The whole process of magnetosome biogenesis can be divided into four sequential steps; (i) cytoplasmic membrane invagination, (ii) magnetosomes alignment, (iii) iron crystal nucleation and (iv) species-dependent mineral size and shape control. Since both magnetite and greigite are a mix of iron (III) and iron (II), iron redox state management within the magnetosome vesicle is a key issue. Recently, studies have started pointing out the importance of a MTB-specific c-type cytochrome domain found in several magnetosome-associated proteins . This magnetochrome (MCR) domain is almost always found in tandem, and this tandem is either found alone (MamT), in combination with a PDZ domain (MamP), a domain of unknown function (MamX) or with a trypsin combined to one or two PDZ domains (MamE). By taking advantage of new genomic data available on MTB and a recent structural study of MamP, which helped define the MCR domain boundaries, we attempt to retrace the evolutionary history within and between the different MCR-containing proteins. We propose that the observed tandem repeat of MCR is the result of a convergent evolution and attempt to explain why this domain is rarely found alone.Magnetotactic bacteria (MTB) can swim along Earth's magnetic field lines, thanks to the alignment of dedicated cytoplasmic organelles. These organelles, termed magnetosomes, are proteolipidic vesicles filled by a 35–120 nm crystal of either magnetite or greigite. The formation and alignment of magnetosomes are mediated by a group of specific genes, the  These magnetosomes are aligned within the cytoplasm of magnetotactic bacteria (MTB), acting as a compass needle for orientation. A tentative selective advantage would be an efficient localization of the cells in vicinity of the oxic-anoxic transition zone in the water column at their preferred position in the oxygen  gradient. Since their first scientific description by RP Blakemore in 1975  the Nitrospirae phylum, including several uncultured strains and, (iii) the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC)  genes. The mam genes are organized in clusters in the genome of MTB, in some cases defining a bona fide magnetosome genomic island (MAI)  the Komeili, . CurrentKomeili, . A core c-type cytochromes motif endemic in MTB and potentially play a role in the iron biocrystallization process that takes place inside the magnetosome , whereas chemical synthesis requires mixing iron(II) and iron(III) in appropriate proportion .In a recent study focused on the biochemistry of MamP and its structural characterization, it was found that MamP displays ferroxidase activity . The phenotypes of the mamX, mamZ and mamH mutants led the authors to propose a functional MamXZH interaction that would form an iron oxidoreductase and transport complex through the magnetosome membrane. The understanding of this system and the role of the MCR-containing protein MamX need further study. For example, electron transfer partners and directionality of electron flow remain unknown for MamX.The situation is somehow clearer in Altogether the bioinformatics and experimental data available on MCR-containing Mam proteins suggest their involvement in iron redox chemistry to ensure the proper mineralization of magnetosomes. By taking advantage of new genomic data available and recent structural data on MCR domain, we attempt to retrace the evolutionary history of this domain within and between the different MCR-containing proteins. We also hypothesize on the reasons why this domain is rarely found alone but rather in tandem repeats.c-type cytochrome domains of MamP were already proposed to define a novel domain that is only found in MTB . The overall fold of MamP in the crystal revealed a dimer with both monomers mainly stabilized by numerous contacts between their PDZ domains. The first magnetochrome domain (MCR1) is in contact with its own PDZ domain, while the second (MCR2) is projected above the PDZ domain of the other monomer. This structural study allowed the first fold description of a MCR domain, substantiating its uniqueness at the structural level. Indeed, a structural homology search with DaliLite v.3 returns no significant hits, demonstrating the specificity and the uniqueness of these domains . A detailed examination of the structure identified two hydrophobic residues, which delineate the N-terminal and C-terminal regions of the MCR domain. In the fold, these two residues interact hydrophobically to close off the domain. Based on these observations, we proposed a more accurate delineation of a typical MCR domain: ψ 1X5−9PHX5−9CX2CHψ2  closes the MCR fold by interacting with the ψ1 residue. Being composed of 19–28 residues, it represents the smallest mono-heme cytochrome known to date (the mono-heme cytochrome c-553 from Bacillus pasteurii contains 71 residues surrounding the heme moiety). Overall, this results in a highly solvent-exposed heme moiety, with all four solvent edges exposed  and separated them into MCR1 and MCR2 as described above. A protein sequences alignment was computed with all the individual MCR using the Muscle algorithm Edgar,  and a phA rather simple evolutionary scheme can be proposed for the MCR-containing Mam proteins where the basic scheme is provided by model #2: an initial sequence divergence event followed by domain duplication. It is interesting to note that even based on short MCR domain sequences, one can relatively easily infer the nature of the protein to which it belongs .Alphaproteobacteria, suggesting that it evolved relatively recently. However, its evolutionary history based on the MCR domain only suggests that it did not emerge from the tandem of another MCR-containing protein like previously existing MamP or MamE. What we may be witnessing here is an example of convergent evolution where the tandem repeat is linked to the functional role of the protein. Indeed, this domain is almost always found in tandem and there are only rare examples where it is found either, alone (MamE of Candidatus Magnetobacterium bavaricum and MamE' of SS-5) or in triplicate (MamE of strain MC-1). It is tempting to link this pattern to iron and magnetite (or greigite) chemistry. Both magnetite and greigite are a mix of one iron(II) and two iron(III) equivalents. The possibility to abstract or give two electrons by a pair of magnetochrome domains suggests its involvement directly in magnetite or greigite crystal production, not just the iron chemistry that requires a single electron. Such use of two monoheme cytochromes was also suggested to evolve in order to adapt to the storage of the two electron generated from sulfite oxidation  and the magnetite/greigite crystals have diversified since then. Because magnetochrome-containing proteins are the only redox proteins associated to the magnetosome this raises interesting possibilities possibly linking magnetite/greigite crystal shape to the evolution of these magnetochrome-containing proteins.A hypothesis concerning the evolution of magnetotactic bacteria suggests they all evolved from a common ancestor 2.5–3.0 billion years ago when levels of atmospheric oxygen were low and anaerobic to microaerobic environments dominated. At this time, magnetite/greigite crystals likely did not serve a role in magnetotaxis but rather in scavenging reactive oxygen species and later, when atmospheric oxygen levels increased, served to aid MTB in navigation , Magnetospirillum gryphiswaldense MSR-1 (GenBank: CU459003.1), Magnetospirillum magnetotacticum MS-1 (JGI project 402922), Magnetococcussp. MC-1 (GenBank: CP000471), Magnetovibrio blakemorei MV-1 (GenBank: FP102531), Magnetospira sp. QH-2 (EMBL: FO538765), strain SS-5 , Candidatus Magnetobacterium bavaricum , Candidatus Desulfamplus magnetomortis BW-1 (GenBank: JN830627–JN830646 and JN845570–JN845575).The complete genomes or contigs discussed in this paper include: c nitrite reductase.From a structure based sequence alignment we devised a protein pattern that harvests almost all the magnetochrome domains without many false positive. This pattern is the following:Initial multiple alignment were generated using the MUSCLE program Edgar, , applyinThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We compared thermally induced denaturation and aggregation of two isoforms of the isolated myosin head  containing different “essential”  light chains, A1 or A2. We applied differential scanning calorimetry (DSC) to investigate the domain structure of these two S1 isoforms. For this purpose, a special calorimetric approach was developed to analyze the DSC profiles of irreversibly denaturing multidomain proteins. Using this approach, we revealed two calorimetric domains in the S1 molecule, the more thermostable domain denaturing in two steps. Comparing the DSC data with temperature dependences of intrinsic fluorescence parameters and S1 ATPase inactivation, we have identified these two calorimetric domains as motor domain and regulatory domain of the myosin head, the motor domain being more thermostable. Some difference between the two S1 isoforms was only revealed by DSC in thermal denaturation of the regulatory domain. We also applied dynamic light scattering (DLS) to analyze the aggregation of S1 isoforms induced by their thermal denaturation. We have found no appreciable difference between these S1 isoforms in their aggregation properties under ionic strength conditions close to those in the muscle fiber (in the presence of 100 mM KCl). Under these conditions kinetics of this process was independent of protein concentration, and the aggregation rate was limited by irreversible denaturation of the S1 motor domain. The case when j = N corresponds to the experiment without preliminary incubation .If the calorimetric experiment with preliminary incubation  of the protein preparation has been carried out -th and i-th calorimetric domains has different values (otherwise these domains should be considered as a single domain), it is possible to select the temperature and time of annealing, at which ix–1, j ≈ 0  and ix, j ≫ 0. From the predicate of the numeration of domains follows that under these conditions of incubation 1st, 2nd, . . . , (i – 2)-th domains are also annealed, i.e., x1, j, x2, j, . . . , ix–2, j ≈ 0. Thus, it is possible to select the set of annealing conditions that makes the system of If domains are numbered in order of increasing thermostability, the following condition holds at i.e., the thermogram of the multidomain protein coincides with the thermogram of the most thermostable domain. In this region of temperatures the function W(T)  and Nk(T). The optimization of the whole thermogram by thermogram corresponding to the most thermostable domain in the coordinates Nk (T) · Nx(T)/ν)  see  is lineaW(T) and conclude that the thermogram is not described by the one-stage model of irreversible denaturation. Using W(T) we estimate parameters of the most thermostable domain: a, NE, NH . With a knowledge of these parameters, we simulate N – 1)-th domain, simulate function N – 2)-th domain, (N – 3)-th domain and so on down to first calorimetric domain.Based on these considerations one can propose the following recursive method for the estimation of parameters of calorimetric domains for multidomain protein. After obtaining of N-th domain, we should take into account that the error will be rather high because the region of temperatures where the function W(T) is linear is small in comparison with the temperature interval for the whole thermogram. Besides, to determine the corresponding parameters, the high-temperature branch of the DSC profile is used where W(T) in this region is even noisier since it contains the term ln N–1)-th domain. Moreover, the additional error, connected with the inexact subtraction of simulated function However, when determining parameters of aE, T* and ΔH for all calorimetric domains.When the corresponding annealing procedures have been carried out and all thermograms have been obtained, the multidimensional non-linear optimization in the parameter space may be used for final calculation of parameters 2.2.i–1)-th domains, where i is a number of domain in the order of increasing thermostability. This provides a means for the determination of parameters of i-th domain. From the theoretical point of view, if the values of only one of parameters aE or T* for two domains are non-identical, the conditions of annealing may be selected at which ix–1 ≈ 0 and ix > 0. However, the acceptable values of ix−1 and ix are the following: ix−1 < 0.01 and ix > 0.1. Otherwise the determination of parameters of i-th domain becomes impossible. The acceptable time of annealing is at least 10 min. If time of annealing is less than 10 min, delayed heating of the sample to the temperature of incubation and following cooling inevitably lead to an unacceptably large error. In other words, the following conditions should be satisfied:T* are about 320 K, after the substituting of the Arrhenius equation in the expressions for the corresponding rate constants and solving the system of inequalities T* values the minimum reasonable difference between parameters aE is ∼150 kJ/mole. For the case of identical values of the activation energy the difference The method is based on annealing of 1st, 2nd, . . . , (Tann and tann. For definiteness we fix the ratio ix−1/ix ≡ A (with the requirements for ix−1 and ix we obtain that A < 0.1). Finally we come to the system of inequalities:tann is a curve tann = f(Tann) established by the conditions:Taking into account the above-mentioned requirements to the annealing conditions, we can propose the following method for the selection of 3.3.1.aE, T* and ΔtransH  and temperature (in K), respectively. Knowing all three parameters of the calorimetric domain, we can simulate its thermogram. Let us consider how the above-described principles of DSC data analysis can be applied to S1(A1) thermal denaturation in the presence of 100 mM KCl. It was proposed from earlier DSC studies that the total heat sorption curve of S1 is composed of three independent calorimetric domains ,19–21. HN and D correspond to native and denatured states of the protein and I is a kinetic intermediate state. First-order rate constants k1 and k2 obey the Arrhenius law . Correspondingly, the thermogram of such a domain can be described as followsNx is the portion of the native state and Ix is the portion of the kinetic intermediate state:T = T0. Obviously, before annealing, k1 and k2 from the Arrhenius equation  undergoes one-stage irreversible denaturation, whereas thermal denaturation of more thermostable domain  is described by two-stage scheme:nius law , and botequation . It is ei.e., to preheating for definite time at definite temperature), as well as the time and temperature of these annealing procedures. The parameters obtained by this way are presented in H) and two parameters of the Arrhenius equation, namely, activation energy (aE) and special temperature .To find the parameters of the S1 calorimetric domains according to the above described two-domain model for the S1 thermal denaturation, we have used multidimensional nonlinear optimization in the space of these parameters. The following input data were simultaneously used for this analysis: the thermograms of native S1(A1) and S1(A2) preparations, the thermograms of these preparations subjected to two different annealing procedures  under low ionic strength conditions, as well as with S1(A2) independently of ionic strength. T* parameter of domain I, we have performed additional optimization procedure, as follows. All calorimetric parameters (except only T* of domain I) were allowed to change and domains’ I T* values were set equal to the average value among S1(A1) and S1(A2) (318.3 K under high ionic strength conditions) and frozen. The parameters obtained after such an optimization were used to simulate the DSC profiles of S1 isoforms after control annealing (17 min at 44 °C)  than in S1(A1). It should be noted that this effect is independent of ionic strength conditions as it is observed both at low ionic strength and in the presence of 100 mM KCl.In order to confirm the difference between S1(A1) and S1(A2) in the 3.2.i.e., to reveal their correspondence to S1 structural domains) and to verify the DSC results by another independent method, we have studied the temperature dependence of the intrinsic tryptophan fluorescence of S1 isoforms. There are five Trp residues in the S1 molecule .–38.2 in 4.5.2 and 100 mM KCl. S1(A1) or S1(A2) aliquots were heated at appropriate temperature for appropriate periods of time, then cooled and subjected to ATPase measurements. Experiments were performed at different temperatures within the range from 39 °C to 47 °C. The ATPase activity of S1 isoforms (K+-EDTA-ATPase) was determined by Pi release [4 to final concentration of 2.5%.The thermally induced inactivation of S1 isoforms’ ATPase was measured after heating the protein (0.5 mg/mL) at constant temperature in the medium containing 20 mM Hepes (pH 7.3), 1 mM MgCl4.6.Rh) upon incubation of S1 in 20 mM Hepes (pH 7.3) containing 1 mM MgCl2 and 100 mM KCl. The kinetics of S1 isoforms’ aggregation was studied either at constant temperature of 44 °C and different protein concentrations, from 0.25 to 1.0 mg/mL, or at constant protein concentration of 0.5 mg/mL and different temperatures, from 39 °C to 47 °C. Temperature dependences of Rh growth were studied upon heating of S1 isoforms’ preparations with constant rate of 1 °C/min in the same buffer as in the kinetic experiments.DLS measurements were performed on a Photocor Complex apparatus  equipped with a temperature controller. The sample was illuminated by a 632.8 nm laser light, and the scattering signal was observed at an angle of 90°. DLS data were accumulated and analyzed with multifunctional real-time correlator Photocor-FC. DynaLS software  was used for polydisperse analysis of DLS data. The kinetics of S1 isoforms aggregation was studied by measuring an increase in the mean hydrodynamic radius of the particles (4.7.W(T) function and appropriate annealing conditions (time and temperature) were realized as scripts running in the Matlab  environment. The optimization procedures used for calculation of en-route and final values of calorimetric domains’ parameters were also performed in the Matlab environment. For this purpose, we have written scripts-wrappers for the lsqcurvefit function of the Optimization Toolbox for Matlab.The algorithms for estimation of chemical base line, calculation of aE and T* values calculated for the same transition of the same S1 preparation never exceeded 40 kJ/mole and 0.4 K, respectively. The main source of the enthalpy error is inexact subtraction of chemical base line. Selection of different linear segments on thermograms before and after the peak of excess heat absorption results in slightly different values of calorimetric enthalpy. In our experiments this difference never exceeded 10%. Thus, we assumed the following error values for the calorimetric parameters presented: 0.4 K for T*, 40 kJ/mole for aE and 10% of the value for ΔH.Optimization algorithms (embedded into the Optimization Toolbox) allow to calculate only optimal values of the parameters, but not their confidence intervals. Moreover, because of specific features of the calorimetric experiment, the systematic error exceeds many times the random error. This renders the use of regression statistics for estimating the confidence intervals impossible. For this reason we used the following (rather rough) approach to estimate the error in the values of the calorimetric parameters. We carried out a number of different optimization procedures in the process of calculation of the values of these parameters. In one case, all the experimental t2R parameters for the S1 aggregates growth were found with 95% confidence interval, using the Trust-Region algorithm embedded into the Curve Fitting Toolbox in the Matlab environment.The optimal values of the rate constants for the heat-induced S1 ATPase inactivation and the optimal values of the Ea and T* values given in The values of the rate constants and half-life times of the S1 calorimetric domains were calculated using Arrhenius equation  and corrRh growth were also estimated using the Trust-Region algorithm embedded into the Curve Fitting Toolbox in the Matlab environment. The errors of these parameters were calculated as indirect errors caused by the errors in calculated values of the t2R parameters and by the error in incubation temperature that was assumed to be equal to 0.5 °C.The optimal values of the parameters of Arrhenius equation  for the 5.N-terminal segment in myosin A1 light chain does not affect the aggregation properties of S1. Furthermore, under these conditions S1 thermal aggregation follows its thermal denaturation and the aggregation rate is limited by irreversible denaturation of the motor domain of S1 molecule. From these data a new mechanism describing the amorphous thermal aggregation of proteins has been proposed, which explains specific aggregation properties of S1. According to this mechanism, the first stage of the aggregation process, nucleation, is directly followed by sticking denatured S1 molecules to these start aggregates, but not by interactions between the start aggregates.In the present study we have found no appreciable difference between S1(A1) and S1(A2) in their aggregation properties under ionic strength conditions close to those in the muscle fiber (in the presence of 100 mM KCl). This means that under these conditions the presence of additional N-terminal extension of the A1 light chain with the motor domain [Some difference between the two S1 isoforms was only revealed by DSC, independently of the ionic strength conditions, in the thermal denaturation of the regulatory domain. This is, however, quite an expected effect as the alkali light chains are associated with this domain of the S1 molecule. Thus, the interaction of the r domain –14 seemsN-terminal segment on the S1 thermal aggregation was observed only at relatively low ionic strength [N-terminal extension appear to be the main factor underlying the aggregation properties of S1. In contrast, the results of the present study indicate that at relatively high ionic strength (close to ionic strength conditions in muscle fibers) these interactions are too weak to have any effect on the S1 thermal aggregation.A noticeable influence of the A1 strength ,16,40. U


Helicobacter pylori were compared. In the specificity subunit of Type I restriction-modification systems, DNA sequence recognition is mediated by target recognition domain 1 (TRD1) and TRD2. To our surprise, several sequences are shared by TRD1 and TRD2 of genes  at the same locus ; these domains appear to have moved between the two positions. The gene/protein organization can be represented as x-(TRD1)-y-x-(TRD2)-y, where x and y represent repeat sequences. Movement probably occurs by recombination at these flanking DNA repeats. In accordance with this hypothesis, recombination at these repeats also appears to decrease two TRDs into one TRD or increase these two TRDs to three TRDs (TRD1-TRD2-TRD2) and to allow TRD movement between genes even at different loci. Similar movement of domains between TRD1 and TRD2 was observed for the specificity subunit of a Type IIG restriction enzyme. Similar movement of domain between TRD1 and TRD2 was observed for Type I restriction-modification enzyme specificity genes in two more eubacterial species, Streptococcus pyogenes and Mycoplasma agalactiae. Lateral domain movements within a protein, which we have designated DOMO (domain movement), represent novel routes for the diversification of proteins.A protein function is carried out by a specific domain localized at a specific position. In the present study, we report that, within a gene, a specific amino acid sequence can move between a certain position and another position. This was discovered when the sequences of restriction-modification systems within the bacterial species  A specific function of a protein molecule can often be ascribed to a specific region within the polypeptide chain called a domain Recognition of a specific DNA sequence by a protein can be mediated by a domain that is often called the target recognition domain (TRD); this has been studied in detail for several restriction (R) modification (M) enzymes RM systems, which limit the horizontal transfer of genes, are themselves mobile as revealed by genome comparison, genome context analyses, phylogenetic analyses and laboratory experiments Helicobacter pylori is present in the human stomach The H. pylori strains. To our surprise, we found that the domain sequences themselves are mobile within a gene  TRD1 and TRD2 (5′GAA), whereas TRD2 recognizes the 3′ half (RTCG). The central repeat determines the relative distance between these two component sequences. For example, the change in the number of the central 10-bp repeat of EcoR124I from 2 to 3 changes the recognition sequence from GAA(N)6RTCG to GAA(N)7RTCG Type I RM systems contain the specificity (S) subunit that determines their recognition sequence and is necessary for both restriction and modification activities  [8]. Theand TRD2 . TRD1 reH. pylori has been assigned many alleles of Type I specificity subunits, from hsdS1 to hsdS6, based on the locus and sequence similarity of the entire ORF (open reading frame) hsdS1, hsdS2, hsdS4, and hsdS5, hsdS3 and hsdS6, b at TRD1 was paired with domain sequence labeled i, g, f, j, and e at TRD2. This was probably mediated by recombination in the central region and conserved flanking sequences. More specifically, the recombination events that replaced TRD2  as well as in TRD2  at locus 1. Domain sequences labeled b, c, d, and f are also shared by TRD1 and TRD2 at locus 1. At locus 2, domain e appears to have moved between TRD1 and TRD2. We also found that some of these domain sequences  are shared by the two loci.We encountered a deviation from this pattern when analyzing Group 2 Type I S genes . These aSuch apparent movements of domain sequences between TRD1 and TRD2 are clearly shown in their phylogenetic tree . The terd was present at TRD1, recombination could occur between the left y sequence of this allele and the right y sequence of another allele. This argument indirectly supports the involvement of repeat-mediated recombination in the domain sequence movements. A Group 2 S allele  in the central region  repeated in tandem between these  in strain 5632, TRD1 at locus 3 (MAGa6340) in strain 5632, and TRD2 at locus 1 (MAG5640). MAGa6310 had three TRDs while MAG5720 had one TRD, likely through recombination at the repeats.In two strains (56323 and PG2) of H. pylori complete genomes revealed the movement of a sequence between domains in different positions within a protein coding gene. We designated such movement of the domain sequence within a protein (a locus) as DOMO (domain movement). DOMO defines a novel route for protein diversification.Sequence analyses of allelic diversities in target recognition domains of restriction-modification systems in various Domain movement is probably mediated by repeat sequences flanking the TRDs. The movement of sequences between specificity genes at different loci sharing the same repeats and the deletion and duplication of the domains are likely mediated by these repeats. This hypothesis is also consistent with the observation of no DOMO in the Group 1 Type I S genes lacking the repeats. The detection of DOMO in Type I S genes with TRDs flanked by repeats in two more species also support the mechanism. The aberrant S genes with only one TRD , Fig. 5 H. pylori and for Type IS locus in Streptococcus pyogenes. This indicates that DOMO can take place within the same locus at least in these cases.Because we analyze complete genome sequences, we are certain that a single locus is involved in DOMO for Type IIG locus in We do not know whether such recombination is mediated by site-specific recombination machinery or homologous recombination machinery. We also do not know whether the restriction-modification activities themselves are involved in such movement.Staphylococcus aureusMycoplasma pneumoniaeMycoplasma pulmonis, two S genes flank the R and M genes in inverted orientation and are prone to inversion at site-specific recombination site within their ORF, resulting in the shuffling of their TRDs Mycoplasma agalactiae (see above) can be represented as S1-M-hypothetical gene-S2-int-R-S3-M, where int indicates an integrase gene homolog. We do not know whether this integrase homolog is involved in recombination between the S loci as found in Mycoplasma pulmonisLactococcus lactis, two copies of the S genes on different plasmids, interact through homologous recombination and create two chimeric S genes for one RM system, resulting in shuffled recognition sequences Type I Group 2 and Group 3 S are present at multiple chromosomal loci  with linRestriction-modification systems are mobile Neisseria gonorrhoeae is achieved by gene conversion in the central region of the pilE locus with silent pilS cassettes, which have hypervariable regions, as the donor Some examples of domain diversification of bacterial genes were observed in the antigenic variation of pathogenic bacteria by the mechanism of gene conversion Protein diversification by domain movement through repeat-mediated recombination is similar to that by exon shuffling through recombination at intron DNAs or by alternative RNA splicing H. pylori were obtained from NCBI Genome as listed in Sequence data for the circularized chromosomes of RM systems were detected by homology search with RM genes registered in REBASE http://tools.neb.com/~vincze/genomes/) , we chose all the species with multiple genome entries and listed all of their S genes. Dotplot was drawn for S genes of each species by polydot in EMBOSS package From the collection of complete genome sequences in REBASE Genomes .(PDF)Click here for additional data file.Figure S2Nucleotide sequence alignment of Group 2 S (See ).(PDF)Click here for additional data file.Figure S3Nucleotide sequence alignment of Group 3 S (See ).(PDF)Click here for additional data file.Figure S4Nucleotide sequence alignment of the S subunit of Type IIG systems (See ).(PDF)Click here for additional data file.Table S1H. pylori complete genomes analyzed.(XLS)Click here for additional data file.


Ascomycota, the complex is smaller and lacks orthologs for a few CSN subunits, but nevertheless contains a conserved Csn5. This feature makes yeast a powerful model to determine the minimal assemblage required for deneddylation activity. Here we report, that Csi1, a diverged S. cerevisiae CSN subunit, displays significant homology with the carboxyl terminal domain of the canonical Csn6, but lacks the amino terminal MPN- domain. Through the comparative and experimental analyses of the budding yeast and the mammalian CSNs, we demonstrate that the MPN− domain of the canonical mouse Csn6 is not part of the CSN deneddylase core. We also show that the carboxyl domain of Csn6 has an indispensable role in maintaining the integrity of the CSN complex. The CSN complex assembled with the carboxyl fragment of Csn6, despite its lack of an MPN− domain, is fully active in deneddylation of cullins. We propose that the budding yeast Csi1 is a functional equivalent of the canonical Csn6, and thus the composition of the CSN across phyla is more conserved than hitherto appreciated.The COP9 signalosome (CSN) is a eukaryotic protein complex, which regulates a wide range of biological processes mainly through modulating the cullin ubiquitin E3 ligases in the ubiquitin-proteasome pathway. The CSN possesses a highly conserved deneddylase activity that centers at the JAMM motif of the Csn5 subunit but requires other subunits in a complex assembly. The classic CSN is composed of 8 subunits (Csn1–8), yet in several  The COP9 signalosome (CSN) is an evolutionarily conserved protein complex with a canonical composition of eight subunits (Csn1–8) proteasome, the CSN, and the eukaryotic translation initiation factor-3 (eIF3)  . Members” domain .S. cerevisiae genome  complexes, both of which are absent in the e genome  [18]. Thomplexes . Little − subunit in the CSN, and it has been speculated to play a role in structural integrity of the complex − domain contributes to the integrity or the deneddylase activity of the CSN is lacking.Csn6 is the MPNS. cerevisiae, the CSN contains four PCI subunits , one MPN+ subunit (Csn5/Rri1), and Csi1, a unique subunit that contains neither MPN nor PCI recognition domains , but not with the characteristic MPNScCSN) consists of six subunits, five of which have direct orthologous relationship to a canonical CSN subunit: ScCsn5 to Csn5, ScCsn11 to Csn1, ScCsn10 to Csn2, ScRpn5 to Csn4, and ScCsn9 to Csn7 , possibly due to breaches in genomic sequence . Nevertheless, most species within the order of Saccharomycetales are systematically deviated to the extent that they no longer bear a recognizable homolog to canonical Csn6. Instead, a group of proteins similar to ScCsi1 that harbor only S6CD, but not an MPN− domain, emerges within several Saccharomyces species  possess neither a recognizable homolog of Csn6 nor of Csi1. A putative Csn6 candidate, Csa1  is entirely rubylated (neddylated), was used as substrate, and Flag-peptide-eluted immunocomplexes that were associated with mammalian Flag-S6CD or Flag-S6MPN were tested for derubylation. Only Flag-S6CD, but not Flag-S6MPN, exhibited derubylation activity on ScCdc53, despite that Flag-S6MPN was present in higher level than Flag-S6CD  of Cdc53, the yeast Cul1 equivalent ScCSN subunits. Csn5 is the most conserved subunit, but expression of human Csn5/Jab1 in budding yeast cannot complement the Cdc53-derubylation defects of the Δcsn5 mutant (data not shown). Similarly, we found that the derubylation defect of Δcsi1 was not rescued when mouse Csn6 fragments were expressed in Δcsi1 yeast cells , an MPN+/JAMM subunit (Csn5), and Csi1  appear to have been separated from the CSN both structurally and functionally N. crassa CSN, while Csn3 exists N. crassa, its deletion does not result in a defect in deneddylation The and Csi1 . Deletioand Csi1 . ScCsi1 terminus , 2C, andCsn6 has been suggested to play an important role in maintaining structural stability of the CSN ScCsi1 interacts with ScRpn5 and ScCsn9 − domain, were active in deneddylation of human cullins  and the eIF3 complex (eIF3f and eIF3h) − domain locates at the carboxyl terminal region, and it can bind ubiquitin with an affinity comparable to other known ubiquitin binding domains − domain of Prp8 represents a “pseudoenzyme” that has lost its enzymatic activity, but has preserved enzyme’s affinity to its substrate To date, a common biological paradigm of mechanistic role for MPNproteins . Interac− protein. It is noted that Rpn8, as well as the MPN+/JAMM subunit Rpn11, undergo significant conformational changes upon integration of the lid into the proteasome holoenzyme, which simultaneously activates the JAMM DUB activity of the proteasome The recent cryo-electron microscopy of the Rpn8 structure in the lid provides an informative picture on this MPN− domain among higher organisms may suggest that it has a function related to CSN regulation in context with the developmental or physiological processes of complex organisms. Csn6 MPN− domain has been shown to mediate interaction with the14-3-3σ protein, but whether and how the CSN is involved is unclear − domain of CSN6 in the COP9 signalosome.Conservation of the Csn6 MPNCSN5 deletion or a genomic Csn5-TAP tag strains were purchased from EUROSCARF . HeLa and HEK293 cells were cultured and transfected as previously documented Yeast cells were grown under standard growth conditions at 28°C. Wild-type, All plasmids are listed in The following antibodies were used in this study: anti-Csn1, anti-Csn2, anti-Csn8 The yeast two-hybrid liquid assay measuring relative ß-galactosidase activity of the reporter plasmid (pSH18-34) was performed as previously described ScCSN complex for http://rsbweb.nih.gov/ij/). In the experiment shown in csn5 cells extract. Derubylation of Cdc53-Rub1 was performed as previously documented CSN-depleted HeLa cell extract Figure S1Orthologs of Csi1 within Saccharomyces. A. Direct orthologs for Csi1 are found within 9 Ascomycete fungal genomes. A ClustalW alignment of the S. cerevisiae Csi1 with identified orthologs in other fungal species. B. Sequence-based gene tree. Figures display information according to the Fungal Orthogroups Repository website: http://www.broadinstitute.org/regev/orthogroups/.(DOCX)Click here for additional data file.Figure S2Yeast two-hybrid pair wise interactions between Csn6 and other CSN subunits. Full-length mouse Csn6 was expressed as a LexA DNA binding domain (LexACSN1) fusion protein and other subunits of the mouse COP9 signalosome were expressed as transcription activation domain (AD-CSNs) fusion proteins . Pair-wise interactions were indicated by relative beta-galactosidase activities of the reporter plasmid (pSH18-34). The values were relative to the positive control, pSH17-4 (at 100) (LexA-AD fusion). Six independently transformed samples were used to calculate the averages and the standard deviations (error bars). In all samples, protein expression for the respective construct was confirmed by immunoblotting using anti-LexA (Clontech) and anti-HA antibodies (Santa Cruz).(DOCX)Click here for additional data file.Figure S3HA-tagged Csn6 and truncation mutants were used in co-immunoprecipitation in HeLa cell extract to map interactions with cullins. Full length Csn6 (3HA-S6FL) and the S6CD fragment, but not the MPN domain, could co-immunoprecipitate Cul1 and Cul2. Note that addition of 330 mM NaCl to the binding buffer interfered with CSN-cullins interactions.(PDF)Click here for additional data file.Figure S4Deneddylation assay. CSN-depleted HeLa cell extracts were used as a source for neddylated cullin substrates, as compared to untreated (UT) extract. These neddylated cullins could be effectively deneddylated by the CSN complex purified from porcine spleen (CSNPS). The reaction mixtures were western blotted using anti-cullin antibodies. The amounts of CSN were detected by immunoblotting with anti-Csn1 and ant-Csn2 antibodies.(PDF)Click here for additional data file.Figure S5Fragments of mouse Csn6 cannot complement derubylation defects of Δcsi1 mutant of the budding yeast. Truncated fragments of mouse Csn6 were ectopically expressed in WT or Δcsi1 yeast strains. Complementation of derubylation by Csi1 was confirmed as well (brackets stand for over-expression). Total cell extracts were used for western blot analysis of Cdc53. Expression of Csn6 proteins was determined by immunoblotting with anti-Flag, and with anti-Csn6, which recognizes antigenic peptide of AA150–200 that is present only in CBP-Flag-S6CD.(PDF)Click here for additional data file.Table S1Bioinformatic identification of two distinct Csn6 domains, in non-fungal organisms. Canonical Csn6, including both MPN- and S6CD domains, is found in most organisms. The absence of Csn6 in a few organisms could be due to fractions in genome sequences. Interestingly, a few protozoans appear to be devoid of all CSN genes including Csn6. Two unicellular species of the green algae Ostreococcus contain Csn6 with a conserved S6CD and a deviated MPN- domain.(DOCX)Click here for additional data file.Table S2List of plasmids used in this study.(DOCX)Click here for additional data file.


The translocation (T)-domain plays a key role in the action of diphtheria toxin and is responsible for transferring the catalytic domain across the endosomal membrane into the cytosol in response to acidification. Deciphering the molecular mechanism of pH-dependent refolding and membrane insertion of the T-domain, which is considered to be a paradigm for cell entry of other bacterial toxins, reveals general physicochemical principles underlying membrane protein assembly and signaling on membrane interfaces. Structure-function studies along the T-domain insertion pathway have been affected by the presence of multiple conformations at the same time, which hinders the application of high-resolution structural techniques. Here, we review recent progress in structural, functional and thermodynamic studies of the T-domain archived using a combination of site-selective fluorescence labeling with an array of spectroscopic techniques and computer simulations. We also discuss the principles of conformational switching along the insertion pathway revealed by studies of a series of T-domain mutants with substitutions of histidine residues. Diphtheria toxin enters the cell via the endosomal pathway , which iThe crystallographic structure of diphtheria toxin T-domain in the water-soluble form ,19 FiguA provide+-state) of the T-domain is the first step along a complex pathway, leading from a soluble conformation with a known crystallographic structure (W-state), ultimately to membrane-inserted states, for which no high-resolution structural information is available. Initially, this state was identified through membrane binding at lipid saturation [+-state has a midpoint at pH 6.2  and is over at pH 5.5 , as well as for increasing the overall insertion rate [The reported non-exponential kinetics of insertion transition  clearly ion rate . The mec+, occurs in solution and depends little on the properties of the membrane [+-state rapidly associates with the bilayer interface (I-state). It is not clear what structural rearrangements are associated with this transition. Final TM insertion requires the formation of the insertion-competent form (I+), which is populated in another pH-dependent transition and depends strongly on the fraction of anionic lipids and less on the nature of lipid headgroups [Various aspects of the pH-triggered bilayer insertion of the T-domain are illustrated using a pathway scheme in membrane . (This imembrane ). The T-membrane ,43. Applmembrane , but is adgroups ,29.+ and I-to-I+, are not sequential, but staggered, i.e., the second transition starts well before the first one is completed [An important aspect of the insertion pathway is that the two pH-dependent transitions, W-to-Wompleted   or the use of short-chained lipids, such as dimyristoylphosphatidylcholine [Ri = 3,000, but in a lipid-dependent manner, with anionic lipids greatly favoring the insertion [One of the possible reasons for the absence of a high-resolution structure of the T-domain in the final inserted conformation could be the fact that there is no single conformation in the transmembrane state, but, rather, a collection of states with different folds and topologies. It is clear that one can hardly expect the T-domain to form a regular large pore , and itlcholine , and canlcholine , but notlcholine . (Unlikelcholine ). In conlcholine  to demonnsertion . (It is nsertion ). In addnsertion . It is cet al. [Two groups of residues are expected to undergo protonation in the range of pH relevant to physiological changes inside the endosome: acidic residues (aspartic and glutamic acid), which will lose negative charge upon acidification, and histidines, which will gain a positive charge. Histidine protonation has been implicated in the biological activity of other toxins, including anthrax  and aeroet al. , who stuet al. . In our et al. ,29,42.i.e., it can be triggered by multiple residues). Consistent with this mechanism, introducing a pH-independent positive charge at this position is expected to result in an increased activity at neutral pH, which is, indeed, observed for the H257R mutant [The effects of systematic replacement (one-by-one and in groups) of all six native histidines of the T-domain with either glutamine or arginine residues on folding in solution was studied by means of circular dichroism (CD) and intrinsic fluorescence . Some reR mutant .The central role of protonation of H257 in destabilizing the folded structure of the T-domain in solution has been confirmed with thermodynamic integration calculations based on a series of MD simulations. The energy penalty for protonation of H257 in the context of the W-state was found to be 6.9 kcal/mole , which is the highest among the six histidines . This peC-terminal histidine residues, H322, H323, and H372, have a peculiar location, flanking the consensus insertion domain, TH8-9. The replacement of the three C-terminal histidine residues in triple-R or triple-Q mutants prevents effective translocation of the N-terminus, while introduction of these mutations in the full-length toxin results in the decrease of its potency [ potency . In the  potency . This in potency .C-terminal histidines in the scheme summarizing membrane insertion of the WT T-domain and the mutants carrying substitutions of the C-terminal histidines . There is no high-resolution structure of the OCS available (or that of any membrane-associated intermediate); however, the electrophysiological data are consistent with helices TH8, TH9 and TH5 adopting a transmembrane conformation [C-terminal histidines are replaced, the protein still undergoes a proper pH-dependent destabilization in solution, binds to membranes [We illustrate the role of stidines . Upon inembranes  and inseembranes  similar embranes  discussein vitro or in silico relate to the process of cellular entry, and what can we learn from them about molecular mechanism of in vivo action of the T-domain? The initial states on the insertion pathway (+-state corresponds to the state inside the endosome. The pH range of 5.5–6.5 measured for the W-to-W+in vitro (+-states.Cellular entry of DT starts with receptor-mediated endocytosis , but the pathway  can be a pathway  in the fin vitro  correspoThe advantage of long microsecond-scale MD simulations is that they allow one to explore in great detail the distribution of conformational states and characterize their thermodynamic properties, such as the pKas of titratable groups. As a result, rather than analyzing a single average pKa available for static crystallographic structure, we have at our disposal entire distributions . It is r+-state. To illustrate the concept of this linked equilibrium, consider a particular T-domain molecule that adopts a conformation with a pKa of 5.5 for H257 and, thus, has a 50% probability of a proton on this histidine at pH 5.5. The molecule can remain in this conformation and, eventually, lose a proton or go to another conformation in the W-state ensemble without major structural rearrangements. Alternatively, it can undergo a proton-triggered conformational change leading to the formation of a membrane-competent W+-state, in which the probability of remaining protonated at pH 5.5 is 100% (+-state at pH 5.5.In the W-state, H223 exhibits a unimodal pKa distribution with a maximum at pKa 6.5 a, which  is 100% . In acco+-state, which is indeed observed experimentally (The pKa distributions calculated for the membrane-competent state c,d indicmentally . AccordiThe Diphtheria toxin T-domain has been shown to implement its function―translocation of the catalytic domain across the endosomal membrane under acidic conditions―by itself, without the help of any additional protein component . It has Biophysical studies of the pH-triggered action of the diphtheria toxin T-domain are expected to impact not only the field of cellular entry of toxins or targeted cellular delivery of therapy, but would also advance our understanding of general physicochemical principles underlying conformational switching in proteins. For example, a number of proteins from the Bcl-2 family, carrying out both pro-apoptotic and anti-apoptotic functions, have been demonstrated to have a solution fold dominated by a hairpin composed of long hydrophobic helices similar to those of the diphtheria toxin T-domain ,69. Addi


Nucleotide-binding domain leucine-rich repeat (NLR) proteins play a central role in the innate immune systems of plants and vertebrates. In plants, NLR proteins function as intracellular receptors that detect pathogen effector proteins directly, or indirectly by recognizing effector-induced modifications to other host proteins. NLR activation triggers a suite of defense responses associated with programed cell death (PCD). The molecular mechanisms underlying NLR activation, and how activation is translated into defense responses, have been particularly challenging to elucidate in plants. Recent reports, however, are beginning to shed some light. It is becoming clear that plant NLR proteins are targeted to diverse sub-cellular locations, likely depending on the locations where the effectors are detected. These reports also indicate that some NLRs re-localize following effector detection, while others do not, and such relocalization may reflect differences in signaling pathways. There have also been recent advances in understanding the structure of plant NLR proteins, with crystal structures now available for the N-terminal domains of two well-studied NLRs, a coiled-coil (CC) domain and a Toll-interleukin Receptor (TIR). Significant improvements in molecular modeling have enabled more informed structure-function studies, illuminating roles of intra- and inter-molecular interactions in NLR activation regulation. Several independent studies also suggest that intracellular trafficking is involved in NLR-mediated resistance. Lastly, progress is being made on identifying transcriptional regulatory complexes activated by NLRs. Current models for how plant NLR proteins are activated and how they induce defenses are discussed, with an emphasis on what remains to be determined. Plants do not have an adaptive immune system like that found in vertebrate animals. Instead, plants depend solely on an innate immune system that bears intriguing similarities to animal innate immune systems, but is likely independently evolved [see review by Jacob et al. ]. Plant Effector triggered immunity is mostly mediated by nucleotide-binding leucine-rich repeat (NLR) proteins. Plant NLR proteins usually contain a C-terminal LRR domain and a central NB-ARC domain  .The NB-AHere, we focus on the advances made in the last 2 years toward understanding how plant NLRs are activated and how signaling is initiated and transduced. We highlight the compartmentalization of plant NLRs, intra-/inter-molecular interactions before and after activation, and structural and genetic insights into NLR downstream signaling.Arabidopsis TNL, RPS4, is required for RPS4-mediated resistance in the presence of its cognate effector AvrRps4 , localizes to both the nucleus and cytosol , 18. SeqArabidopsis require PM localization to function  but not an unrecognized derivative AVR3a(EM)  . MoreoveArabidopsis RPP1 LRR domain and the oomycete ATR1 effector have been demonstrated using co-immunoprecipitation analysis  containing 4] provides additional insights into the physical interactions between the NB-ARC domain and the LRR domain that function to inhibit NLR auto-activation . NLRC4 dAn open question in plant NLR studies is which domain(s) of plant NLRs is/are directly responsible for downstream signaling. In mammalian cells, the NLR activation usually results in the recruitment and activation of pro-caspase-1 through homotypic interaction with the N-terminal CARD . This leArabidopsis RPS5 proteins. In the case of Rx, overexpression of the NB subdomain alone was found to be sufficient for inducing cell death, suggesting that this domain plays a roll in engaging downstream signaling components  by Arabidopsis RPS2. In this example, RPS2 is activated by the degradation of RIN4 following cleavage by AvrRpt2 . NB-ARC domains form a deep nucleotide-binding pocket. In the “off” state, the NB-ARC domain adopts a “closed” structure where ADP is preferentially bound and coordinates intramolecular interactions to stabilize this structure , 50, 51.Apaf-1 and CED-4 are known to form oligomers through NB-ARC-mediated interaction , 56. TheArabidopsis RPS5 self-associates prior to activation  from the fungus Colletotrichum higginsianum. It is not yet known whether RRS1 and RPS4 physically associate, but both at least partially localize to the nucleus in the presence of effectors  family, which contributes to defense responses activated by Arabidopsis RPS2 (a CNL), RPP2, and RPP4 (TNLs)  . There accessful . In addilic acid . TogetheAquilegia caerulea and the dicotyledonous order Lamiales, as well as from the grass family (Poaceae), suggesting that the TNL family may be dependent on ADR1 family members for activating resistance  . Consistsistance . Since gfunction .Arabidopsis, which is required for resistance mediated by TNLs but not CNLs  in not CNLs , 71. EDSand SNC1 . These cbidopsis , 74. Furbidopsis . This stbidopsis . This fiSaccharomyces cerevisiae and Caenorhabditis elegans , was shown to interact with the WRKY45 transcription factor in the nucleus  interacts with SNC1, a TNL protein, and knocking out TPR1 compromises immunity mediated by SNC1 . Signifiesponses . Most re nucleus . This in2+, as cell death induced by RPS2 and RPM1 can be eliminated by the calcium channel blocker LaCl3 ; Nicotiana benthamiana and Solanum lycopersicum (tomato) (Solanaceae), and Cucumis sativus  (N. benthamiana and/or N. tabacum (tobacco) by transient expression of several different TNL and CNL proteins from diverse plant species, including Arabidopsis, flax, and barley  and Arabidopsis (a dicot and member of the mustard family).Although it is clear that different plant NLRs employ different signaling pathways, these signaling pathways appear to be broadly conserved across plant species, as evidenced by functional transfer of NLRs between species. For example, the itaceae) . In addid barley , 86–88. . hordei . InteresP. syringae  protein, has recently been shown to be required for RPS2- and RPS5-mediated resistance, but not for HR cell death  and N-ethylmaleimide-sensitive factor vesicle fusing ATPase, and soluble N-ethylmaleimide-sensitive factor adaptor protein 33 (SNAP33). RPS2 has also been shown to upregulate production of miR393b, a microRNA that targets at least three different genes likely involved in endomembrane trafficking  is the dual role of the LRR domain in keeping the NLR in the “off state” in the absence of pathogen, and promoting the switch to the “on state” in the presence of pathogen (via binding to effectors or effector-modified host proteins). We have very little insight, however, into how the LRR domain accomplishes either of these roles. The recent structure of mouse NLRC4 indicates that in animal NLRs, the LRR folds back across NB-ARC domain with the N-terminal portion of the LRR making multiple contacts with the NB and ARC3 domains, effectively placing a lid over the ADP/ATP binding pocket. The absence of the ARC3 domain in plant NLRs makes it a certainty that the contacts between the LRR and NB-ARC will differ from NLRC4, but based on the locations of autoactivating mutations and on deletion analyses, the general structure is likely to be similar, with just the N-terminal portion of the LRR (approximately four repeats) required to form the lid . The C-tA second holy grail is identifying the immediate downstream interacting proteins for PM associated CNLs. Although several transcription factors have now been identified that interact with nuclear-localized TNLs and CNLs, we still lack good candidates for downstream signaling proteins for NLRs that signal from the PM such as RPM1, RPS2, and RPS5. Proteomic approaches hold some promise for shedding light on this unknown , 97, butP. syringae, the HR is still a hallmark of NLR activation. One study has implicated fusion of the vacuolar and PM as the primary event leading to cell death (A third holy grail is a better understanding of how cell death is executed during NLR-mediated HR. Although cell death is apparently not required for resistance, at least to ll death . If thisIn summary, although much has been learned in the nearly 20 years since the first NLR was identified, major questions remain. Providing answers to these questions will require both creativity and improvements in technology, but will no doubt come.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Copper is an essential nutrient for most life forms, however in excess it can be harmful. The ATP-driven copper pumps (Copper-ATPases) play critical role in living organisms by maintaining appropriate copper levels in cells and tissues. These evolutionary conserved polytopic membrane proteins are present in all phyla from simplest life forms (bacteria) to highly evolved eukaryotes (Homo sapiens). The presumed early function in metal detoxification remains the main function of Copper-ATPases in prokaryotic kingdom. In eukaryotes, in addition to removing excess copper from the cell, Copper-ATPases have another equally important function - to supply copper to copper dependent enzymes within the secretory pathway. This review focuses on the origin and diversification of Copper ATPases in eukaryotic organisms. From a single Copper ATPase in protozoans, a divergence into two functionally distinct ATPases is observed with the evolutionary appearance of chordates. Among the key functional domains of Copper-ATPases, the metal-binding N-terminal domain could be responsible for functional diversification of the copper ATPases during the course of evolution. At the same time, the levels of bioavailable copper increased [Copper-containing proteins appeared early in evolution, likely in response to increasing need to utilize oxygen and oxygen containing molecules. The abundance and biologic availability of metals through geological periods along with solubility of their sulphide salts are thought to have influenced the appearance and evolution of metalloproteins. In an ancient ocean, the metals existed mostly as sulfides with the following relative solubility: Fe>Mn, Ni, Co>>Cd, Zn, Cu . Gradualncreased . Ocean oIn vitro, copper can bind to various synthetic and biological molecules ; however, in cells copper is used and handled mostly by proteins, which employ this metal as a cofactor to maintain their structure or function. In addition to cuproenzymes, there are four classes of proteins that regulate copper levels in a cell without using copper for enzymatic reactions. In eukaryotic cells, such proteins can be divided into membrane bound importers, ATP-driven copper exporters, the soluble copper-delivery molecules, or copper-chaperones, and intracellular copper chelators . It has been hypothesized that in evolution copper exporters appeared prior to copper chaperones or copper importers [Recent studies suggest that the utilization of oxygen and copper by early prokaryotes followed parallel evolution. Comparative genome analysis of copper transporters and cuproproteomes has found that most aerobic prokaryotes employ copper for their physiologic chemistry, whereas anaerobes do not . In vitrmporters . The discovery of microfossils in deep sea volcanic rocks led to the proposal that the first life on Earth appeared ~3.2 billion years ago . These vThe Copper-ATPases (or ATP driven copper pumps) play the major role in maintaining copper balance in cells. Copper-ATPases have been studied in significant detail in bacteria and archea, and several recent reviews provide comprehensive summary of this work ,5,6. By Archeoglobus fulgidus is one of the phylogenetically primitive organisms, in which copper transporters have been studied in detail. A. fulgidus is the sulphur-metabolising archeobacteria and a hyperthermophile found in hydrothermal vents, oil deposits, and hot springs. Copper ATPases (CopA and CopB) in this organism contain the same functional domains as Copper-ATPases of eukaryotes (see details below); however, they show maximum activity at 75–85°C [A. fulgidus, CopA is activated by Ag+>Cu+ whereas CopB is activated by Cu2+>Ag+>Cu+ [Enterococcus hirae [A. fulgidus, these two transporters show high sequence homology to each other except in regions associated with metal binding and to Copper-ATPases of higher organisms . Disruption of CopB or both CopB and CopA drastically decreases resistance of the bacteria to high copper. In contrast, bacteria lacking only CopA ceased to grow after a few generations in media lacking copper, whereas normal growth was observed in wt cells. It is thought that CopA facilitates copper acquisition under copper limiting conditions, whereas CopB extrudes excess copper. The physiologic need for copper uptake system is not entirely clear, as in these gram-positive bacteria no copper dependent cytoplasmic enzymes are known [E. hirae of a soluble copper-chaperone CopZ and a copper-dependent transcription factor CopY [CopA and CopB were also identified and studied in mesophilic bacteria, us hirae -11. Simire known  However,tor CopY ,13 may avia intestine or the delivery of copper into the brain through a blood brain barrier). In addition to the export function, new roles for Copper ATPase appeared and were retained in evolution. An opportunistic human pathogen, Pseudomonas aeruginosa, which survives in the atmosphere as well as in hypoxic conditions of human body, has two Copper-ATPases (CopA1 and CopA2). Gonzalez-Guerrero and colleagues demonstrated that these two structurally similar Copper-ATPases are both vital for virulence, but have distinct functions [2O2 and reduced cytochrome c oxidase activity, whereas no alteration in copper tolerance is observed [The original function of Copper-ATPases, the maintenance of proper intracellular concentration of copper and protection against metal toxicity, has been preserved throughout evolution. In high eukaryotes, it has been adopted to provide a vectorial transfer of copper through complex barriers  is present. With a growing complexity of organisms (in chordates), ATP7 branched out as ATP7A and ATP7B. These homologues, though sharing significant sequence similarity, have different expression in tissues, unique functions and distinct intracellular trafficking properties . Saccharomyeces cerevisiae, the Copper-ATPase Ccc2p permanently resides in the Golgi network, where it transfers copper to Fet3p, a copper-dependent ferroxidase [trans-Golgi network where these transporters deliver copper into the lumen of secretory pathway for biosynthetic incorporation into copper-dependent enzymes [In a eukaryotic cell, under normal conditions, Copper-ATPases may localize primarily to the Golgi membranes, intracellular vesicles, or the plasma membrane. The subcellular localization of Copper-ATPases correlates with their primary function in the cell, such as sequestration of excess copper for efflux (homeostatic function) or copper transfer to copper binding proteins for functional maturation (biosynthetic function) . In yeasroxidase . The cop enzymes ,19-24. T enzymes -27. It iin vitro using membrane preparations containing Copper-ATPase [ccc2 cells do not grow on iron-limited medium; expressing the active Copper-ATPase restores copper transport to Fet3 and cell viability [The ATPase-driven copper transport is a multi-step process that involves the acquisition of copper from the cytosol, the delivery of cytosolic copper to the intramembraneous site of the transporter and a subsequent release of the metal at the luminal side of the membrane. Copper release occurs in synchrony to the binding and hydrolysis of ATP by the protein. The Copper-ATPases are members of a large family of P-type ATPases. The characteristic step in their catalytic cycle is the transfer of ɣ-phosphate of ATP to the invariant Asp residue in the DKTG motif with the formation of a transient phosphorylated intermediate . This rer-ATPase . For ther-ATPase . This asr-ATPase ,31. In tr-ATPase  and the iability .Legionella pneumophila (LCopA) has recently been solved and yielded important insights into the molecular architecture of this class of proteins [Copper-ATPases from bacterial to humans share the same domain disposition. Although, the structure of eukaryotic Copper-ATPases is not yet available, the structure of prokaryotic CopA of proteins . LCopA i1a) illustrates the domain organization of the human copper transporter ATP7B. The domain composition of the core and the overall fold of individual domains are highly conserved. The Copper-ATPases have eight trans-membrane segments (TMS) with the N- and C-termini of the protein both oriented towards the cytosol. The cytosolic portion of the Copper-ATPases contains several functional domains: the N-terminal copper-binding domain, the actuator (A-domain), the ATP binding domain (which includes the nucleotide binding (or N-) domain, and the phosphorylation (or P) - domain, and the C-terminus . The N-t1b) [Sequence comparison of functional domains in evolutionarily distinct organisms demonstrates very different evolutionary trends for these domains. The P-domain  is most conserved, both with respect to its sequence and size. Similarly, high conservation and little variation in size are found for the A-domain, which plays the central role in protein dephosphorylation and conformational transitions -39. In c1b) .Caenorhabditis elegans, Xenopus laevis and higher eukaryotes, the length of the insert increases, reaching about 30% of the entire N-domain sequence in human ATP7A. Interestingly, the size of insert in ATP7A across species tends to be bigger than that of ATP7B irrespective of their evolutionary position, e.g. the insert in the N-domain of Zebrafish ATP7A is longer than the insert in human ATP7B N-domain. Functional studies indicate that the additional sequence present in the insert is not important for binding of ATP , sugges1b) illustrates that in human ATPases more than 50% of protein structure is employed in regulation of the transporter.The highest sequence variability and increased complexity of overall design are found in the N-terminal metal-binding domain of Copper-ATPases. There is a significant evidence for the complex regulatory role of this region, particularly in eukaryotic Copper-ATPases, which we will discuss in detail below. Fig. (E.coli) CopA, Protista (Dictyostelium), Yeast (Candida & Saccharo-myeces) Nematoda (Cenorhabditis), Arthropoda (Droso-phila), Cephalochordata: , Chordata  illustrates  that yeast and bacterial Copper-ATPases branched out early in evolution and evolved independently. It is interesting that the ATP7A-like proteins in phyla with large evolutionary distances share higher homology between each other than ATP7A and ATP7B of the same organism . For example, human ATP7A has 56% identity to a human ATP7B, but 65% identity with Danio rerio ATP7A (GenBank: AAZ07896.1). Similarly, human ATP7B shared 66% homology with a predicted amphibian ATP7B . This pattern suggests that the functional specialization of the two transporters appeared soon after the gene duplication event, which is thought to produce two Copper-ATPases in the same organism, and that the structural differences between 7A and 7B transporters are governed by their function and are not organism-specific. Similarly, in the phylogram of yeast Copper-ATPases, Ccc2p proteins of Candida and Saccharomyeces are closer to each other than Candida’s CaCRP1 and Ccc2p. Interestingly, C. albicans is estimated to be about 108 years removed in evolution from S. cerevisiae [. During this period, as a result of adaptation to very different environments - an animal body  and rotting fruit (S.cerevisiae), Candida diverged by evolving a novel Copper-ATPases (CaCRP1) presumably from the already existing Ccc2p, however maintaining high homology with the common Copper-ATPase (Ccc2p).Phylogram analysis of Copper-ATPases in representative organisms from different groups , a second copper-transporting P-type ATPase, CaCRP1, is expressed and contributes to high copper resistance [CRP1 had a dramatic effect on organism sensitivity to copper. The minimum inhibitory concentration of copper for the Cacrp1∆ strain is reduced to 0.5 mM (compared to 25 mM for the wild-type), whereas resistance to Ag+, Cd2+ and Zn2+ remains unaltered. This observation suggests that CaCrp1 plays the major role in copper detoxification via copper efflux. The diploid opportunistic fungi,sistance . Studies+1 as compared to 25 µM Cu+1 for the wt strain. As an opportunistic pathogen, C. albicans is found in the digestive tract of warm-blooded animals where the environment is largely anaerobic and predominantly acidic. Under these conditions even relatively low levels of copper found in food may become toxic to the yeast further emphasizing the physiologic importance of CaCRP1. It appears that cellular pH homeostasis is linked to copper export. It has been observed that yeast sensitivity to copper markedly increases with decreasing pH of the growth media. The yeast becomes particularly sensitive at pH 3.0 and can only tolerate 25 µM copper in the medium, possibly due to due to inability of copper binding proteins to retain copper. The ∆CaCRP1 mutant is even more sensitive and fails to grow in 2uM CuCandida albicans was designated as Crd1 [C. albicans is a frequent nosocomical infectious agent in burn patients and patients with catheters. Since silver was used as a microbiocidal agent for many years, and silver products are commonly used as a burn antiseptic, it was suggested that Crd1p may enhance the chances of C albicans’ survival as a nosocomical pathogen by increasing resistance to silver. In another study, the CRP1 gene of  as Crd1 . Crd1 waOnly limited information is available about Copper-ATPases in unicellular eukaryotes. Copper-ATPases have been studied in soil amoeba Dictyostelium and parasitic protozoan apicomplexa Cryptosporidium ,46. HighCryptosporidium parvum, the immunostaining of Copper-ATPase, CpATPase2 [Similarly, in the apicomplexan pathogen, pATPase2  yielded pATPase2 ) and theCUA1, containing three N-terminal copper binding sites has been cloned from the nematode worm, Cenorhabditis elegans [CUA1 cDNA showed that CUA1 can rescue ∆ccc2p mutant of S. cerevisiae and hence it can function as a copper transporter. Due to limited studies, the role of CUA1 in normal physiological functions of the nematode is unknown. However, in S. cerevisiae, the HA-tagged CUA1 was shown to localize to large intracellular vesicles, possibly Golgi membranes, which can be further substantiated by the fact that, CUA1 cDNA rescues ∆ccc2p mutant, as already mentioned.Information on the function and structure of Copper ATPases in lower metazoans are limited. A single copper ATPase (1238 amino acids),  elegans . Yeast cMore detailed information on the function of Copper ATPase in non-chordate metazoans is available for Drosophila. Drosophila posses a single Copper-ATPase DmATP7 (1254 amino acids) and has four metal binding GMxCxxC motifs in the N-terminal domain . Though DmATP7 mutants showed limited mobility as compared to the wt flies and had reduced pigmentation [et al. have shown that DmATP7 participates in (a) delivering copper to cuproenzymes, such as tyrosinase that is required for pigmentation, (b) normal development of neuronal functions, and (c) removing excess cellular copper via facilitated efflux [Among the non-chordates Copper-ATPases, phenotypic studies had been carried out only on DmATP7 ,50. DmATentation . Norgated efflux . These oDanio rerio). Copper chelation was found to disrupt the notochord and vascular development in this organism [Cal) lacks melanin pigment and has a wavy notochord, mimicking the phenotypes induced by copper deficiency [Cal mutants rescued the embryos, confirming the primary role of Copper-ATPase in the phenotype and functional conservation of ATP7A. Besides ATP7A, genomic sequence of zebrafish predicted a second copper transporter (XP_684415), which is thought to be an orthologue of human ATP7B. In this Copper-ATPase, only 4 copper binding sites were identified in the N-terminus, as compared to six in Zebrafish ATP7A. This may reflect functional difference between ATP7A and ATP7B, though further sequence verification and functional studies are needed to substantiate this conclusion.Among early chordates, physiologic significance of Copper-ATPases was ascertained in zebrafish , each with one copper-binding site. Also in the amoeba, Dictyostelium, only two MBD has been found. In yeast (Saccharomyeces and Candida), two MBDs are present. However, as mentioned earlier, CaCRP1 has three MBDs along with two proximal GMxCxxC motifs, which might have a role in copper binding and its plasma membrane localization. CUA1 of C.elegans has three MBDs. Further up the evolutionary ladder, Drosophila ATP7 has four MBDs. Among chordates  six MBDS are observed. Interestingly, in Branchistoma (amphioxus), in the hypothetical protein BRAFLDRAFT_63720, seven MBDS are detected. Phylogenetic analyses among the chordates using the N-terminal sequence of ATP7B revealed further interesting trend. The evolutionary position of the organism matched well with the evolutionary clustering and divergence of the N-terminal domain . This again points towards the fact that the evolutionary signatures are embedded within the N-termini of Copper-ATPases.As described above, the transmembrane portion of the Copper-ATPase and the functional domains involved in ATP binding and hydrolysis are well conserved. In contrast, the N-terminal metal-binding domain seems to be the most altered during evolution. A general trend of increasing the number of copper binding sites is observed from lower to higher organisms. The How can the N-terminal influence the properties of evolutionary different Copper-ATPase? Recent studies indicate that besides the number of MBDs (which may modulate the ATPase sensitivity to copper), the N-terminal domain is a subject of multiple regulatory activity. This increasingly multifaceted function of the N-terminus imparts uniqueness on the corresponding Copper-ATPases allowing them to adapt to a large spectrum of conditions.1b). The first four metal binding sites in MBD1–4 are present in chordate Cu-ATPases and have been reported to have a regulatory function in mammals [Significant experimental evidence illustrates that the N-terminus is the major site of Copper-ATPase regulation  determine the redox state and copper binding efficiency of the N-terminal of ATP7B . Mutatin1Met-63Arg) is critical for the apical targeting of ATP7B. Deletion of the N-terminal sequence encompassing the first 4 or 5 MBDs and the terminal 63 amino acids (ΔMBD1–4 and ΔMBD1–5) resulted in protein being targeted constitutively to the basolateral surface. However, fusing the 1-63 N-terminal sequence to ATP7B with only MBD 5 and 6 restored the wild type behavior of the protein. Within the 1-63 sequence a stretch of nine amino acids, F37AFDNVGYE45, is an essential copper dependent apical targeting determinant [AFDNRGY). This similarity suggests that apical trafficking behavior ATP7B may also be conserved in lower chordates.Copper binding and subsequent changes in domain-domain interactions most likely allow Copper-ATPases to adopt specific conformations that are critical for its targeting and trafficking. Besides the MBDs, the N-terminal also harbors structural elements that are critical for the directionality of Copper-ATPase trafficking in polarized epithelium. The very N-terminal of the protein has been implicated in determining the apical localization of the protein in high copper . This sterminant . This sein vitro and in cells. Analysis of phosphorylated peptides by mass spectrometry reveals that the loop connecting MBD3 and MBD4 contains phosphorylated residues. Interestingly, the region encompassing MBD1-3 (29 kDa fragments) was shown to have a inhibitory effect on phosphorylation [Another mechanism by which the N-terminal domain may regulate the ATPase is through a kinase mediated phosphorylation. The N-terminus contains site(s) for kinase mediated phosphorylation; copper binding to ATP7B in cells increases protein phosphorylation and stimulates trafficking . The recrylation . In anotrylation , suggestThe phenomenon of regulatory phosphorylation of Copper ATPases is also observed in lower eukaryotes. The N-terminus of Ccc2p in yeast (Ser258) is a target of a kinase mediated phosphorylation, and this phosphorylation is higher in case of catalytically inactive protein containing mutated Asp residue (Asp627) . The funIn conclusion, the copper ATPases provides an interesting case to study evolution of a large and complex proteins, in parallel to the evolution of its harboring organism. Originating as a plasma membrane protein in thermophilic bacteria whose exclusive function was probably to export copper, Copper-ATPases have evolved in higher organisms as a transporter with more than one function and a very complex regulation. The increased functional complexity has been associated with structural changes. These changes did not affect equally all functional domains. Instead we show that that the N-terminus of the copper ATPases underwent the major diversification both structurally and functionally in comparison to other conserved domains.


Actinomyces oris type-1 pili are important for the initial formation of dental plaque by binding to salivary proteins that adhere to the tooth surface. Here we present the X-ray structure of FimP, the protein that is polymerized into the type-1 pilus stalk, assisted by a pili-specific sortase. FimP consists of three tandem IgG-like domains. The middle and C-terminal domains contain one autocatalyzed intramolecular isopeptide bond each, a feature used by Gram-positive bacteria for stabilization of surface proteins. While the N-terminal domain harbours all the residues necessary for forming an isopeptide bond, no such bond is observed in the crystal structure of this unpolymerized form of FimP. The monomer is further stabilized by one disulfide bond each in the N- and C-terminal domains as well as by a metal-coordinated loop protruding from the C-terminal domain. A lysine, predicted to be crucial for FimP polymerization by covalent attachment to a threonine from another subunit, is located at the rim of a groove lined with conserved residues. The groove may function as a docking site for the sortase-FimP complex. We also present sequence analyses performed on the genes encoding FimP as well as the related FimA, obtained from clinical isolates.The  C. diphtheriaeActinomyces orisStreptococcus pyogenesStreptococcus pneumoniaeStreptococcus agalactiaeBacillus cereusPili are hair-like organelles on the surface of bacteria. They are essential for functions such as biofilm formation, host-pathogen interactions and attachment to surfaces. Gram-negative pili are well studied both regarding structure and function Gram-positive proteins that function as building blocks for pili polymerization share some common characteristics. There is a signal peptide located in the N-terminus and an LPXTG motif in the C-terminus, followed by a transmembrane segment. The LPXTG motif is a sorting signal recognized by a sortase (a cysteine transpeptidase) that cleaves the protein between the threonine and the glycine in the motif. In the next step the threonine is covalently attached either to the cell-wall peptidoglycan if the sortase is a housekeeping sortase or to a lysine of a central pilin motif (WXXXVXVYPK) Actinomyces spp together with streptococci are among the first colonizers of the oral biofilm and promote further biofilm formation by their interaction with a wide variety of proteins and carbohydrates on microorganisms and host cells, or from saliva. A. oris  is involved in the co-aggregation reaction with carbohydrates 31–491) from A. oris strain T14V was expressed in E. coli, purified and crystallized. The N-terminal signal peptide, the C-terminal transmembrane helix and the cell-wall anchoring motif LPLTG were not included in the construct , middle (M) and C-terminal (C) domains . The IgGThe N-domain comprises a β-sandwich of one three-stranded, mixed β-sheet (S1) and one four-stranded (S2) anti-parallel sheet. Two helices (HA and HB) pack against the S2 sheet. In addition, two short anti-parallel β-strands, β7-β8, connected by a long loop, are located perpendicular to the β-sandwich .The M-domain comprises a β-sandwich of two five-stranded anti-parallel sheets, S3 and S4. A helix (HC) is wedged in between the upper part of the sandwich. Below the S4 sheet two anti-parallel strands, β26 and β27, are located. A 20-residue long loop that connects β18 with β19 packs against the S4 sheet. The continuation of β19, β20 connects the M- and C-domains .2+ ion , is located at the top of the S5 sheet. A long segment connects strands β21 and β24 and contains a β-hairpin (β22/β23) packed against the S6 sheet, as well as a long loop that coordinates a Ca2+ ion .S. pyogenes pilin Spy0128 The presence of intramolecular isopeptide bonds in Gram-positive surface proteins was first described for the 31–491. The M-domain is stabilized by a bond between Lys-190 and Asn-319, linking two strands that run anti-parallel to each other , H2O (C-domain) or both (wild type) upon isopeptide bond formation compared to the masses calculated from the primary FimP sequence (The presence of the two isopeptide bonds was evident from the continuous electron density between the lysines and the asparagine (in the M-domain) or the aspartic acid (in the C-domain). Their existence was also confirmed by ESI-TOF-MS. Site directed mutagenesis was applied to generate two mutants, D230A and E452A, each replacing the catalytic residue of the isopeptide triads with alanine. Mass analyses of the mutant and wild type proteins confirmed the approximate mass losses of NHsequence . Thus th31–491 structure and interpretable electron density is missing for residues 57–63 and 70–72.Not all Gram-positive bacteria use disulfide bonds to stabilize secreted proteins although it is a common tool used by Actinobacteria In the C-domain a disulfide bond between C385 and C449 joins the S6 sheet of the β-sandwich and the β22-β23 β-hairpin, the structural segment that is followed by the protruding metal-coordinating loop. .31–491 structure and they are modeled as calcium due to the high concentration of calcium in the crystallization conditions. Three of them are likely to be present due to the crystallization solution; one is located between symmetry-related molecules and the other two are coordinated by only three protein atoms each. The fourth metal seems to have a structural role and is coordinated by a long loop in the C-domain, protruding from the S6 sheet. This metal is coordinated by seven oxygen atoms: Asp-396 (OD1 and OD2), Asp-398 (O), Thr-401 (OG1 and O), Thr-403 (O) and Asp-405 (OD2) (2+ (2.33–2.39Å) than to for instance Mg2+ (2.05–2.26Å) 2 with is similar to the surrounding oxygen atoms that are refined to an average of 26.4 Å2.Four metal ions are found in the FimP05 (OD2) . The dis31–491 structure in the Protein Data Bank using the DALI server C. diphteriae  is exposed on the hinge that connects the N- and M-domains, and is thereby available for the sortase mediated pili polymerization reaction  and 5. TS. pneumoniae pilin RrgB give an explanation of the function of the N-domain cleft and what dictates its isopeptide bond formation. When RrgB was crystallized in a form devoid of the sorting motif, an isopeptide bond in the N-terminal domain was not formed The recent structures of the In concurrence with the RrgB structures we propose a similar role for the FimP N-terminal groove; that the cleft and mobile loop (residues 56–64) function as recognition and interaction sites for the sortase-FimP complex during pili polymerization and that a preformed isopeptide bond (juxtaposed to the pilin Lys-182) results in a protein too rigid to properly present the pilin lysine to the sortase. However, during pili polymerization more strain may be imposed on the flexible hinge between the N- and M-domains and Asn-183 and Lys-52 may come close enough to form a bond. Indeed, in the BcpA pilin the N-domain isopeptide bond is observed only after BcpA polymerization In the quest for new drug candidates against pili-bearing Gram-positive bacteria, the N-domain groove may constitute a target for the development of pili inhibiting peptides or chemicals, with the sorting signal as the original template.Actinomyces spp can express two different forms of pili, encoded by separate gene clusters. The respective shaft proteins, FimP and FimA, are similar in terms of size and sequence. FimP consists of 533 residues and FimA of 535. A sequence analysis reveals that the major features; the pilin motif, the number of cysteines, the residues involved in isopeptide bond formation and the LPXTG motif are strictly conserved. Thus FimP and FimA are expected to have very similar structures. On the other hand, some differences are expected to be found, e.g., in the organization of loops and metal binding ( binding .A. naeslundii strain 12104) was cut at the pilin motif lysine as well as in the C-domain (Lys-372). Mishra et alA. oris strain T14V is similarly cut at the pilin lysine. This difference in sensitivity to trypsin implies that FimP is more rigid than FimA in which the pilin motif is more exposed.Limited proteolysis experiments digesting FimA and FimP with trypsin gave different results which indicate some differences in exposure of certain regions (data not shown). Trypsin treatment of FimP generated a cut at Lys-62, located in the mobile loop of the N-domain whereas FimA (from 31–491 and FimA (M- and C-domains) revealed that the major differences between the two structures are the loops connecting the β-strands. The most prominent difference is the segments that pack against the S4 sheet of their respective M-domains. In FimA this constitutes a proline-rich segment (332PPTPETPPTDPENPP347) that runs diagonally over half the sheet. In FimP the equivalent segment is a long, coiled region, located between β18 and β19, that folds over the whole S4 sheet like a clamp  or host-bacteria adhesion partnerships. Notably, Actinomyces has multiple co-aggregation partners and hosts, therefore their pilin proteins are expected to possess multiple binding activities.Sequence comparison of the FimP and FimA N-domains indicates different positions of the disulfide bridges. In FimP the first cysteine of the N-domain is located directly after the presumed isopeptide forming lysine and participate in a disulfide bond that unites the ends of a flexible loop. Also in FimA, the presumed disulfide bond is located near the putative isopeptide bond but with a different organization where its second cysteine is located directly before the catalytic glutamic acid. When the FimA sequence is applied on the FimP template, the first cysteine is located in the coil after the two helices A and B and the second in the S2 sheet, next to the isopeptide bond glutamic acid, as discussed. Assuming that a disulfide bridge is formed also in FimA, we suggest that its S2 sheet is more occluded due to tighter packing with the N-domain helices. The overall appearance of the N-domains may therefore be quite different, which indeed is reflected by their different sensitivity to trypsin. The structural differences are consistent with the differential functional roles of the two pili. The evolution and adaptation of type-1 (FimP) and type-2 (FimA) pili to different intraoral niches and tropisms, suggest their pilin proteins to be organized and function more differently than assumed from their co-presence in the fimP gene from six A. oris reference strains  expressing FimP pili of defined binding profiles Sequencing of the fimA from A. oris isolates (n = 14). The FimP and FimA proteins showed 31% identity/45% similarity and fully conserved isopeptide bond triads, number of cysteines, pilin and LPLTG motifs. The metal binding loop was proved to be unique for FimP and the proline-rich segment unique for FimA. In addition, FimA from A. oris (n = 14) and A. naeslundii (n = 17) were highly related (64% identity/76% similarity) with fully conserved isopeptide bond triads, position of cysteines, pilin and LPLTG motifs as well as proline residues in the proline-rich segment. The sequence alignment of FimA from A. oris is presented in FimP was also compared to FimA, deduced from 31–491was cloned from A. oris strain T14V, expressed and crystallized as described in situ proteolysis with 1% (w/w) α-chymotrypsin immediately before crystallization set up. Crystals of the native protein were obtained in 100 mM sodium acetate pH 5.0, 100 mM CaCl2 and 20% PEG4000. Crystals of the SeMet containing protein were obtained in the same conditions after seeding with the native crystals.FimPGeneration of the mutants D230A and E452A was performed using the overlap extension PCR technique Crystals were flash-cooled in liquid nitrogen after a 30 s soak in the crystallization solution supplemented with 20% glycerol. X-ray diffraction data of the native crystals were collected at beamline ID14-1 and of the SeMet crystals at beamline ID-23 at the European Synchrotron Radiation Facility, ESRF, in Grenoble, France to 1.6 and 2.0 Å resolution respectively. Data were processed with XDS The native structure was solved with MOLREP The X-ray coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 3UXF.Buffer solutions of FimP, FimP-D230A, and FimP-E452A were exchanged for water by dialysis. Accurate molecular masses were determined by ESI-TOF mass spectrometry at Proteomics Karolinska (PK) Institute, Stockholm, Sweden.A. oris strain T14V) and FimA (A. naeslundii strain 12104) in 20 mM CaCl2, 50 mM Tris-HCl pH 8.0, 1% glycerol and 2 mM DTT were incubated with trypsin at a ratio 1∶100 trypsin: FimP/FimA at 293 K for 1 h. The cleavage products were separated on by SDS-PAGE and transferred to a PVDF membrane. N-terminal sequencing of the cleavage products was performed.Purified recombinant FimP  and reference strains . The fimP genes were amplified by PCR using purified genomic DNA and the Ready-To-Go PCR polymerase kit .The fimA genes were sequenced from A. oris (n = 14) and A. naeslundii (n = 17) isolates. The fimA genes were amplified by PCR from purified DNA using the Iproof High-Fidelity enzyme (BioRad). Purified fragments were cloned into Zero Blunt® TOPO® PCR Cloning Kit (Invitrogen) and sequenced. All sequencing was performed by Eurofins MWG Operon. The fimP (corresponding to amino acids 31–516) and fimA (corresponding to the full length protein) sequences were assembled and analyzed using the CodonCode Aligner and MEGA5 software. Identity/similarity values were generated using the NCBI web site.The


Domains are predicted using a library of profile HMMs from 2738 CATH superfamilies. Gene3D assigns domain annotations to Ensembl and UniProt sequence sets including >6000 cellular genomes and >20 million unique protein sequences. This represents an increase of 45% in the number of protein sequences since our last publication. Thanks to improvements in the underlying data and pipeline, we see large increases in the domain coverage of sequences. We have expanded this coverage by integrating Pfam and SUPERFAMILY domain annotations, and we now resolve domain overlaps to provide highly comprehensive composite multi-domain architectures. To make these data more accessible for comparative genome analyses, we have developed novel search algorithms for searching genomes to identify related multi-domain architectures. In addition to providing domain family annotations, we have now developed a pipeline for 3D homology modelling of domains in Gene3D. This has been applied to the human genome and will be rolled out to other major organisms over the next year.Gene3D ( Proteins commonly contain ≥1 discrete independently folding domains. Similar to these folded domains, it is becoming increasingly clear that many proteins can also contain long disordered sections that are also of functional importance. The CATH database which focuses on the folded domain, classifies structures in the PDB into their constituent domains, and each domain is subsequently assigned to a single superfamily by homology . The claAs a summary of the principal updates for this article, we have expanded Gene3D domain assignments considerably by using CATH version 4.0 (updated from CATH version 3.5). As CATH version 4.0 contains nearly 50% more domain structures this has resulted in greater sequence coverage. We have also updated our core sequence and genome databases, increasing the number of unique sequences to >20 million. The FunFams have been updated using the new CATH version 4.0 superfamily assignments for Gene3D version 12. To provide 3D models for selected model organisms, we have developed a homology modelling pipeline that exploits the FunFam multiple sequence alignments to improve accuracy and coverage. For the first time in Gene3D, we have fully integrated Pfam  and SUPEThe number of sequences in the database has increased by 45% since our last publication with >6000 cellular genomes now present. We report a significant increase in domain coverage of sequences in Gene3D, over the previous NAR database publication . The newIn our previous Gene3D publication , we intrAs mentioned earlier in the text, Gene3D now incorporates FunFams, CATH, Pfam and SUPERFAMILY domain assignments to produce highly comprehensive MDAs. We have added in a new inter genome domain architecture similarity search algorithm. This method uses the Needleman–Wunsch (NW) dynamic programming algorithm, modified to align domain strings (MDA) between two proteins  . AligninAnother advantage of the FunFams is that as well as being functionally more pure than CATH superfamily clusters, they also represent structurally cohesive clusters that can be used for homology modelling. Analysis has shown that we can build significantly more 3D models, at a certain high level of accuracy, than by using simple pairwise thresholds on sequence similarity i.e. building models for target sequences sharing ≥30% sequence identity with a protein of known structure . TherefoAs another new feature in Gene3D database, we now integrate PTMs from the Database of Post translational modifications [dbPTM ]. This iThe website has been redesigned to provide better and more unique analysis tools. For brief descriptions of each page see the following sections.The principal results on an individual protein page are the domain assignments which show the MDA with no overlapping domain assignments. Among other things the Protein View page shows details of predicted functions, OMIM mutations , known dFrom the Protein page, it is possible to link out to the individual domain sequence pages . StructuThe structural data and sequence conservation information can be useful both for functional interpretation and for increasing the confidence in various sequence features. For example most ELMs must be exposed on the surface to be functional. From the FunFam alignment of the modelled domain it is possible to colour residues in the structure based on their conservation. In a similar manner, it is possible to colour residues according to the number of phosphorylation sites that have been identified at a given point in the alignment across all members of the alignment. In the near future, we will be adding complementary data on known interactions and the Catalytic Site Atlas for displaying on the structure.From the GO terms associated with each FunFam, we are able to annotate the putative functions of a protein as a whole. This provides a high-throughput means of protein functional annotation. Sometimes the GO predictions from FunFams are extensive, so we provide links to visualize the results using the Revigo resource .We provide details on the physical protein interactions for all organisms from IntAct . The intWe have improved our genome summary pages to better identify the most highly enriched domain families for a given genome by using the Fisher's exact test. The enrichments are calculated relative to the closest set of genomes as judged by the NCBI taxonomy. We also provide a list of the most distinct MDAs for a given genome using the alignment methods described earlier in the text.The average MDA length (the number of domains assigned to a protein) varies greatly between different taxonomic levels, and we now provide the MDA length distribution plots in the summary page. Where multiple transcripts are assigned to a gene we select the longest transcript.Apart from a genome summary, it is also possible for the user to input a higher taxonomic level ID to get similar summary pages. In this case, the NCBI taxonomy is used to group all genomes at the specified taxonomic level before producing summary outputs. In this way, it is possible to get summaries for groups such as Metazoa.For the genome comparison pages, we identify domain families with significant differences in gene counts. We also provide information on those MDAs that are most distinctive between the genomes, both in terms of MDA alignment distances and unique domain compositions. We have also updated our genome comparison tool, so any two taxonomic levels can be easily compared.For a domain family summary page, we provide information on how the family is distributed across the Tree of Life and at which points in the taxonomic tree the family shows expansions in the number of genes in which it occurs. We also provide a table detailing the list of MDAs in which the domain family is found. If the MDA has a known Drug or reliable UniProt-GO annotation, then this data is displayed. Further details on the domain family e.g. structural variation of relatives are available from the equivalent pages at the CATH resource.ftp://ftp.biochem.ucl.ac.uk/pub/gene3d_data/CURRENT_RELEASE/). We have added the fully resolved MDAs Gene3D_12_MDAs.gz. The domain assignments for the Ensembl genomes can be found at Gene3D_12_MDA-genomes.gz.We provide the same downloads as for previous releases at the usual ftp site ; Wellcome Trust (to I.S.); BBSRC (to S.D.); MRC (to N.L.D.); Fondation du 450ème anniversaire de l’Université de Lausanne; and Swiss National Science Foundation [132476 and 136477 to R.A.S.]; This project has been funded in whole or in part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services [HHSN272200700058C and HHSN272201200026C to B.H.D. and C.Y.]. Funding for open access charge: Equally contributed by the Wellcome Trust and the MRC.Conflict of interest statement. None declared.