unique protein sequences; (right), surface rendering of the groove in the LysGH15 amidase-2 domain. (C) The SH3b domain. (Left), alignment of the 26 unique protein sequences; (right), surface rendering of the groove in the LysGH15 SH3b domain. This figure was generated using the ConSurf server (http://consurftest.tau.ac.il/) and PyMOL (TIF)Click here for additional data file.Figure S5AGGGGG”.Titration of the LysGH15 SH3b domain with the peptide “ (A) 1H-15N HSQC spectra of the titration of the SH3b domain with the peptide “AGGGGG”. Residues exhibiting chemical shift perturbations are labeled. (B) KD values for binding of the SH3b domain to “AGGGGG”. The fit of the four residues exhibiting the largest chemical shift perturbations are shown with the determined KD values. (C) A detailed view of the residues in the SH3b domain that interact with the “AGGGGG” peptide. The residues involved in the interaction with the peptide are shown as sticks.(TIF)Click here for additional data file.Table S1Data collection and refinement statistics.(DOC)Click here for additional data file.Table S2The experimental restraints and structural statistics for the 20 lowest energy structures of the LysGH15 SH3b domain.(DOC)Click here for additional data file.


Prolyl oligopeptidases (POPs) are proteolytic enzymes, widely distributed in all the kingdoms of life. Bacterial POPs are pharmaceutically important enzymes, yet their functional and evolutionary details are not fully explored. Therefore, current analysis is aimed at understanding the distribution, domain architecture, probable biological functions and gene family expansion of POPs in bacterial and archaeal lineages.Exhaustive sequence analysis of 1,202 bacterial and 91 archaeal genomes revealed ~3,000 POP homologs, with only 638 annotated POPs. We observed wide distribution of POPs in all the analysed bacterial lineages. Phylogenetic analysis and co-clustering of POPs of different phyla suggested their common functions in all the prokaryotic species. Further, on the basis of unique sequence motifs we could classify bacterial POPs into eight subtypes. Analysis of coexisting domains in POPs highlighted their involvement in protein-protein interactions and cellular signaling. We proposed significant extension of this gene family by characterizing 39 new POPs and 158 new α/β hydrolase members.Our study reflects diversity and functional importance of POPs in bacterial species. Many genomes with multiple POPs were identified with high sequence variations and different cellular localizations. Such anomalous distribution of POP genes in different bacterial genomes shows differential expansion of POP gene family primarily by multiple horizontal gene transfer events.The online version of this article (doi:10.1186/1471-2164-15-985) contains supplementary material, which is available to authorized users. Proteases are degradation machines that aid in the proper functioning of cells by sustaining the balance between protein synthesis and hydrolysis. The proteolytic enzymes are also involved in post-translational modifications and generation of active peptide molecules in the cell . In factA particular type of serine protease that can hydrolyse internal proline residues distinctly is referred as prolyl oligopeptidase  , 10, 11.Myxococcus xanthus, Sphingomonas capsulata and Aeromonus punctata suggested two domain architecture with a sequentially discontinuous catalytic α/β hydrolase and a β-propeller domain [Aeromonus punctata suggested induced-fit mechanism of substrate entry, where addition of a substrate induces large-scale conformational changes in two domains along with alterations at the active site [In most of the species, POPs are ~700 residues long with a cylindrical structure . Crystalr domain , 16. Theive site . POPs frive site .POP family’ (S9 in MEROPS), which also includes dipeptidyl peptidase IV , acylaminoacyl peptidase  and oligopeptidase B  [POPs are one of the members of the larger ‘PB, S9A) , 11. AllPB, S9A) , DPP libPB, S9A) . OPB clePB, S9A)  and ACC PB, S9A) . DPPs arPB, S9A) –23. POP PB, S9A) , 25.Flavobacterium meningosepticum, Sphingomonas capsulatum and Myxococcus xanthus has shown rapid cleavage of them [Porphyromonas gingivalis, which is a major pathogen associated with adult periodontitis [T. brucei, the causative agent of African trypanosomiasis [POPs and POP family members are pharmaceutically important enzymes. bPOPs are considered as therapeutic agents for the oral treatment of celiac sprue, which is a small-intestinal disorder caused by abnormal response to gluten proteins . High pr of them . Physioldontitis . Similarsomiasis . In trypsomiasis .The availability of genomic information of many bacterial and archaeal species offers a great opportunity to understand the detailed distribution and biochemical role of POPs in prokaryotic lineages. Motivated by the clinical importance of POPs, we have carried out genomic identification of POPs and its homologs using exhaustive sequence search procedures. We found POPs to be widely distributed in all the classes of bacteria and archaea with diverse domain architectures. These bPOPs were depicted to be involved in protein-protein interactions and cellular signaling. Rigorous sequence searches employed in this study aided the identification of many additional POPs, which were not characterized earlier. Detailed clustering and identification of class specific sequence motifs allowed classification of bPOPs into eight subtypes. We found that multiple horizontal gene transfer events were responsible for the differential expansion of POP gene family in bacteria. To our knowledge, this is the first analysis that reports the presence of multiple POPs in many bacterial genomes.-10, only 1,791 POP homologs could be identified, while relaxing the E-value to 10-3 could capture 3,387 additional POP homologs  and archaeal (Nanoarchaeota and Thaumarchaeota) lineages no POP homologs could be identified. BLAST searches also failed to capture POP homologs in these phyla except in Dictyoglomi[. However, sequence searches against appended-PALI + database could pick at least one POP homologue in the above phyla except for Nanoarchaeota[The collected hits included annotated POPs, POP family members and nearby hydrolases of α/β hydrolase superfamily. Altogether, they are referred as ‘POP homologs’ in this report. In certain bacterial  in prokaryotic lineages than DPPs (24%) and OPBs (10%)  Figure . We coulBacteroides (78%) and Acidobacteria (75%) had maximum number of POP homologs with signal peptides, while in some bacterial phyla  signal peptides were completely absent. POP homologs from gram-positive bacterial phyla (Actinobacteria and Firmicutes) showed relatively less number of signal peptides.Earlier studies have shown that bPOPs are associated with the signal peptides . Signal Spirochaetes and Fusobacteria.Recently, membrane-bound forms of POPs isolated from synaptosomal membranes of bovine brain were also reported , 34. CytProteobacteria, while in mycobacterial species POPs were replaced by other hydrolases. Within a phylum, anomalous distribution of POPs was observed. Mapping of domain architecture on archaeal species tree depicted presence of only C-terminal POP domain in most of the organisms, while full-length POP domains were observed in a few species of Crenarchaeota  repeats. Two of the ‘C-terminal processing peptidases’ (S41) had PDZ domains, which are associated with signaling proteins of bacteria, plants and higher order organisms. PDZ domains are involved in assembly of large protein complexes, thereby coordinating and guiding the flow of regulatory information [Candidatus Solibacter usitatus (YP_821861) and Roseiflexus (YP_001276641) were associated with WD40 and DPP domains. TPR repeat motifs facilitate interaction with other proteins. These motifs were also related with hydrolase domain in Candidatus Solibacter usitatus (YP_824720). TPR-proteins are also associated with multi-protein complexes, and are involved in functioning of chaperones, cell-cycle, transcription and protein transport complexes [POP homologs were frequently associated with protein-protein interaction domains ormation , 37. PDZomplexes , 39.Ferrimonas balearica (YP_003914375) was predicted to be associated with Sel1 repeats.POP homologs were also associated with signaling modules such as WD-repeats. Proteins with WD-repeat exhibit high degree of functional diversity –42. Somevia a helix-turn-helix motif [Candidatus Solibacter usitatus (YP_827731) and Caulobacter segnis (YP_003594106), and those with transcription regulatory domain include four homologs (two each from Actinobacteria and Gammaproteobacteria (YP_888147 (Mycobacterium smegmatis), YP_001759306 (Shewanella woodyi), YP_735011 (Shewanella sp. MR-4) and YP_954217 ). Targeted deletions of the predicted accessory domains will be beneficial to understand the related biological functions.Bacterial POP homologs were also found to co-exist with several DNA-binding modules of transcription regulatory domains. Numerous bacterial transcription regulatory proteins bind DNA ix motif . These aix motif , 47. POPProteobacteria, most of the POPs were periplasmic in nature. Clustering analysis of the predicted cytoplasmic and periplasmic POPs resulted in a clear separation of cytoplasmic and periplasmic POPs with a few exceptions.We have also examined the cellular localization of annotated bPOPs to infer the possible functions of POP in more detail. Prediction of cellular localization using PSORT-b also revealed cytoplasmic nature of the annotated POPs (176 versus 115 POPs which were predicted to be periplasmic) , which are located at the interface of two domains. These sites were replaced by non-polar and positively charged amino acids in most of the subtypes of bPOPs  were considered as an outgroup. We observed that OPBs and DPPs were distinctly clustered, while the ACCs and POPs were dispersed all over the tree . Moreover, we could identify 12 copies of POP gene in Shewanella piezotolerans, and 10 copies each in Shewanella pealeana and Shewanella sediminis. Besides genus Shewanella, 15 POP genes were also identified in Solibacter usitatus. High sequence variations in paralogs of POP suggested that they are not closely related to each other, except in S. thermophilus genome  and gene duplication are the two driving forces, which may lead to expansion of gene families in prokaryotic systems . We haveer lakes . We exam-3 and it had similar domain architecture (with both α/β hydrolase and β-propeller domains). During this analysis some partial POPs comprising of only catalytic domain were also identified  or with incomplete propeller domain  or with only hydrolase domain (e.g. Mycoplasma genomes). These partial POPs could be due to errors in the available gene prediction algorithms. Additionally, wrong or incomplete annotation of collected protein sequences could also lead to another source of error. Experimental validation of these reported sequences would help in improving the current annotations of the corresponding genomes.During this study, we have also encountered many incomplete POP sequences with either missing N-terminal hydrolase domain  proteomes were downloaded from NCBI. These proteomes were further classified according to NCBI Taxonomy database into 23 bacterial and 4 archaeal phyla. These bacterial species include diverse groups of extremophiles, pathogens, model organisms and symbiotic bacteria.All the prokaryotic proteomes were scanned for the presence of POPs using BLAST and HMM-SEARCH  of the HMMER suite were used at stringent E-values of 10and 10-10. Homologand 10-10. This ap-3 and 10-10. This approach helped in picking homologs from proteomes, where annotated POPs were absent.Profile based approach: Bacterial and archaeal specific profiles were generated with a member (POP) from each phylum using HMMbuild. This could help in picking nearest POP homologs from all the collected bacterial proteomes. Besides archaeal and bacterial-specific profiles, an integrated profile was also generated using bacterial, archaeal and eukaryotic POPs. Furthermore, HMMsearch was performed using these three different profiles at two different E-values of 10-3 were further confirmed as ‘true homologs’ by constructing the database of sequences obtained at stringent E-value of 10-10. All the sequences found at E-value of 10-3 were considered as query and BLAST was performed against the database of ‘true homologs’. Domain definitions were identified using HMMScan against Pfam database for all the collected hits [Homologs collected at the relaxed E-value of 10ted hits . A hit wTo be sure that none of the POPs were missed, BLASTP was also performed against some of the selected proteomes. Furthermore, some phyla were also screened using an indirect approach of appending bacterial proteomes to PALI + database, which comprises of trusted homologs of proteins of known three-dimensional structures .Relative abundance of POP homologs was calculated using relative density. Relative density is defined as the total number of serine proteases (POP homologs) identified in a taxonomic lineage by the total number of genomes of that lineage, which were considered for the study. Relative occurrence is defined as the total number of POP homologs identified in a taxonomic lineage by the total number of gene products in a taxonomic lineage.-3), which scans the sequences against HMMs of Pfam database. Transmembrane regions of all the hits were examined using TMHMM [Domain assignments were mapped using HMMPfam Additional file 1: Table S1a: Sequenced bacterial and archaeal genomes analysed in this study. Table S1b: BLAST searches of Distribution of the sequenced genomes of bacterial and archaeal lineages. Distribution of sequenced bacterial (A) and archaeal (B) genomes. (PDF 2 MB)Additional file 2: Schematic representation of the pipeline followed to collect true homologs.(PDF 2 MB)Additional file 3:A. Enrichment of true homologs. B. Relative abundance and occurrence. C. Other domain architectures of POP homologs. D. Different cellular localization. E. Cluster-wise sequence identity. F. Structural mapping of sequence motifs of each cluster. G. Functional domains of annotated bacterial POPs are conserved and glycine rich. H. Divergence of POP family members. I. Detailed analysis of POPs of Shewanella. J. Sequence similarity searches to understand HGT events. (DOCX 2 MB)Additional file 4: Distribution of POP-family members in different bacterial and archaeal phyla.(PDF 76 KB)Additional file 5:Domain architecture of POP homologs.Abbreviations: POP_C-prolyl oligopeptidase C-terminal, DPP_N-Dipeptidyl peptidase N-terminal, WD-WD domain, ABH-α/β hydrolase, DLH-Dienelactone hydrolase, EstPHB-Esterase PHB, Xpro -X-Pro dipeptidyl-peptidase, ABC- ABC transporter, TFB- transcription regulatory domain, TAP- TAP-like protein, TPR- Tetratrico peptide repeats, CNB- cyclicnucleotide binding, Osmc- OsmC-like protein, ASST- Arylsulfo transferase, KAS- beta-ketoacyl synthase, AT- Acyl transferase, AD- Alcohol dehydrogenase, ZnD- Zinc binding dehydrogenase, Branched chain aa- Branched chain amino acid, PRT- Phosphoribosyl transferase, DUF- Domain of unidentified function, BRP- Bacterial regulatory protein, FGS- Formylglycine generating sulfatase, S-layer- S-layer homology, TRD- Transcriptional regulatory domain, SMP-SMP-30/gluconolactonase/LRE-like region.(PDF 432 KB)Additional file 6:Cellular localization of annotated bPOPs.(PDF 3 MB)Additional file 7:Detailed phylogeny of annotated bPOPs. Color code: Thermotogae-cyan, Firmicutes-lime, Chloroflexi-green, Deinococcus-thermus-blue, Chlorobi-magenta, Actinobacteria-blue, Acidobacteria-yellow, Alphaproteobacteria-teal, Betaproteobacteria-grey, Gammaproteobacteria-olive, Deltaproteobacteria-blue, Bacteriodetes-black, Planctomycetes-black, Cyanobacteria-purple, Gemmatimonadetes-Red branch with species name in black, Spirochaetes-pink branch with species name in black, Fibrobacteres-light grey, Archaebacteria-red. (PDF 62 KB)Additional file 8: Sequence alignment and class specific motifs of each cluster. An arrow represents class specific motifs. (PDF 20 MB)Additional file 9: Cluster-wise mapping of sequence motifs on the structure of POPs.(PDF 920 KB)Additional file 10:A) Mapping of non-permissible amino acid replacement sites on bPOP structure. Color code: Amino acid replacement sites-red, functionally important residues-cyan and green, active site-magenta, catalytic domain-yellow and propeller domain-blue. B) Cluster-wise conservation and replacement of functionally important residues. Top row shows functionally important residues as reported in mammalian POPs. Active site residues are represented in pink. Non-permissible amino acid replacements are shown in yellow. Numbers represent percentage conservation in different clusters. (PDF 345 KB)Additional file 11: Phylogenetic analysis of POP family members. Color code: POP-blue, DPP-red, ACC-magenta, OPB-green, carboxyesterase-brown. (PDF 2 MB)Additional file 12: Detailed phylogeny of POP family members. Color code: POP-blue, DPP-red, ACC-magenta, OPB-green, carboxyesterase-brown. (PDF 89 KB)Additional file 13: Chromosomal mapping of 16 POP genes ofShewanella woodyi. Color code: Purple and green color represents GC content and GC skew in this genome. (PDF 173 KB)Additional file 14: Schematic of sequence searches followed in this work.(PDF 684 KB)Additional file 15:


For the past four decades the compositional organization of the mammalian genome posed a formidable challenge to molecular evolutionists attempting to explain it from an evolutionary perspective. Unfortunately, most of the explanations adhered to the “isochore theory,” which has long been rebutted. Recently, an alternative compositional domain model was proposed depicting the human and cow genomes as composed mostly of short compositionally homogeneous and nonhomogeneous domains and a few long ones. We test the validity of this model through a rigorous sequence-based analysis of eleven completely sequenced mammalian and avian genomes. Seven attributes of compositional domains are used in the analyses: (1) the number of compositional domains, (2) compositional domain-length distribution, (3) density of compositional domains, (4) genome coverage by the different domain types, (5) degree of fit to a power-law distribution, (6) compositional domain GC content, and (7) the joint distribution of GC content and length of the different domain types. We discuss the evolution of these attributes in light of two competing phylogenetic hypotheses that differ from each other in the validity of clade Euarchontoglires. If valid, the murid genome compositional organization would be a derived state and exhibit a high similarity to that of other mammals. If invalid, the murid genome compositional organization would be closer to an ancestral state. We demonstrate that the compositional organization of the murid genome differs from those of primates and laurasiatherians, a phenomenon previously termed the “murid shift,” and in many ways resembles the genome of opossum. We find no support to the “isochore theory.” Instead, our findings depict the mammalian genome as a tapestry of mostly short homogeneous and nonhomogeneous domains and few long ones thus providing strong evidence in favor of the compositional domain model and seem to invalidate clade Euarchontoglires. The non-uniformity of DNA composition in mammalian genomes has been known for over four decades. Many attempts have been made to provide a concise description of this heterogeneity and to identify the evolutionary driving forces behind this compositional phenomenology. The first concise description of the genome suggested an isochoric structure according to which the mammalian genome consists of a mosaic of long, compositionally homogenous DNA sequences. With the advent of genome sequencing, this description was found to be inappropriate. We have recently proposed an alternative “compositional domains” model that depicts the human and cow genomes as composed of mixture of compositionally homogeneous and nonhomogeneous domains. Most of these domains are very short. Since its proposal, this model has been validated in plethora of invertebrate genomes. Here, we test the validity of this model on eleven mammalian and avian genomes using seven attributes of compositional domains and discuss their evolution. We also use these attributes to decide between two competing phylogenetic hypotheses. Our findings provide strong supporting evidence for the “compositional domains” model and indicate that rodents are not as close to primates as envisioned by the Euarchontoglires hypothesis. Human and cow genomes have been shown to possess a complex architecture, in which compositionally homogeneous and nonhomogeneous domains of varying lengths and nucleotide composition are interspersed with one another JSJS failed to partition sequences with low compositional dispersion and had difficulties in identifying short homogeneous domains. To rectify these inadequacies, Elhaik et al. Numerous methods for segmenting DNA sequences into contiguous compositionally-coherent domains have been proposed in the literature. These methods differ from one another in the number and types of parameters used in the segmentation process, as well as in the levels of user intervention. Unfortunately, even methods that limit user input to a few parameters yield incongruent results with one another The compositional domains identified by IsoPlotter are contiguous genomic segments, each with a characteristic GC content that differs significantly from the GC contents of its adjacent upstream and downstream compositional domains. By comparing the GC content variance of compositional domains with that of the chromosomes on which they reside, compositional domains can be further classified into two types: “compositionally homogeneous domains,” or simply “homogeneous domains,” and “compositionally nonhomogeneous domains” or “nonhomogeneous domains.” A subset of long homogeneous domains, where “long” is arbitrarily defined as ≥300 kb, are termed “isochoric” domains sensu . By segmHere, we characterize the compositional architecture of ten completely sequenced mammalian genomes and an avian outgroup, and attempt to identify quantitative trends in the evolution of homogeneous and nonhomogeneous domains. Seven attributes of compositional domains are used, many of which were previously used to characterize compositional architectures This evolutionary study represents a dramatic departure from earlier studies that either extrapolated from a few genes to the entire genome All mammalian genomes in our study are similar in size, ranging from 2 Gb in horse to 3.4 Gb in opossum. At 1 Gb, the size of the chicken genome is considerably smaller than the average mammalian genome. The genomic characteristics of the compositional domains for the 11 species under study are listed in Genome statistics for compositional, homogeneous, nonhomogeneous, and “isochoric” domains are shown in On average, over two thirds of all mammalian domains are homogeneous, but this proportion varies with taxon . OpossumThe mean compositional-domain length varies from ∼25,700 bp in primates to ∼38,500 bp in murids . The medp<0.01), however, this is due to the large sample sizes. The magnitude of the differences between homogeneous and nonhomogeneous domain lengths is very small in all species  with the chicken genome exhibiting borderline similarity .Almost all the distributions of homogeneous domain lengths in all studied species  are signA comparison of the cumulative distributions of domain lengths indicates that the top percentile in murids consists of domains larger than 511 kb, whereas the top percentile in the laurasiatherian genomes consists of domains larger than 281 kb . In mammDomain density measures the average number of compositional domains per Mb. When divided into GC-poor and GC-rich compositional domains it ranges from 0 to 90 domains/Mb for GC-poor domains and up to 121 domains/Mb for GC-rich domains . HomogenOn average, murid chromosomes are the least dense (26 domains/Mb), whereas the horse genome is the most dense (49 domains/Mb). The chromosomal domain densities of opossum are as low as murids for homogeneous domains  and as high as primates for nonhomogeneous domains (13 domains/Mb). The overall chromosomal domain densities position opossum (34 domains/Mb) between murids (26 domains/Mb) and other mammals (43 domains/Mb).Similar patterns were observed when comparing the compositional domain densities of GC- rich and GC-poor domains ; the opoDomain density largely varies among chromosomes and chromosome types. Density differences between chromosomes can reach 100% , S2 withIn P(x) and their maximum likelihood power-law fits deviate from a straight line, and the p-value is sufficiently small  that the power-law model can be ruled out  with a mean of 42–44% and a standard deviation of 5.7–8.5%. The GC distributions of compositional domains of the same type are significantly different from one another, particularly between related taxa ; however, this is due to the large sample sizes. Similar to the patterns observed in compositional domain lengths, the small differences in the GC contents of homogeneous and nonhomogeneous domains allow grouping the species into five taxonomic groups: Primates, Laurasiatheria, Muridae, opossum, and chicken , the low frequency of short GC-poor domains and the abundance of medium GC-rich domains causes mammalian GC distributions to be highly right-skewed (0.56<γ<0.77) (γ≈1.12) and chicken (γ≈0.86) are the most right-skewed of all species, due to the high abundance of short GC-rich and medium-short GC-rich domains, respectively.With the exception of the murid genomes (<γ<0.77) . OpossumGCσ) of the GC content of each domain calculated over short nonoverlapping windows within the domain  than GC-poor domains (low GCσ). The high compositional variability of horse and dog is also reflected in the wide range of GCσ values compared with those of the Cetartiodactyla species.To further study the GC content fluctuations within compositional domains, we looked at their compositional variability. Compositional variability is measured from the standard deviation . As shown before, the majority of domains in all genomes are short (6–8 kb), and their GC content distributes close to the mammalian genome mean GC content. With the exception of murids, homogeneous domains are significantly more AT-rich than nonhomogeneous domains . The genomic landscape topologies of primates, laurasiatherian, and murids are remarkably similar with short (103–104 bp) GC-rich domains 1.3–1.7 times more frequent than GC-poor domains and medium-large (105–106 bp) GC-rich domains 1–2 times more frequent than GC-poor domains  and long (>106 bp) GC-rich domains compared to all other genomes   . By comp0–1,591) .de facto abandoned by most scientists (with the exception of its proponents), leaving open the basic questions: how, when, and why in the course of evolution, did mammalian genomes acquire their current composition and organization?One of the most fascinating features of mammalian genomes is the uniformity of GC content over hundreds and hundreds of thousands base-pairs termed short- and long-range correlations, respectively. Although these structures have been known for over three decades The most effective approach to understanding the compositional organization of human and mammalian genomes is by comparative analysis – preferably a large-scale one. In a previous analysis of the human genome, Elhaik et al. 3–104 bp) homogeneous domains (36%), medium-short (104–105 bp) homogeneous domains (26%), medium-long (105–106 bp) homogeneous domains (4%), and only a miniscule fraction of 0.16% long (106–107 bp) homogeneous domains , short  differ slightly from those of cetartiodactyls (cow and pig), possibly adding support for the validity of clade Pegasoferae . HoweverOverall, the laurasiatherian genomes are more similar to the primate genomes than the murid genomes, which, in turn, are more similar to the opossum genome than to any other genome . The murThe two phylogenetic hypotheses tested here differ in the validity of clade Euarchontoglires. According to the first hypothesis , murids According to the second hypothesis , murids Unfortunately, the representation of marsupial mammal as outgroup yielded more questions than answers as opossum reflected either unique genomic characteristics or oscillated between murid and non-murid characteristics –2. Thus,Homo sapiens build 37.1), chimpanzee (Pan troglodytes build 2.1), orangutan (Pongo abelii build 1.2), mouse (Mus musculus build 37.1), rat (Rattus norvegicus build 4.1), horse , dog (Canis familiaris build 2.1), pig (Sus scrofa build 2.1), and cow (Bos taurus build 4.1). The gray short-tailed opossum (Monodelphis domestica build 2.1) was used as an outgroup to the eutherians, and chicken  was used as an outgroup to the mammals. Genomes were downloaded from http://www.ncbi.nlm.nih.gov/Genomes/. Nulls, i.e., unknown, undetermined, or ambiguous characters in the genomic sequences, were discarded.Nine eutherian genomes that are either fully sequenced or have reliable genomic drafts were included in this study: human , medium-short (104–105 bp), medium-long (105–106 bp), and long (106–107 bp) domains.Version 2 of IsoPlotter The mean GC content of all mammalian genomes in this study (40.9%) was used as a critical value. A compositional domain was defined as GC-rich or GC-poor if its GC content was higher or lower, respectively, than the critical value.g estimator of Cohen's dd was smaller than 0.05, we considered the magnitude of the difference between the two distributions to be too small to be biologically meaningful.For each species and for each domain category, log domain-lengths were sorted and smoothed. Smoothing was carried by dividing the log domain-lengths into 1,000 groups of equal size and then using the mean domain length of each group to calculate a histogram with 38 bins ranging from 8 to 16. To test whether or not two distributions are different, we used the Kolmogorov-Smirnov goodness-of-fit test and the False Discovery Rate (FDR) correction for multiple tests The distributions of domain GC contents were calculated in a similar manner. To smooth the GC content distributions, domain GC contents were divided into 1,000 groups of equal size, and the mean domain GC content of each group was used to calculate a histogram with 38 bins ranging from 0 to 1. The remaining calculations were carried as described above.To test whether the GC-content distributions of homogeneous and nonhomogeneous domains fit a normal distribution, we used the Lilliefors (1967) test. This test is a two-sided goodness-of-fit test suitable when a fully-specified null distribution is unknown and its parameters must be estimated. It tests the null hypothesis that domain GC contents come from a distribution in the normal family, against the alternative that they do not come from a normal distribution.γ) of the GC content distributions using the “skewness” function in Matlab, which first centralizes the distribution by subtracting it from its mean, calculates its third (3k) and second (2k) moments, and then computes the skewness, so that: 0GC = GC – μ(GC), 3k = μ(0GC3), 2k = μ(0GC2), and γ = 3k/2k1.5.We also estimated the standardized skewness , plpva.m (version 1.0.6), and plplot.m (version 1.0) in www.santafe.edu/~aaronc/powerlaws/ and medium-long (105–106) nonhomogeneous (a) and compositional (b) domain sizes in log scale.The cumulative distribution of medium-short Click here for additional data file.Figure S2Compositional domain densities of A) homogeneous and B) nonhomogeneous domains over all chromosomes. Box plots summarize medians, quartiles, and range.(TIF)Click here for additional data file.Figure S3Frequency of domain density for (a) homogeneous domains, (b) nonhomogeneous domains, and (c) compositional domains. GC-poor domains (red), GC-rich domains (blue), and all domains (black) are plotted.(TIFF)Click here for additional data file.Figure S4The cumulative density function P(x) of compositional domain (a) and nonhomogeneous domain (b) sizes (x) (points) plotted on a log-log scale. The solid lines represent the maximum likelihood power-law fits to the data.(TIF)Click here for additional data file.Figure S5A comparison of compositional (a), homogeneous (b), and nonhomogeneous (c) domain lengths in the cumulative representation C(k). The horizontal axis is plotted as k/k0, where k0, is the size of the smallest domain size group. Although the distributions are clearly different for all the animals, the deviation from a power-law (dashed line) and the “fat tails” are shared features.(TIFF)Click here for additional data file.Figure S6A two dimensional joint distribution of nonhomogeneous domain GC content and its standard deviation (GCσ). Each domain GC content and GCσ are represented by a point on the map. The frequency of different points is represented by colors ranging from red (highest frequency) to blue (lowest frequency). The mean GC content of the mammalian genome is marked by horizontal line.(TIF)Click here for additional data file.Figure S7A two dimensional joint distribution of nonhomogeneous domain GC content and size in a log scale. Each domain GC content and its size are represented by a point in the map. The frequency of different points is represented by colors ranging from red (highest frequency) to blue (lowest frequency).(TIF)Click here for additional data file.Table S1Chromosome statistics for compositionally homogeneous, nonhomogeneous, and “isochoric” domains.(DOC)Click here for additional data file.Table S2Categories of compositional domains by length and GC content.(DOC)Click here for additional data file.Table S3List of 49 publications by Professor Giorgio Bernardi and colleagues in which isochores are defined as compositionally homogeneous genomic stretches longer than 300 kb*#.(DOC)Click here for additional data file.


The type 1 N-degron recognizing UBR-box domain of the eukaryotic Arg/N-end rule pathway is known to possess a novel three-zinc-stabilized heart-shaped fold.The N-end rule pathway is a part of the ubiquitin–dependent proteolytic system wherein N-recognin proteins recognize the amino terminal degradation signals (N-degrons) of the substrateUsing sequence and structure analysis we argue that the UBR-box fold emerged from a binuclear RING-like treble clef zinc finger. The RING-like core is preserved in the UBR-box and the metal-chelating motifs display significant sequence and structural similarity to B-box and ZZ domains. UBR-box domains retrieved in our analysis co-occur with a variety of other protein domains, suggestive of its involvement in diverse biological roles. The UBR-box is a unique family of RING-like treble clefs as it displays a distinct circular permutation at the zinc-knuckle of the first zinc-binding site unlike other documented permutations of the RING-like domains which occur at the second zinc-binding site. The circular permutation of the RING-like treble clef scaffold has possibly aided the gain of a novel and relatively deep cleft suited for binding N-degrons. The N- and C-terminal extensions to the circularly permuted RING-like region bind a third zinc ion, which likely provides additional stability to the domain by keeping the two halves of the permuted zinc-knuckle together.Structural modifications and extensions to the RING-like core have resulted in a novel UBR-box fold, which can recognize and target the type 1 N-degron containing proteins for ubiquitin-mediated proteolysis. The UBR-box appears to have emerged during the expansion of ubiquitin system pathway-related functions in eukaryotes, but is also likely to have other non-N-recognin functions as well.This article was reviewed by Eugene Koonin, Balaji Santhanam, Kira S. Makarova.The online version of this article (doi:10.1186/s13062-015-0066-5) contains supplementary material, which is available to authorized users. A p. A pi.e.lix Fig.  8–10]. . i.e., tlix Fig. . The terlix Fig. .Fig. 2CoMany binuclear treble clefs such as the B-box, ZZ, Zf-UBP, TFIIH-p44 and C1-type zinc fingers share two characteristic features with the RING finger. First, these domains contain a three-stranded β-sheet comprising of the primary β-hairpin and the C-terminal β-strand . Second,The UBR-box may be regarded as a novel three zinc-stabilized fold , 5, but S. cerevisiae UBR1 (PDB identifier 3NIH_A) was able to find matches to the Pfam B-box zinc finger family , B-box domain of tripartite motif-containing protein 5  and to the Pfam ZZ zinc finger family  and ZZ domain of ZZZ3 protein . Similarly, FFAS [S. cerevisiae UBR1 (PDB identifier 3NIH_A) retrieved B-box of Midline-2  and ZZ domain of zinc finger SWIM domain-containing protein 2  as top-scoring matches. A weak resemblance of a part of the UBR-box motif to the ZZ domain of PRT1 has also been noted previously [Sequence similarity searches initiated with the sequences of the complete UBR-box, and independently of the region corresponding to the binuclear RING-like region, are able to retrieve several B-box and ZZ domains as statistically-significant matches. For example, a HHpred  search ieviously  and Jackeviously  database).The sequences of the UBR-box classified in Pfam PF02207) and those obtained by the JackHMMER search were analyzed for the presence of co-occurring domains and their distribution across eukaryotes  [) identified in our analysis is suggestive of diverse biological roles for the UBR-box.Previous reports have shown that UBR-box containing proteins function in diverse biological processes , 19, 20  domains , 34. Cur (DIAP1) . The larS. cerevisiae UBR-box against the PDB did not find matches to any other proteins as reported earlier [α atoms with a single permutation at the zinc-knuckle. Interestingly, TopSearch [α atoms with a TM-Align score of 0.44 . The circularly permuted binuclear RING-like region of UBR-box (PDB identifier 3NY1_A) and the B-box domain (PDB identifier 3DDT_A) could be manually superimposed using the pair fitting command of PyMOL with an RMSD of 2.46 Å over 40 pairs of Cα atoms   while being present at an equivalent spatial location, is circularly permuted with respect to its position in the sequence  and other treble clef domains were retrieved from the PDB. Dali [Structures of the human and DB. Dali , TopSearDB. Dali , TM-aligDB. Dali  and Fr-TDB. Dali  tools weSequence similarity searches initiated with the UBR-box domain were performed using iterative PSI-BLAST  , JackHMMReport form: Kaur and Subramanian examine the structure of the UBR box and show that it is a circular permutation of the binuclear treble clef Zn finger rather than a truly novel Zn-binding fold. To the best of my understanding, this is a valid relationship and as such, quite a useful observation. In my opinion, the authors miss out on phylogenomic analysis of the UBR box. I think it would have been quite informative to identify UBR box-containing proteins across the entire eukaryotic diversity and reconstruct the scenario of evolution. Is N-degron recognition the only function of the UBR box or are there additional exaptations? This question could be addressed through careful analysis of the domain architectures of the UBR box-containing proteins. As it stands, the manuscript reads more like a Discovery Note although the format is that of a research article. Expansion along the above lines could rectify this mismatch between content and form.The manuscript is not carefully written or edited, careful editing is a must.Quality of written English: Needs some language corrections before being publishedWe have now analysed and reconstructed phylogeny for UBR-box domains across all eukaryotes. A phylogenetic tree of representative UBR-box domains is included as an additional figure. Domains co-occurring with the UBR-box are provided besides the branch labels. Our analysis, similar to earlier studies, reveals that the UBR-box co-occurs with a diverse set of protein domains. Most domain architectures are suggestive of a likely involvement of the UBR-box containing proteins in ubiquitin-related pathways. However, it is also known that many UBR-box containing proteins lack the N-recognin function and do not bind N-degrons. In such scenarios, the most plausible function of the UBR-box may be in mediating protein-protein interactions, as discussed in the manuscript.Report form: In this manuscript the authors argue, based on sequence and structure analyses, for the relationship between UBR-box and binuclear RING-like treble clef zinc fingers such as RING, B-BOX, ZZ, PHD and MYND fingers. The authors provide convincing arguments for how UBR-box is a unique circularly permuted version of binuclear treble clef zinc finger and indeed related to other binuclear treble clef fingers. Their proposal appears scientifically sound and convincing. However, I have a few comments listed below:There seems to some intertwined arguments for relationship between UBR-box and binuclear treble clef fingers. These include systematic structural searches, followed by sequence searches and also manual analyses (Line # 107 to 152). The organization and writing of this part is confounding the understanding of the strategies to link UBR to binuclear treble clef fingers.I found it rather strange that the authors narrate their structural analyses first and discussed the sequence analyses later. Sequence searches clearly show clear link between UBR and ZZ, B-Box (at least for significant parts of the domains). This also possibly adds to the confusion stated above. I would have naturally expected sequence searches to be the first straightforward way to get an evolutionary link between domains. Hence, has to be the first one to be discussed here.3.There was no detailed discussion on their observation of evolutionary link between ZZ, B-box and UBR later as well.4.Given there is strong sequence/structure relationship between UBR-box and ZZ and B-box, I was expecting an evolutionary model similar to the Ref. 9 . Ref. 9 suggests that FYVE finger is likely to have evolved from a mononuclear version through LIM like intermediate through circular permutations (CP). Is it possible that UBR-box evolved by CP of either ZZ or B-box precursor? Did the authors look for UBR-like proteins in prokaryotes?The relationship between UBR-box and RING-like domains has been established using a combination of statistically-supported sequence and structural similarities. Interestingly, the similarity of the UBR-box to RING-like domains can be made only upon assuming a circular permutation of the structures. The evidence for the circular permutation is primarily based on structural similarity, i.e., assuming this permutation allows us to superpose the entire core of the RING-like domain on the UBR-box. While sequence similarity statistics between the UBR-box and RING-like domains is convincing, however none of the automated tools can recapitulate the alignment over the entire length of the RING-like core because of the circular permutation and remote similarity. Hence, the structural similarity to RING-like domains was discussed first followed by sequence similarity which helps us to establish the evolutionary link between UBR-box and RING-like domains. We have nevertheless taken into consideration the comments of the reviewer and discuss sequence similarity before the structural evidence in the revised manuscript.5.As articulated in Ref. 9 binuclear treble clef fingers have independently or by convergence acquired Ub-related functions. UBR seems to be no exception; do the authors have any comment?In the current manuscript we provide a detailed discussion about the relationships between the ZZ, B-box and UBR-box domains. It must be noted that these domains are distantly related. Given the evidence from sequence and structural similarities, we can speculate that the UBR-box emerged via circular permutation of a RING-like domain such as the ZZ or B-box. However, there is no clear cut evidence to pinpoint to the exact precursor. Our sequence similarity searches do not retrieve any UBR-box sequences, which are not circularly permuted with respect to the RING-like treble clefs. Also, we are not able to identify any UBR-like proteins in prokaryotes at this time.A. thaliana, a ZZ domain is suggested to interact with the N-terminal destabilizing residue . Thus, 7.It is rather strange to me that structural searches using TopSearch yielded best hit to PARP1 and not to ZZ or B-box as suggested by sequence searches. Do the authors have any explanation?Our structural search strategy primarily involves the TopSearch tool to scan the entire PDB for structures similar to the UBR-box. Dali and TM-Align were used for reconfirming the structural matches. In our opinion, TopSearch is presently one of the best programs to detect structural similarities between proteins related by circular permutations. Our manual structural analysis also support the results obtained by TopSearch. Our primary purpose in this manuscript was to determine if the UBR-box is indeed a new fold or if it is evolutionarily related to any other well classified zinc finger domain. So we did not deem it necessary to run Dali locally on an expanded database of structures.TopSearch perhaps finds highest similarity to PARP1 because of an extensive structural similarity of the protein domains, especially in the regions involved in ligand binding. We notice that the UBR-box and the treble clef domain of PARP1 have a similar twist in their respective primary β-hairpins. Also, the additional β-strand which forms a three stranded β-sheet with the primary β-hairpins in both the proteins is similarly extended into the knuckle-containing β-strand and helps sculpt the respective ligand binding regions.Report form: The paper presents a relatively straightforward sequence and structure comparative analysis of several families of RING treble clef zinc finger domain. Based on this analysis authors hypothesized that UBR box domain originated though a circular permutation of the ancestral RING-like domain. This hypothesis considering presented data appears plausible and the text and figures are generally clear. I don’t have any significant criticism for this work. The only problem I have with the paper is its length and relevance of some parts of the text and some Figures. I believe that the arguments in support of this hypothesis could be described in a text half of the current size and rather in a format of a Discovery Note paper than the original research paper.Below I have a list of relatively minor suggestions how to improve the clarity of the paper and what parts could be dispensable in the main text.For a better evolutionary perspective and understanding the UBR-box family I’d recommend to start Result and Discussion with a brief description of diversity of this domain in different eukaryotic lineage. It can be described together with results of other sequence similarity searches and the Fig. four should be moved forward accordingly. Furthermore the details of sequence similarity searches could be moved to Additional material and only essence of that searches mentioned in the text. In the Fig. four organisms to which aligned sequences belong should be indicated and more UBR-box sequences from different eukaryotic lineages should be shown in the multiple alignment accordingly. Otherwise it not clear when the permutation happened during the evolution of this domain.We have taken all suggestions of the reviewer to improve the manuscript. We have added details relating to the distribution of the UBR-domain in eukaryotes in the There is no need to mentioned other cases of the permuted treble clef zinc finger domain, especially if they are not described as such in the literature . So if authors find it interesting then they should write a different a paper showing this explicitly or a review describing this phenomenon in general.Examples of circular permutations in the treble clef domain are rather rare. We have only mentioned well documented cases of circular permutations with the exception of the CRISPR system Cascade subunit CasA. The purpose of this section was to illustrate that the circular permutation observed in UBR-box is not unprecedented and a similar permutation is observed in the third treble clef domain of the triquetra knot containing protein Rds3p. For this reason, we would like to retain this section.On the Fig.2a please indicate respective cysteines and histidines for Zn ligands. Indicate the fact that pink strands are actually not structured in UBR-box structures.The zinc-chelating residues have not been labelled in Fig. The representation of the UBR-box in Fig. Comparison with PARP1 mononuclear treble clef does not add anything to the story, as the Fig. 5. It can be either moved to the supplement or omitted entirely. The same applies to the Fig.3.We have moved these figures to the Additional files section as suggested by the reviewer. However, we would like to retain the text which compares PARP1 and the UBR-box. PARP1 is among the best scoring hits in TopSearch and binds DNA in a similar region to that used by UBR-box to bind the N-degron peptide. Analysis of binding sites in all treble clef domains suggest that this region is unique to these two proteins and we feel it is appropriate to discuss this in the context of the UBR-box.Report form: The paper presents a relatively straightforward sequence and structure comparative analysis of several families of RING treble clef zinc finger domain. Based on this analysis authors hypothesized that UBR box domain originated though a circular permutation of the ancestral RING-like domain. This hypothesis considering presented data appears plausible and the text and figures are generally clear. I don’t have any significant criticism for this work.After the first round of revision the clarity of the paper improved, although there are still some parts of the paper that are not explained in sufficient details and are not directly relevant for the main point of the paper. As I mentioned before this concerns circular permutations in the treble clef domain of CasA protein and the triquetra knot in Rds3p protein which were “noticed” but not elaborated by the authors (now both are shown in the supplement). Since no analysis of these cases were provided this leaves a reader with the only option to believe that the authors are correct in their interpretations.Taking into consideration the concerns of the reviewer, we have removed the text and figure illustrating the circularly permuted treble clef motif which we observed in the CasA protein. However, we retain the prolyl-tRNA synthetase and Rds3p examples as these treble clef motifs have already been documented and discussed in previously available literature , 47.I also still have an issue with the authors’ response to my request to indicate aminoacids involved in Zn binding on the Fig.2a in order to have a better understanding of the amino acid signatures of the 1st and 2nd Zn binding motif. I expect that each family shown in the Fig.2a has a typical signature (consensus) for Zn binding ligands. These residues don’t have to be 100 % conserved. Showing this consensus is the same as showing a generalized representation of the secondary structure elements for the respective domains , although some proteins could have deviations from this typical organization.We have added the metal-chelating aminoacids for the domains as per Pfam consensus in Fig.