Post-translational modification by Small Ubiquitin-like Modifiers (SUMO) has been implicated in protein targeting, in the maintenance of genomic integrity and in transcriptional control. But the specific molecular effects of SUMO modification on many target proteins remain to be elucidated. Recent findings point at the importance of SUMO-mediated histone NAD-dependent deacetylase (HDAC) recruitment in transcriptional regulation.Rad60, Esc2 and mouse NIP45 . Different proteins of the novel family are known to interact directly with histone NAD-dependent deacetylases (HDACs), structural maintenance of chromosomes (SMC) proteins, and transcription factors. In particular, the highly non-trivial designation of the first of the two successive SUMO-domains in non-plant RENi provides a rationale for previously published functionally impaired mutant variants.We describe the RENi family of SUMO-like domain proteins (SDP) with the unique feature of typically containing two carboxy-terminal SUMO-like domains. Using sequence analytic evidence, we collect family members from animals, fungi and plants, most prominent being yeast Till now, SUMO-like proteins have been studied exclusively in the context of their covalent conjugation to target proteins. Here, we present the exciting possibility that SUMO domain proteins, similarly to ubiquitin modifiers, have also evolved in a second line – namely as multi-domain proteins that are non-covalently attached to their target proteins. We suggest that the SUMO stable fusion proteins of the RENi family, which we introduce in this work, might mimic SUMO and share its interaction motifs . This presumption is supported by parallels in the spectrum of modified or bound proteins e.g. transcription factors and chromatin-associated proteins and in the recruitment of HDAC-activity. Among ubiquitin-related proteins, containing at least one domain with a ubiquitin-like fold, one can distinguish ubiquitin-like modifiers (UBLs) and ubiquitin domain proteins (UDPs) .UBLs can be covalently attached to target proteins analogously to ubiquitin. Unlike ubiquitin, UBLs mostly do not directly target proteins for degradation , althougIn contrast to UBLs, UDPs are not conjugated to other proteins and lack the C-terminal double glycine motif characteristic for ubiquitin and ubiquitin-like modifiers. They are a heterogeneous class of usually multi-domain proteins, which are unrelated outside of their ubiquitin-like domain . In seveThe biological relevance of non-conjugatable multi-domain proteins having a domain with clear relationship to UBLs like SUMO, rather than ubiquitin, is yet unknown. Here, we present a detailed sequence analysis of a family of SUMO-like domain proteins (SDPs) containing one or two SUMO-like domains. Members of the proposed RENi family act as factors in transcriptional regulation, chromatin silencing and genomic stability.During the study of the predicted nuclear subset of the Drosophila proteome, we encountered the unknown 424 amino acids long protein CG4449 NP_651134). Initial analysis of its sequence complexity shows that the disordered N-terminal half of the protein is followed by a likely globular segment . Indeed, a compositionally biased, polar low-complexity region (LCR) spans almost the entire N-terminal 220 amino acids (AA) as reported by CAST  [1134. IniThe C-terminal half of CG4449 turns out to contain an internally repeated segment identifiable with RADAR  region .In conclusion, we found that the Drosophila protein CG4449 NP_651134) has a tripartite architecture: with a N-terminal LCR followed by two globular domains with a SUMO-like fold (termed SD1 and SD2). Whereas SD2's similarity to single domain SUMO-like sequences can be easily detected with BLAST tools, the identification of SD1 is non-trivial , H. sapiens , M. musculus  and C. elegans . All these proteins contain a LCR at the N-terminus followed by two SUMO-like domains, the first of which has mostly diverged away beyond recognition thresholds using traditional sequence-profile searches  including the two SUMO-like domains, collects a family of animal proteins with the same tripartite organization in D. melanogaster CG4449 (220–424) and the corresponding sequences derived from A. gambie, X. laevis, C. elegans, C. briggsae, M. musculus and H. sapiens  A. thaliana , O. sativa , S. pombe , S. cerevisiae , Y. lipolytica , C. glabrata  and in other recently published fungi proteomes [S. cerevisiae and S. pombe homologs correspond to the studied Rad60 and Esc2 proteins, respectively [A multiple sequence alignment of the globular C-terminal half of s Figure  was usedroteomes . The S. ectively ,15; the These HMM-search results suggest the most likely plant and lower eukaryote orthologues to the animal NIP45-like proteins. For establishing the orthology relationship, these initial results need to be confirmed by reciprocal searches independently performed for fungal and plant proteins. Further below, we present this evidence for the homology between the C-terminal part of the proteins found in the various taxonomic groups.C. glabrata representative (CAG57776) and retrieving the best and significant hits in the proteomes of S. cerevisiae, S. pombe, K. lactis, C. albicans, Y. lipolytica, D. hansenii, A. nidulans [M. musculus , A. thaliana , C. elegans .The set of fungal RENi proteins can be autonomously collected using a BLASTP search started with the nidulans . The domnidulans ). A HMM A. thaliana  against the TIGR Gene indices of barley, maize, rice, potato and soybean [A. thaliana in Y. lipolytica  and H. sapiens proteomes  [Potential plant RENi homologs, derived in a full-length TBLASTN search with  soybean , show a  soybean ) and a ct 8e-05) .We propose to name the collected group of protein sequences the RENi-family after its most studied members Rad60, Esc2 and NIP45. All representatives have a similar sequence architecture involving a N-terminal low complexity region with many polar and (positively) charged residues and a C-terminal globular part with one (plant proteins) or two  SUMO-like domains.The use of a model representing the complete globular region of RENi proteins was essential for the successful collection of the family. A global HMM spanning the SD1 and SD2 domains tests for homology in the whole globular part and, correspondingly, directly collects the RENi family. In contrast, when using the C-terminal half of various RENi family members as query sequence in PSI-BLAST , the seaWhile RENi proteins of the fungal, metazoan and mycetozoan taxa contain two C-terminal SUMO-like domains SD1 and SD2), only the second one can be clearly defined in plant representatives   and rad663E  and A graph representation of the pair-wise similarity relationship for SD1 and SD2 sequences to other known ubiquitin-like domains  is involved in chromatin silencing via the recruitment or stabilization of the Sir (silent information regulators) complex [ complex ,28. It i complex  and, thu complex  and act  complex  as well  complex ,32. With complex  but also complex .NIP45, the one studied RENi in metazoa, has been implied in gene regulation, where it needs its DNA-binding partner NFATp for this activity . StrikinThere is little experimental data on the importance of the predicted SUMO-like domains in RENi proteins. Nevertheless, all listed functions of RENi proteins conform with the known role of SUMO in transcriptional regulation and the control of genome integrity . In the In this report, we use sequence-analytical methods to infer the homology relationships between RENi family members and determine their tripartite (bipartite for plant homologs) domain architecture. A N-terminal polar low-complexity segment and two consecutive SUMO-like domains in the C-terminal half characterize the functionally described fungal and metazoan RENi proteins. While the more C-terminal SD2 is easily detectable, it is the particularly divergent SD1 that was shown in fungi to be essential for the assayed molecular functions. Due to the likely limited sequential-  requirements, this SUMO-like domain is difficult to detect and has been missed in previous analyses of individual family members. The identification of the more N-terminal SUMO-like domain SD1 helps rationalizing experimental findings for mutant fungal RENi family members.RPS-BLAST  and FFASThe sequence analytic work was executed by MN. All authors  contributed to evaluating the results and making the discoveries reported here. MN prepared all the figures and, together with FE, the manuscript text. All authors read and approved the final manuscript.


Many proteins, especially in eukaryotes, contain tandem repeats of several domains from the same family. These repeats have a variety of binding properties and are involved in protein–protein interactions as well as binding to other ligands such as DNA and RNA. The rapid expansion of protein domain repeats is assumed to have evolved through internal tandem duplications. However, the exact mechanisms behind these tandem duplications are not well-understood. Here, we have studied the evolution, function, protein structure, gene structure, and phylogenetic distribution of domain repeats. For this purpose we have assigned Pfam-A domain families to 24 proteomes with more sensitive domain assignments in the repeat regions. These assignments confirmed previous findings that eukaryotes, and in particular vertebrates, contain a much higher fraction of proteins with repeats compared with prokaryotes. The internal sequence similarity in each protein revealed that the domain repeats are often expanded through duplications of several domains at a time, while the duplication of one domain is less common. Many of the repeats appear to have been duplicated in the middle of the repeat region. This is in strong contrast to the evolution of other proteins that mainly works through additions of single domains at either terminus. Further, we found that some domain families show distinct duplication patterns, e.g., nebulin domains have mainly been expanded with a unit of seven domains at a time, while duplications of other domain families involve varying numbers of domains. Finally, no common mechanism for the expansion of all repeats could be detected. We found that the duplication patterns show no dependence on the size of the domains. Further, repeat expansion in some families can possibly be explained by shuffling of exons. However, exon shuffling could not have created all repeats. The building blocks that create proteins are called domains, and domains are often combined to create multidomain proteins. In many vertebrate proteins, repeats with several adjacent domains from the same family can be found. The authors have investigated how these repeats may have evolved. It is believed that the repeats are created through internal duplications where the duplicated region is inserted next to its origin. Therefore, the pairwise sequence similarity between all repeated domains in a protein was used to identify recent duplications, and a method based on autocorrelation vectors was employed to distinguish patterns of duplication. The authors found that repeat regions are often created from the duplication of several domains at a time while duplication of one domain is less common. Further, the internal duplications often occur in the middle of the repeats. This is in contrast to the evolution of nonrepeating, multidomain proteins, which are thought to evolve by the addition of a single domain at the N-termini or C-termini. A preference for duplication of a certain number of domains was found for some of the domain families. Finally, the authors discuss some of the possible mechanisms for repeat expansion. However, the exact mechanism remains to be discovered. Proteins are composed of domains, recurrent protein fragments with distinct structure, function, and evolutionary history. Protein domains may occur alone, but are more frequently found in combination with other domains in multidomain proteins. While the creation of new multidomain architectures through shuffling of protein domains has been studied extensively during the last few years –4, one thttp://gln.ibms.sinica.edu.tw/product/repeat/). Single amino acids or short peptide motifs may be repeated in proteins, too. However, in this study we have focused on larger repeating units, domains. Therefore, when repeats are mentioned in this text, it refers to repeats of protein domains.Repeating domains are often short, such as the leucine rich repeat (LRR) family with a repeating unit of 30 residues. Some repeated domain families are mainly found in repeats, e.g., LRR and C2H2 zinc fingers, while other families are also frequently found as a single unit. The repeats may form regular structures, such as antiparallel β-sheets or solenoids, while others form filaments or are only structured upon binding to their ligands . Some exDomain repeats are often involved in interactions with proteins or other ligands such as DNA or RNA. Even if the repeated domains have a well-defined and conserved structure, the sequence conservation is often low, with only a few conserved residues required for the correct fold. Their variable sequences and the variation in number of domains provide flexible binding to multiple binding partners. Hence, repeats are found in proteins with highly diverse functions such as the tetratrico peptide repeats (TPR) that are involved in cell-cycle regulation, transcriptional regulation, protein transport, and assisting protein folding . In addiThe domain repeats are found in all kingdoms of life, and long repeats, containing several domains in tandem, have been observed to be particularly common in multicellular species ,8. RepeaDomain repeats are thought to arise via tandem duplications within a gene , where aIn addition to internal duplications, frequent duplications of repeat-containing genes have occurred in the mammalian genomes . This caArabidopsis thaliana and especially Caenorhabditis elegans have fewer repeats than expected from their proteome size. Actually, the worm and the two yeast species have a similar fraction of proteins with repeats, hence multicellular organisms are not always distinguished by more repeats than unicellular ones. In addition, some prokaryotes, such as Escherichia coli and Pseudomonas aeruginosa, with similar proteome size as yeast, contain very few proteins with repeats. Thus, having many repeats is a feature of eukaryotes rather than of multicellular species.It has been demonstrated that protein domain repeats are particularly abundant in multicellular organisms ,8. HowevAs many proteins with repeats of more than two domains are found in vertebrates, they should provide functions that are required in complex organisms. Consistently, the proteins with repeats mainly have important binding functions in protein–protein interactions and complex assembly as demonstrated for the largest domain families in The repeated domains are more abundant than nonrepeated domains. In fact, nearly half of the assigned domains in the vertebrates are found in repeats . Still, C. elegans. In addition, further expansions may have taken place after the splits between fish, birds, and mammals, since the chicken proteins contain fewer repeated domains than the zebrafish ortholog.Further evidence of the frequent duplication in repeats is that orthologs appear to have expanded independently . We founTreponema pallidium. This domain family is found in other bacteria, but never with more than five consecutive domains, while in metazoa, the domain family is commonly repeated. Hence, a likely scenario is that this repeat has been horizontally transferred from a eukaryotic host, rather than expanded in the bacteria.Expansion of repeats through internal duplication is not unique to eukaryotes since some prokaryote-specific repeats can be found, e.g., the bacterial immunoglobulin (IG)–like domain and haemaglutinin repeats. Other prokaryotic repeats may be explained by horizontal transfer . For insThe formation of repeats is not well-understood, therefore we aim to understand some of the underlying mechanisms of repeat expansion by studying the number of domains that is duplicated each time. Since domain repeats are assumed to be created through internal duplications , sequencDistinct patterns of repetition could often be distinguished, and in many proteins, units containing multiple domains have been duplicated in tandem. For instance, in the human zinc finger protein found in For many proteins, however, no clear pattern was seen since all domain pairs had similar alignment scores. In other proteins, there were mixed patterns within the protein as distinct parts of the protein have been expanded with duplication units of different sizes. Therefore, autocorrelation vectors (ACVs) were used to get a general view of the relative frequency of duplication units of different sizes in each protein. We have defined ACV as the average alignment score between domains at each distance, i.e., the alignment score between neighboring domains, domains at distance two, three, etc. . The peaThe most common duplication pattern for a domain family can be elucidated when the average ACV for all repeats containing the family is calculated. As an example, the chicken nebulin protein  has beenSuch clear patterns could not be found for all domain families, as can be seen in The ACVs show that duplication units of a few different sizes are dominant in each family. However, duplications of many different unit sizes may occur within a family. To get a view of how the patterns are distributed among the domain families, hierarchical clustering of the ACVs from all proteins was performed . ProteinIn conclusion, the domain repeats are most often created from the duplication of several domains at a time, while duplication of one domain appears to be less common. Further, the number of domains involved in each duplication event differs considerably within the domain families. However, for some domain families, there may be selection for duplication of a certain number of domains due to some functional or structural constraint, as is likely in the case of the nebulin domain. In addition, the most commonly repeated domains, the C2H2 zinc fingers, show the most diverse distribution of duplication patterns.To determine if duplication at either end of a protein is preferred, the most recent duplications were identified and their positions were determined, revealing that a large proportion of the repeats have been expanded in the middle of the protein. The fraction of duplications we observe in the middle is slightly, but significantly, higher than expected by chance . In addiRepeated domain families are on average shorter than nonrepeated domain families . HoweverAnother possibility is that there is a preference for duplication of certain sizes due to functional constraints, where a fixed number of domains are required for function. In that case, short repeats with that particular length may also be common. This seems to be true for cadherin domains, which have a peak in the ACV at distance five and are also abundant in five domain repeats  and S3. Exon shuffling, i.e., nonhomologous recombination in the intron regions, can create new exon combinations and new proteins. As a consequence, exon shuffling is responsible for many new domain combinations, and it has been demonstrated that exon-bordering domains often combine with other domains . HoweverTo verify if the exon junctions are enriched in repeated domains or in linkers between the domains, simulations with random positioning of the junctions were performed. As a result, it was evident that more exon junctions are located in linkers than is expected at random . FurtherOur results are consistent with findings that extracellular domains, such as IG and EGF, are often recombined through exon shuffling . HoweverInterestingly, the exon structures revealed that 30% of the repeats with ten or more domains are located within one large exon, excluding the possibility of exon shuffling as the mechanism for their expansion. This was especially evident for human C2H2 zinc finger proteins, where 78% of the long repeats were found within one exon. The corresponding number of one-exon zinc finger repeats was lower in the other species, e.g., 11% in zebrafish. Also, LRR had many repeats in one exon, while other domain families always have the repeats spread over several exons . NeverthIn conclusion, exon shuffling may be responsible for the expansion of some domain repeats, especially the extracellular ones that are often expanded one domain at a time. However, all repeat duplications cannot have been created by exon shuffling.A complication in this analysis is deletions within proteins, since our method does not detect domain deletions. However, protein evolution tends to generate longer proteins, and it has been shown that proteins are more often extended by fusion than truncated by fission in protein evolution ,25. FurtWright and coworkers recently published a study on protein aggregation where they found that neighboring domains, in repeats of IG and fibronectin domains, have lower sequence identity compared with more distant domains, and suggest that this may prevent protein aggregation . For IG Whether repeat expansion is a random process or a controlled mechanism, where specific segments are selectively duplicated, remains to be discovered. Internal duplications may take place in all proteins, but it is likely that such duplications are lost if the protein does not contain domains that have a repeat-forming characteristic. On the other hand, an increase in the number of repeated domains might not alter the protein structure drastically and can actually promote protein stability ,27. The Short protein repeats may be created from DNA hairpin formation and strand slippage while the hypermutability of minisatellite loci (repeating units of more than ten nucleotides) is thought to be due to recombination events . The expIdentification of such hotspots would require exact identification of the gene segments that have been duplicated, which is difficult in most cases. Further, a method that would distinguish overrepresented DNA motifs at their flanks is needed. Finally, detection of such motifs would require that the motifs are conserved after the duplication has occurred. Still, many challenges lie ahead before the tandem duplication of protein domains can be fully understood.In this work, we show that repeat regions are most often created from the duplication of several domains at a time while duplication of one domain is less common. Further, we found that the internal duplications often occur in the middle of the repeats. Hence, the internal duplications in repeats evolve differently from other domain recombinations, which mainly involve the addition of a single domain at either terminus. Preference for duplication of a certain number of domains could be seen for some of the domain families. However, most domain families show broad distribution of duplication patterns and can be expanded with different numbers of domains, even if certain duplication sizes are more common. The exact mechanism behind these duplications is not well-understood. We found no correlation between the size of each duplicated fragment and the domain sizes. For some domain families, however, selection for functional units containing a certain number of domains may favor the duplication of that unit. In addition, exon shuffling could partly explain the duplications of some domain families, especially the extracellular domains. However, many repeats are found within one large exon, hence it is highly unlikely that they have evolved via exon shuffling.Homo sapiens, Mus musculus, Rattus norvegicus, Gallus gallus, Danio rerio, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Saccharomyces cerevisiae, and Schizosaccharomyces pombe; seven bacteria: Escherichia coli K12, Pseudomonas aeruginosa, Bacillus subtilis, Rickettsia conorii, Mycoplasma pulmonis, Prochlorococcus marinus, and Treponema pallidum; and seven archaea: Aeropyrum pernix, Methanococcus jannaschii, Nanoarchaeum equitans, Pyrococcus abyssi, Thermoplasma volcanium, Archaeoglobus fulgidus, and Methanosarcina mazei.We have analyzed the proteomes of 24 species; ten eukaryotes: ftp://ftp.ncbi.nlm.nih.gov/genbank/genomes/Bacteria/) and the eukaryotic genomes from Ensembl (ftp://ftp.ensembl.org/), except for S. cerevisiae and S. pombe, which were collected from Saccharomyces Genome Database [http://yeastgenome.org/). In the eukaryotic species, the longest transcript from each gene was used.The microbial sequences have been collected from the National Center for Biotechnology Information (NCBI) (Database  (http://ftp://ftp.ensembl.org/).Exon and intron information for the seven metazoan species were extracted from Ensembl  with a cutoff for assignments at an E-value of 0.1. This rather high cutoff was used to increase the number of assigned repeating domains. As we in this study focused on repeating domains , the number of false positives is effectively reduced compared with when single domains are considered. After domain assignments, gaps within the repeats were evident in many cases, as shown in ftp://ftp.ensembl.org/). When gaps of the same size or larger than the surrounding domains were found in the repeat regions, the proteins were subjected to additional assignments with HMMER as described above. All these additional assignments increased the number of domains in long repeats quite drastically, with additional domains in 40% of the proteins with more than ten repeating domains.Pfam-A domains were assigned to the prokaryotic proteomes, the yeast species, and ftp://ftp.sanger.ac.uk/pub/databases/Pfam/). For a better view of repeat expansions, all families from the same clan were grouped together, rendering a slight increase in the number of repeated proteins, from 12.2% to 12.6%, and also an increase in the number of domains in each repeat, especially for proteins with LRR.In addition, many repeats with alternating domains from related domain families were found, e.g., the different Pfam families of TPR or the IG-like domains. Such related domains are grouped together in the Pfam Clans  was calculated, i.e., the average alignment score between all domains at each distance k, where the distance between domain one and two is k = 1, between one and three is k = 2, and so on. If the alignment score between domains id and jd is defined as i,dj)S. When the autocorrelation for all the repeating proteins with the same domain family was calculated, the average alignment score for all domains at distance n in all proteins with that family were used to calculate ACV(k). Before these calculations, the dataset was homology-reduced using nrdb90 [Our analysis is based on the assumption that the most recently duplicated domains have the highest sequence similarity to their originating domains. To quantify the duplication patterns, an g nrdb90 , removinA = 17 , nDA = 460 (number of domains for ACV), nPs = 16 ); C2H2 Zinc finger ; IG ; EGF ; Fibronectin 3 ; LRR ; Ankyrin ; Cadherin ; All .The ACVs presented in r and ns are the number of objects in clusters r and s, and ACVs of length nine were created for all proteins with ten or more repeats. As longer vectors cannot be created for proteins with repeat length ten, we used this cutoff to be able to compare the whole dataset. Hierarchical clustering of the ACVs was performed using the Ward incremental sum of squares distance measure, in Matlab , to measure similarity between the vectors. The distance is defined asIn addition, the domain families were also clustered with the same method using the distribution of domain families in the 20 ACV clusters.The position of latest duplication was determined for all proteins with repeats of five or more domains. To identify where in the repeat the most recent duplication took place, a matrix was created similar to the one in Z = |x − μ|/σ, where x is the observed value and μ is the average value obtained from simulations with standard deviation σ. Assuming a normal distribution of the data, the p-value was then derived from the Z-score using normal distribution p-value tables.To estimate the statistical significance of our results, Z-scores were calculated from randomization in 10,000 iterations. The Z-score was calculated as In the simulation of ACVs, the positions of the domains in a protein were shuffled while maintaining their individual alignment scores. In each iteration, an ACV for proteins with each domain family was calculated, and finally the Z-score for each position of the vector was calculated from these randomized values.In the case of enrichment of exon boundaries in linker regions, the domain and linker positions in each protein were kept constant. The number of exon boundaries in each protein was also conserved, but they were positioned randomly along the protein sequence. In each iteration, the fraction of linkers that contained exon boundaries was calculated.The enrichment of the domain families in each cluster in For estimation of the position of latest duplication, the domain order was shuffled in each protein while maintaining individual alignment scores. In each iteration, the fraction at each position was estimated, as described in the previous section. Finally, the Z-scores for fraction at N/C-terminal or middle were calculated.Figure S1A repeated domain family is defined as a family found in a repeat of at least three domains, and nonrepeated families are never found as repeated. The copy numbers for repeated domains have been calculated as the total number of copies (Rep. Copies) or counting each protein with the repeat only once (Rep. Compressed).(24 KB EPS)Click here for additional data file.Figure S2p-value for each datapoint was calculated from random shuffling of domains, and peaks with p-values below 10−5 are indicated with an asterisk.The domain family name is followed by the number of proteins (nP) and number of domains (nD) used in the calculations. The autocorrelation for each family was normalized around zero, hence the dashed line at zero is the mean bit score between all domains in the family. The (61 KB EPS)Click here for additional data file.Figure S3(47 KB EPS)Click here for additional data file.Figure S4(A) The alignment scores between all domains in a human zinc finger protein with darker color for higher scores.(B) All scores that are one standard deviation over the mean score are set to one (gray). Then the longest diagonal of “ones” is identified (black) and the position of that diagonal is determined. In this case the latest duplication is estimated to occur in the end.(168 KB EPS)Click here for additional data file.Figure S5The fraction of different regions that contain disordered regions or different secondary structures. The first bar shows the distribution in all of the proteins followed by repeated domains (RepDom), non-repeated domains (NRDom), and regions without domain assignments (Unass).(25 KB EPS)Click here for additional data file.Figure S6The size of most duplicated units, i.e., the number of domains involved in most duplications, was determined from the highest peak in the ACVs  for the (884 KB EPS)Click here for additional data file.Figure S7D. melanogaster, C. elegans, and S. cerevisiae are displayed, and they all show higher connectivity with increasing repeat length.The connectivity is displayed for proteins with no repeat (repeatlength 1), two-domain repeats, etc., up to repeats of length nine or more. The networks for three eukaryotic species, (26 KB EPS)Click here for additional data file.Table S1(21 KB DOC)Click here for additional data file.Table S2(28 KB DOC)Click here for additional data file.Protocol S1(60 KB DOC)Click here for additional data file.


Lactococcus lactis recognizes an asymmetric, complementary DNA sequence, consisting of 5'GACGC'3 in one strand and 5'GCGTC'3 in the other and provides a prodigious barrier to bacteriophage infection. LlaJI is comprised of four similarly oriented genes, encoding two 5mC-MTases (M1.LlaJI and M2.LlaJI) and two subunits responsible for restriction activity (R1.LlaJI and R2.LlaJI). Here we employ a detailed genetic analysis of the LlaJI restriction determinants in an attempt to characterize mechanistic features of this unusual hetero-oligomeric endonuclease.Restriction/modification systems provide the dual function of protecting host DNA against restriction by methylation of appropriate bases within their recognition sequences, and restriction of foreign invading un-methylated DNA, such as promiscuous plasmids or infecting bacteriphage. The plasmid-encoded LlaJI restriction/modification system from E.coli K-12. R1.LlaJI did not appear to contain a catalytic centre, whereas this conserved motif; PD....D/EXK, was clearly identified within the amino acid sequence for R2.LlaJI. Both R1.LlaJI and R2.LlaJI were found to be absolutely required for detectable LlaJI activity in vivo. The LlaJI restriction subunits were purified and examined in vitro, which allowed the assignment of R1.LlaJI as the sole specificity determining subunit, whilst R2.LlaJI is believed to mediate DNA cleavage.Detailed bioinformatics analysis confirmed the presence of a conserved GTP binding and hydrolysis domain within the C-terminal half of the R1.LlaJI amino acid sequence whilst the N-terminal half appeared to be entirely unique. This domain architecture was homologous with that of the "B" subunit of the GTP-dependent, methyl-specific McrBC endonuclease from in vivo, in that it restricts un-methylated DNA, it resembles the Type IV McrBC endonuclease, an enzyme specific for methylated DNA. A number of similar restriction determinants were identified in the database and it is likely LlaJI together with these homologous systems, comprise a new subtype of the Type II class incorporating features of Type II and Type IV systems.The hetero-subunit structure of LlaJI, wherein one subunit mediates DNA binding whilst the other subunit is predicted to catalyze strand hydrolysis distinguishes LlaJI from previously characterized restriction-modification systems. Furthermore, this distinction is accentuated by the fact that whilst LlaJI behaves as a conventional Type IIA system  The diverse structural and functional activities described for restriction endonucleases (REases) is reflected by their ubiquitous abundance among prokaryotic organisms . These e+2 dependent manner.Four distinct groups of REases are known, with the "Type II" group being the most richly represented . MembersAlthough generally lacking significant amino acid sequence homology, structural analysis of many of these REases has revealed in some instances a conserved core, consisting of a four/five-stranded mixed β-sheet flanked by α-helices, reviewed in . This coFor double-stranded scission this hydrolysis event must occur on both strands of the target DNA thereby requiring two catalytic sites , althougOne such characterized enzyme, BbvCI, has been shown to be heterodimeric, with each individual subunit independently responsible for cleavage of separate strands of its asymmetric recognition sequence ,19. SimiPreviously we have described the cloning and transcriptional regulation of LlaJI R/M system ,20. The mcrBC locus encodes two major products, McrB (53 kDa) and McrC (39 kDa), which mediate the restriction phenotype [mC sites (separated by 40 bp to up to 3000 bp) and cleaves at random positions between the two [in vitro has been suggested as 3–5:1, McrB:McrC , with troup [+2 .The McrB subunit harbours the specific DNA binding activity (which resides within its N-terminus), in addition to GTP binding and hydrolysis activities ,30,34. TIn this report, we execute a detailed genetic and phenotypic assessment of the McrBC-like LlaJI endonuclease which serves to portray the complexity of this unique enzyme.R1.LlaJI is predicted to be a 67.2 kDa protein which exhibits sequence homology to a number of partially characterized and putative REases in the database  .Additional conserved modules (of identity) can be discerned within R1.LlaJI and its homologues; however, at this point no functional significance can be assigned to any of these regions Fig . It was For most of the identified R1.LlaJI homologues, significant alignment was only achieved with portions of the sequences corresponding to the C-terminal half of each, with the N-terminal portions apparently representing non-conserved sequence. The two exceptions to this, namely LlaIB and BsuMIB Fig  and 1B, Database searches with R2.LlaJI (48.8 kDa) revealed a set of similar proteins that are encoded by genes located adjacent to those that had been identified as encoding homologues of R1.LlaJI see Fig  and 1C. These data cumulatively suggest that LlaJI, and indeed other restriction systems exhibit structural and therefore likely mechanistic similarities with that of the McrBC methyl-specific endonuclease (see discussion).llaJIR1 and llaJIR2 genes were amplified individually, and in tandem, and placed under the control of the strong nisin-inducible promoter of plasmid pNZ8020 to facilitate their over-expression.Previously, we have described the bacteriophage resistance phenotype conferred by the LlaJI operon . To fullThe resulting constructs, pNZ-R1, pNZ-R2, and pNZ-R1R2 were subsequently introduced into the expression host NZ9000, containing the plasmid pJO-M1M2 (which encodes both LlaJI MTases), and analyzed for their ability to confer phage resistance. As can be seen from Table The fact that this optimal level of resistance may be achieved at 30°C by mere over-expression of R1.LlaJI and R2.LlaJI suggests that the temperature sensitivity of the LlaJI system previously reported, may be attributable to the intracellular concentration of active LlaJI at the respective temperatures , rather than to the temperature "activity" spectrum of assembled functional LlaJI, as had previously been speculated [As stated, the results obtained with the hosts expressing R1.LlaJI and R2.LlaJI individually  indicated that neither subunit alone was capable of restricting the growth of phage. However, despite the lack of significant decrease in EOP at either sampling temperature used, a delicate but visibly obvious variable plaque phenotype was observed for the host expressing R1.LlaJI alone . Over-expression with pQE-R1 allowed purification of R1.LlaJI as a C-terminal 6xhis tagged derivative. Mass spectrometry and N-terminal sequencing confirmed the predicted molecular mass (68.139 kDa inclusive of the mass of the 6x His tag) and identity of purified R1.LlaJI.As it was established that both LlaJI subunits were required for "complete" activity, it was assumed that expression of the individual subunits separately for purification purposes could be achieved in the absence of the LlaJI MTases. To this end, both llaJIR2 was inserted downstream of the malE gene of plasmid pMal-c2x under the control of the Ptac promoter  which, upon induction resulted in the expression of an R2-MBP fusion of the predicted molecular mass (~90.8 kDa). This fusion was purified by affinity chromatography using amylose resin and the R2.LlaJI subunit was subsequently released from the R2-MBP preparation by cleavage with factor Xa protease. Rebinding of MBP to amylose resin resulted in purification of R2.LlaJI to ~60 % homogeneity.Purification of R2.LlaJI by this approach failed, possibly due to instability of the resulting recombinant protein. To overcome this presumed instability, Given the domain architecture shared between R1.LlaJI and McrB, and the established DNA binding activity of the latter, we investigated the possibility of R1.LlaJI constituting the DNA recognition element of LlaJI.+2 in the binding buffer had no discernable effect on R1.LlaJI specific binding, nor did its addition post protein:DNA complex formation. Interestingly however, and in contrast to McrB, inclusion of GTP did not stimulate the R1.LlaJI DNA interaction expressing the LlaJI system (as measured by its phage resistance phenotype). Subsequent attempts to reconstitute active LlaJI by combination of the partially purified subunits in various ratios, and with an assortment of cofactors  failed to reveal any specific nuclease activity. Furthermore, neither subunit was found to exhibit a DNA nicking activity. Possible explanations for these observations are discussed below.The presence of the putative GTPase domain in R1.LlaJI prompted us to investigate whether or not this subunit displayed such an activity. Analysis of GTP hydrolysis, conducted by colorimetric assay  suggested that R1.LlaJI did not exhibit an intrinsic GTPase activity. Surprisingly, R1.LlaJI dependent GTP hydrolysis could not be activated by the inclusion of "specifc" DNA and/or partially purified R2.LlaJI in the reaction. Similar results were obtained with alternative nucleotides.E. coli host was performed. As can be seen from Table In an attempt to explain the lack of endonuclease activity in the protein preparations, a phenotypic investigation of the efficacy of LlaJI in the heterologous in vivo LlaJI restriction in this host.When both subunits were over-expressed in the same host (plasmid pUC-R1R2) however, and in agreement with results for the lactococcal over-expression host above, a phage resistance phenotype was apparent Table . The degE.coli further revealed that, although the observed resistance (with plasmid pUC-R1R2 at 30°C) was not evident at 37°C, it was not significantly enhanced with growth at low temperatures (data not shown). Again, as with the lactococcal hosts, crude extracts of E.coli over-expressing R1.LlaJI and R2.LlaJI (from plasmid pUC-R1R2) did not exhibit detectable endonuclease activity.Investigation of the temperature induced efficacy of LlaJI in The molecular mechanism by which the lactococcal plasmid-encoded LlaJI R/M system achieves DNA restriction has remained largely elusive. Previous studies have characterized the phage resistance capabilities of this four-gene cluster which is accompanied by an intricate transcriptional regulatory mechanism ,20.In the present study we offer additional phenotypic and genetic data which alludes to an unusual mode of action for LlaJI. Analysis of the deduced amino acid sequence for R1.LlaJI revealed a segmented architecture, with a C-terminally located putative GTP binding and hydrolysis domain, and a largely unique N-terminal portion. This organization was similar to that observed for the McrB subunit of the McrBC methyl-specific endonuclease ,37.A mutational analysis of the three motifs which comprise the GTPase domain of McrB confirmed the assignment for motif I only, which is the most highly conserved of the three with respect to R1.LlaJI and its homologues see Fig . ConservIn addition, the relative spacing between motif's II and III on McrB (29 amino acids), appeared too short to be structurally analogous to the classical GTPases (>49 amino acids). HoweverIt has been shown that it is the N-terminal 160 amino acids of McrB which, like R1.LlaJI did not exhibit significant homology to the database, specified the DNA binding activity of McrBC . This, iInterestingly, and in contrast to McrB, R1.LlaJI did not exhibit an intrinsic GTPase activity, which could neither be stimulated by specific DNA nor partially purified R2.LlaJI. This latter finding was again in contrast to McrB, whose intrinsic GTPase activity was stimulated 30-fold by McrC . A putatin vivo activity was clearly demonstrated (Table Lactococcus), although this was most likely due to DNA binding only, which may have served to partially hinder the phage replication machinery.The absolute requirement for both R1.LlaJI and R2.LlaJI for complete E.coli provided resistance to phage infection where, again an absolute requirement for both R1.LlaJI and R2.LlaJI was observed  of R1.LlaJI would only become apparent following assembly of the "active" heterooligomer on its target DNA. It must be noted however, that an additional lactococcal derived cofactor, proteinacious or otherwise, can not be entirely excluded at this point, despite the absence of endonuclease activity in LlaJI-expressing lactococcal crude extracts.In summary, LlaJI is a heterooligomeric endonuclease wherein the R1.LlaJI subunit is responsible for determining specificity. It is suggested that R2.LlaJI harbours the catalytic centre for DNA cleavage, whose site, although undetermined, is likely to be defined by the relative "placement" of R2.LlaJI on the target DNA (due to its proposed interaction with R1.LlaJI).in vivo however; unlike any characterized enzyme of its kind it exhibits striking genetic similarities with the Type IV enzyme McrBC, which is specific for methylated DNA. The presence of an McrBC-like enzyme as a component of an R/M system suggests two mutually exclusive modes of action for these enzyme types whereby they are either in isolation, cleaving methylated DNA (McrBC), or in combination with MTases cleaving unmethylated DNA (LlaJI). Whilst unusual, this is not altogether surprising as such contrasting activities have been described for some conventional endonucleases (DpnI and DpnII) [LlaJI was shown to retain behavioral characteristics of a conventional Type IIA system, in that unmethylated DNA is targeted d DpnII) ,40. SimiL. lactis strains were grown in M17 broth  containing 0.5 % glucose at 30°C. E. coli was grown at 37°C in Luria-Bertani media [L. lactis, 5 μg ml-1 tetracycline, and 10 μg ml-1 chloramphenicol. For E. coli, 100 μg ml-1 ampicilin, 25 μg ml-1kanamycin and 10 μg ml-1 chloramphenicol. Induction of the nisin-inducible promoter of pNZ8020 in the NZ9000 host background was achieved by inclusion of nisin (1 ng ml-1) in growth media [E.coli hosts. Lactococcal bacteriophage were propagated as described previously [Details of bacterial strains, bacteriophage and plasmids used in this study are summarized in Table ni media . Where ath media . Inductieviously  and the eviously .E.coli plasmid DNA was isolated using the SV wizard plasmid miniprep kit  according to the manufacturer's instructions. Lactococcal plasmid DNA was isolated as described previously [E.coli was performed using standard techniques [L. lactis as described previously [Taq DNA polymerase  was used. For plasmid construction, where high fidelity was required, PCR's were conducted using Phusion DNA polymerase (New England Biolabs) according to the manufacturer's instructions. All PCR products were purified using the Jetquick PCR purification kit . The correct orientation and integrity of all constructs was verified by sequencing, performed at MWG Biotech . Sequence assembly, verification and analysis were achieved using the seqman program from the DNASTAR package . Database searches were performed using the BLAST suite of programs [AY530537.Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs , and used according to the manufacturer's instructions. eviously . Electrochniques  and in Leviously . For rouprograms  and the programs . The nucllaJIR1 encompassing PCR product  was inserted into the BamHI – XhoI sites of pNZ8020. Plasmid pNZ-R2 was constructed by insertion of an llaJIR2 encompassing PCR product  into the BamHI – XhoI sites of pNZ8020. Similarly, for plasmid pNZ-R1R2, an llaJIR1R2 encompassing PCR product  was inserted into the BamHI – XhoI sites of pNZ8020.For the construction of plasmid pNZ-R1, an llaJIR1 encompassing PCR product  was inserted into the NcoI – BglII sites of pQE60. For plasmid pQE-R2, an llaJIR2 encompassing PCR product  was inserted into the NcoI – BglII sites of pQE60. Plasmid pMal-R2 was constructed by insertion of an llaJIR2 encompassing PCR product  into the XmnI – XbaI sites of pMal-c2x. Plasmid pUC-R1 was constructed by insertion of an llaJIR1 encompassing PCR product  into the SalI – BamHI site of pUC19. For plasmid pUC-R2, an llaJIR2 encompassing PCR product  was inserted into the SalI – BamHI sites of pUC19. Plasmid pUC-R1R1 was constructed by insertion of an llaJIR1R2 encompassing PCR product  into the SalI – BamHI sites of pUC19.For plasmid pQE-R1, an R1.LlaJI was expressed as a C-terminal 6xhis tagged derivative, overproduced from plasmid pQE-R1 in the M15/pRep4 host background and purified essentially to homogeneity using Ni-NTA Agarose (Qiagen) affinity chromatography, according to the manufacturer's instructions. R2.LlaJI was overproduced from plasmid pMal-R2 in ER2683 and purified by affinity chromatography using amlyose resin (New England Biolabs), according to the manufacturer's instructions. Purified proteins were subsequently dialyzed against buffer containing 10 mM Tris (pH 7.5), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA and 50 % glycerol, and stored at -20°C. Initial mass and purity estimations were achieved by SDS-PAGE  under de-1 calf thymus DNA and approximately 2.5–5 nM final concentration of a probe. All probes were generated by PCR amplification of a section of the LlaJI promoter (coordinates 309–539 using primers akjF 5'ctggagactatattgctcatcg'3 and P1R 5'gggattgtgtttgaaatcacacc'3) which contained two LlaJI recognition sites. Probe J represented the "wild type" sequence. Disruption of the 5' proximal and 3'proximal sites individually  to produce probes J1 and J2 respectively, was achieved by oligonucleotide-directed mutagenesis. Both sites were disrupted in this manner to produce probe J3, which was devoid of any LlaJI recognition sites. All probes were subsequently end-labelled with (γ-32P)ATP by use of polynucleotide kinase (New England Biolabs). Binding reactions were allowed to proceed at room temperature for 30 minutes, followed by the addition of 5 μl 50 % glycerol. Samples were then subjected to electrophoresis on a 4 % polyacrylamide gel containing 2.5 % glycerol in 1 × TAE buffer for 3 hours at 120 V. The protein-DNA complexes were detected by autoradiography of the dried gel.DNA binding assays were carried out in 20 μl volumes containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM β-mercaptoethanol, 8 % glycerol and 50 μg mlL. lactis strains were prepared from 1 l of culture grown in GM17 broth, concentrated by centrifugation  and washed in lysis buffer (10 mM Tris-HCl (pH 7.6), 1 mM EDTA, 100 mM NaCl and 1 mM DTT). The cell pellets were resuspended in 10 ml of lysis buffer and disrupted with a French press (at 2000 lb/in2) after which cell debris and ribosomes were removed by centrifugation . E. coli crude extracts were generated essentially as above from 1 l of culture grown in LB broth, where lysis was achieved by sonication (5 × 10 sec bursts punctuated by 30 sec on ice). Cleavage activity of LlaJI was surveyed in all 4 NEBuffers (New England Biolabs) and at temperatures ranging from 19 – 30°C.Crude cell extracts of -1 BSA, and 2 μl crude extract in addition to 5 mM GTP in a final volume of 20 μl. For assays with purified proteins, typically reactions contained 0.5 μg R1.LlaJI or R2.LlaJI .Typically the reactions contained 1 μg substrate DNA (λ DNA and Sk1 DNA for cleavage assays or pBR322 DNA for nicking assays), 100 μg mlGTP hydrolysis activity of R1.LlaJI was investigated via quantification of the release of inorganic phosphate by colorimetric assay based on spectrophotometric quantification of a phosphomolybdate-malachite green complex using the Malachite Green Phosphate Assay kit  according to the manufacturer's instructions.Financial competing interests: RR, GGW, and DFH are employed at New England Biolabs from which enzymes for routine molecular techniques and certain purification reagents were purchased.The author(s) declare that there are no competing interests. Non-financial competing interests: The authors declare that there are no non-financial competing interests.JOD conducted all bioinformatic, genetic, molecular, and biochemical experiments and drafted the manuscript. DFH participated in endonuclease activity screening of crude extracts. GGW, GFF, RR, and DVS participated in data interpretation and critical revision whilst JOD and DVS conceived of the study. All authors have read and approved the final manuscript.


Arabidopsis thaliana and rice (Oryza sativa) genome sequences has permitted the identification and comparison of the repertoire of serine protease-like proteins in the two plant species.Serine proteases are one of the largest groups of proteolytic enzymes found across all kingdoms of life and are associated with several essential physiological pathways. The availability of Arabidopsis and rice, we identified a very similar number of serine protease-like proteins in the two plant species . Nearly 40% of the above sequences were identified as potential orthologues. Atypical members could be identified in the plant genomes for Deg, Clp, Lon, rhomboid proteases and species-specific members were observed for the highly populated subtilisin and serine carboxypeptidase families suggesting multiple lateral gene transfers. DegP proteases, prolyl oligopeptidases, Clp proteases and rhomboids share a significantly higher percentage orthology between the two genomes indicating substantial evolutionary divergence was set prior to speciation. Single domain architectures and paralogues for several putative subtilisins, serine carboxypeptidases and rhomboids suggest they may have been recruited for additional roles in secondary metabolism with spatial and temporal regulation. The analysis reveals some domain architectures unique to either or both of the plant species and some inactive proteases, like in rhomboids and Clp proteases, which could be involved in chaperone function.Despite the differences in genome sizes between The systematic analysis of the serine protease-like proteins in the two plant species has provided some insight into the possible functional associations of previously uncharacterised serine protease-like proteins. Further investigation of these aspects may prove beneficial in our understanding of similar processes in commercially significant crop plant species. The proper functioning of a cell is ensured by the precise regulation of protein levels that in turn are regulated by a balance between the rates of protein synthesis and degradation. Protein degradation is mediated by proteolysis, which paves the way for recycling of amino acids into the cellular pool. In addition to degradation, regulated proteolysis plays a major role in diverse cellular processes by regulating biochemical activities of proteins in concert with other cellular mechanisms such as post-translational modification,2. SerinArabidopsis thaliana, most of these studies are either restricted to specific families [Arabidopsis thaliana and Oryza sativa constitute model systems for Dicotyledons and Monocotyledons respectively, the major evolutionary lineages in angiosperms[Serine proteases appear to be the largest class of proteases in plants. Consistfamilies -13 or spgiosperms,15. The Arabidopsis and rice serine protease-like proteins, phylogenetic analysis was performed based at their protease domains. Cross genome comparison of the serine proteases across the two genomes has led to the identification of members likely to be conserved across the two species. Predictions of the sub-cellular localizations of serine protease-like proteins to obtain further insight into their possible functional associations were carried out using TargetP[Arabidopsis and rice serine proteases that are likely to be involved in basic functions associated with various physiological processes in plants would be useful in expanding the current understanding of these processes in regulating plant growth and development. This would prove beneficial in understanding protease processes in plants and application of this knowledge to important crop species.The current analysis aims at the identification and analysis of serine protease families encoded in the genomes of the two plant species. The domain organization of the putative serine proteases in the two plant genomes have been analysed employing several sensitive sequence search methods -18 that g TargetP. CompariArabidopsis and rice (125 Mb and 389 Mb respectively) and encoded number of genes  from rice are predicted to contain PDZ-like domains C-terminal to the protease domain, while the rest appear to be a single protease domain containing proteins. The absence of PDZ-like domains in majority of plant DegP-like proteins has been suggested to have as of yet unclear functional implications for these proteins[Arabidopsis DegP1 (At3g27925) was found to be chloroplast localised and was rapidly upregulated in response to elevated temperatures[16 DegP-like proteins could be identified in peratures. We idenArabidopsis and rice DegP-like proteins showed that a majority of sequences cluster into three major clades, supported by high bootstrap values, with additional members less related to the three clusters  with no other structural similarity posing aArabidopsis and rice are characterised by significantly larger number of subtilases in comparison to other organisms such as human, Caenorahabditis and Drosophila, suggesting that expansion of the subtilase gene family in the two plant species is due to multiple duplication events[A large number of subtilisin-like serine proteases have been identified in various plant species, where they have been implicated in diverse processes  domain, that occurs as a C-terminal insertion in the Peptidase_S8 domain of a majority of subtilisin-like proteins identified in these two plant species . However, the presence of gene products in both the proteomes with only the protease domain suggests that this domain alone is sufficient for the activity of these enzymes  family of serine peptidases belong to the α/β hydrolase fold grouped into SC clan of serine peptidases. The family includes members of different types and with distinct specificities. For example, prolyl oligopeptidase is an endopeptidase localised to the cytosol, while dipeptidyl peptidase IV (DPPIV) is a membrane bound exopeptidase. According to MEROPS database, POPs maPOPs have been implicated in degradation of biologically important peptides such as peptide hormones and neuropeptides associated with learning and memory and therefore have become significant targets for drug design. They haArabidopsis and 23 in rice. A majority of these either lack a predicted signal sequence or are predicted to localise to chloroplast and mitochondria. Only few gene products identified here are predicted to carry a β-propeller N-terminal to the protease domain ; At5g24260 ; At2g47390, LOC_Os03g19410, LOC_Os07g48970  [Serine carboxypeptidase-like proteins (SCPLs) have been identified in several plant species, where they form large and diverse gene families in contrast with prokaryotes and other eukaryotes. Plant SCPLs have been implicated in several biochemical processes including secondary metabolism  and SMT/SNG1 (At5g09640) have been shown to function as acyltransferases in phenylpropanoid metabolism[Arabidopsis SMT is a heavily glycosylated protein that localises to central vacoules of the leaf tissue, suggesting that some SCP-like proteins identified in the two plant species may also be vacoule localised[We identified 54 genes coding for serine-carboxypeptidase (SCP)-like proteins in CHC; Pfam accessioCHC; Pfam accessioetabolism,43. Studlocalised.Arabidopsis and rice SCPL proteins cluster into two major clades, but there are some additional SCPL proteins that are less closely related to members of the clades (See Arabidopsis SCPL proteins), Clade IB (includes only rice SCPL proteins) and Clade IC (includes a pair of Arabidopsis and rice SCPL proteins). Clade IA includes known SCPL acyltransferases such as SMT (At2g22990) and SCT (At5g09640). Clade IA and IB consist exclusively of Arabidopsis and rice sequences respectively that have no identifiable orthologues in either subcluster. Therefore these two subclades may represent early diversification of certain SCPL proteins followed by their lineage specific delineation and expansion subsequent to speciation  proteins were the first to be identified and are the most common beta-lactamases. All these proteins share sequence similarity within the class but not with members of other classes. However, proteins of different classes share significant structural similarity and are believed to be homologues that have undergone divergent evolution. Structure-based phylogeny studies suggest that beta-lactamases are ancient enzymes that probably evolved as means of protection against beta-lactam antibiotics. Site-diSerine beta-lactamases are widespread in bacterial genomes, and their corresponding genes may occur on bacterial chromosomes or on plasmids. This allows for their transfer to distant species and may account for their distribution and diversity. Serine Arabidopsis and rice proteomes, both of which are predicted to localise to mitochondria. An interesting feature is the presence of a single ABC1 domain . Though the protease domain is capable of proteolysis on its own, the ATPase subunits are essential for effective levels of proteolysis. Structural analysis  that share comparatively low pairwise sequence identity with each other, as opposed to the members within a cluster that share significant sequence similarity with each other  fall into a single gene cluster (Clade I) and share between 36 and 72% pairwise sequence identity with each other , a central ATPase domain (AAA+ module) and a C-terminal proteolytic domain (Lon_C). The N-terminal LON domain along with the AAA module is believed to impart substrate specificity to Lon proteases[Lon proteases are also a group of ATP-dependent serine proteases. Unlike the Clp proteases, the catalytic and ATPase domains of the Lon proteases reside in the same polypeptide. They are conserved in prokaryotes and eukaryotic organelles such as chloroplast and mitochondria. proteases,53. StruArabidopsis has been implicated in the control of cytoplasmic male sterility[Unlike Clp proteases, Lon proteases appear to be present in organisms from all forms of life. They are believed to play a significant role in removal of aberrant proteins and thereby maintain cellular stability. Interestingly, an archaeal membrane bound Lon protease was found to have both ATP-dependent and ATP-independent proteolytic activities towards folded and unfolded proteins respectively. Little sterility.Arabidopsis and an equal number in rice. In addition, we also identified five protein sequences containing only the Lon N-terminal domain in Arabidopsis and four in rice (data not shown). Of the Arabidopsis Lon protease-like proteins, At3g05790 and At5g26860 gene products are predicted to localise to chloroplast, while At3g05780 and At5g47040 gene products are predicted to be mitochondria localised. All four Lon protease-like proteins identified in rice are predicted to localise to chloroplasts. The Lon-protease like proteins identified in the two plant species share higher similarities with the LonA subfamily . The largest of these clusters consists of five sequences , all of which, except At3g05780 are predicted to localise to chloroplasts and share between 76 and 91% pairwise sequence identity with each other. The second cluster consists of two sequences (At5g47040 and LOC_Os09g36300) that are 82% identical to each other but predicted to have different subcellular localizations. At5g47040 is predicted to localise to mitochondria and LOC_Os09g36300 is predicted to localise to chloroplasts. The members of the two clusters display between 40 and 45% pairwise sequence identity with each other. A Lon protease like gene product from rice, LOC_Os06g05820, is less closely related and shares between 19 and 25% pairwise sequence identity with the rest of the Lon protease like gene products from the two species.Sequence analysis reveals that Signal peptidases (SPases or leader peptidases) are a diverse group of serine endopeptidases, that belong to MEROPS family SE.coli lepB) and homologues have subsequently been identified in archaea, mitochondrial inner membrane, the thylakoid membrane of chloroplasts and the endoplasmic reticulum membranes of yeast and higher eukaryotes[The Type I SPases are membrane-bound serine endopeptidases that were first identified in bacteria  has been found to be required for complete maturation of the plastid protein translocation channel Toc75. It is proposed that Plsp1 may have multiple functions and is believed to be essential for biogenesis of plastid internal membranes[In bacteria, Type I SPases appear to be essential for cell viability and their inactivation invariably leads to cell death. Therefore they have been attractive targets for antibacterial drugs,58. In henal side,60. An Amembranes.Arabidopsis and seven in rice. A majority of these gene products are predicted to localise to mitochondria, though some are predicted to be chloroplast localised, membrane bound and even secreted , though predicted by TargetP[We have identified nine genes coding for signal peptidase-like proteins in y TargetP to be miy TargetP, suggestArabidopsis and rice Type I SPase-like proteins cluster into two major clades supported by high bootstrap values and high pairwise sequence identity , dipeptidyl-peptidase II and thymus specific serine peptidase and is grouped in MEROPS clan SC Members of this family have been identified exclusively in eukaryotes and have been implicated in several processes  belong to MEROPS family SE. coli Tsp) are believed to play an important role in degradation of improperly synthesised proteins. For instance, proteins translated from damaged mRNA are modified by addition of a C-terminal tag, which enables their recognition and degradation by CPPs[In bacteria, CPPs  are predicted to be mitochondria localised, while a single gene product LOC_Os06g21380 is predicted to be translocated to chloroplasts , an inner membrane protein was the first protein identified with signal peptide peptidase activity  is a light-inducible membrane-bound protein with an unusual monotropic arrangement, associated with thylakoid membranes and has been proposed to be involved in light dependent degradation of photosystem II and LHCII antenna proteins[Protease IV family appears to be represented by single members in y TargetP to be mi proteins. A singls SppA Atg73390 isRhomboid proteins are a family of serine proteases that cleave substrates within transmembrane domains,70. RhomArabidopsis AtRBL2 was recently isolated and shown to have proteolytic activity and substrate specificity[Drosophila ligands Spitz and Keren but not similar proteins like TGFα, when expressed in mammalian cells, thereby releasing soluble ligands from the cell into the media. AtRBL2 along with AtRBL1, another Arabidopsis rhomboid homologue was found to localise to Golgi apparatus in plant cells consistent with previous observations where several eukaryotic rhomboid proteins have been predicted to localise within Golgi apparatus[Rhomboid like proteins have been identified in plants, however, there was no experimental information available about them till recently. A rhomboid homologue from ecificity. AtRBL2 apparatus,74. InteArabidopsis and 18 in rice. Subsequent to multiple sequence alignment, it became evident that several proteins appeared to be catalytically inactive, owing to loss or mutation(s) in residues believed to be essential for catalysis. While the role of these catalytically compromised proteins remains unclear, it is believed that they may function as regulators of active rhomboid proteases[Arabidopsis gene products  and two gene products from rice  are predicted to possess a ubiquitin associated (UBA) domain, C-terminal to the rhomboid protease domain. An Arabidopsis gene product At3g17611 is predicted to have a zinc finger-like module C-terminal to the predicted rhomboid protease domain  that were earlier identified as members of the PARL subfamily, the other major subfamily of rhomboid-like proteins[At1g18600, At1g74130, At1g74140 and LOC_Os01g55740; 29–56% pairwise sequence identity), Clade IV, consisting of two , Clade V, consisting of two  and Clade VI consisting of two  sequences respectively  were found to carry an additional UBA domain and At3g17611 was found to carry a zf-RAN domain C-terminal to the rhomboid protease domain  share high pairwise sequence identities with each other (see above) but share low sequence identity with other rhomboid-like proteins identified in the two plant genomes and are likely to have diverged early in evolution. Our results may in part be explained by previous observations by Koonin and coworkers that reveal a polyphyletic origin for eukaryotic rhomboids and the possibility of a bacterial origin for rhomboid-like proteins and their subsequent dissemination in other lineages through multiple horizontal gene transfer events followed by extensive gene duplication.HisPheSer motif and an intein-like mechanism for autoproteolysis - Domain architecture SN-S8-PA-zfCCHC - Domain architecture Ex2-S8-zfCCHC-rve - Domain architecture S8-zfCCHC-rve. The putative gene product carries a zhCCHC domain and an rve domain C-terminal to the protease domain - Domain architecture SN-S8-PA-Arf2-C2. The putative gene product carries an Arf2 and a C2 domain C-terminal to the subtilisin protease domain - Domain architecture S10-Transposase_21 - Domain architecture S10-Retrotrans_gag - Domain architecture ABC1-S12. The putative gene products are associated with an ABC1 domain N-terminal to the serine β-lactamase domain - Domain architecture S54-UBA. The putative gene products are associated with an UBA domain C-terminal to the rhomboid protease domain - Domain architecture S54-zfRanBP. The putative gene product is associated with a zf-RanBP domain C-terminal to the rhomboid protease domain , which has been associated with lateral root emergence[Arabidopsis counterpart and whether they display redundancy in function. Similarly, Arabidopsis ARA12 [Arabidopsis.When comparing multi-gene families between species it is frequently observed that several genes in one species may collectively be orthologues of a single gene in the other, indicating recent duplications exclusive to the former. In such cases, knowledge of gene function of certain members allows confirmation of orthologous and paralogous relationships. One such instance is emergence and has Arabidopsis and rice genomes and were able to identify instances of segmentally duplicated genes corresponding to certain families of serine proteases in Arabidopsis and rice genomes  proteomes were obtained from The Institute for Genomic research (TIGR[Sequences encoded by the complete rch (TIGR).Arabidopsis and rice proteomes, using Type sequences for each serine-protease family as classified by MEROPS[-3.a. A preliminary search for serine-proteases was performed using BLASTP on Arabiby MEROPS as queriby MEROPS employs b. The protein sequences thus obtained were scanned further against a dataset of hidden Markov models (HMMs) obtained from the Pfam A database, employiArabidopsis and rice genome sequence was matched to sensitive protein family profiles obtained from the PfamA database[-5 by IMPALA[c. A complimentary approach of matching sequences to a database of annotated profiles was also employed. Each  database, using I database. A sequeby IMPALA was consArabidopsis and rice genome sequences was scanned against protein family profiles obtained from the NCBI  conserved domain database (NCBI-CDD[d. A similar approach was carried out where each of (NCBI-CDD), a comp. The list includes gene identifiers, predicted subcellular localization, chromosome location, chromosomal nucleotide position and domain architectures of serine proteases identified in current analysisTable S1. An inventory of Click here for fileTable S2. An inventory of rice serine protease-like proteins. An inventory of rice serine protease-like proteins identified by multifold approach (see methods for details). The list includes gene identifiers, predicted subcellular localization, chromosome location, chromosomal nucleotide position and domain architectures of serine proteases identified in current analysisClick here for fileTable S3. Background information on serine proteases. Additional literature information on serine protease families taken up for study in current analysis. The information is categorized into three parts namely a brief structural overview, enzyme characteristics and functional information where known. Additional references for the material contained in the file have been provided at the end.Click here for fileArabidopsis thaliana serine protease-like proteins and their putative orthologues in rice genome (see methods for details). Functional information gathered from literature has been provided for Arabidopsis gene products where possibleTable S4. Orthologous serine protease-like proteins identified in the two plant species. A list of Click here for fileArabidopsis and rice DegP protease-like proteins. A: Multiple sequence alignment of Arabidopsis and rice DegP protease-like proteins. Multiple sequence alignment of the DegP domain region of the annotated Arabidopsis and rice DegP protease-like proteins. The catalytic triad residues are indicated. Gene names correspond to those in Additional files B: Phylogenetic analysis of Arabidopsis and rice DegP protease-like proteins. Unrooted N-J tree computed from multiple sequence alignments of Arabidopsis (red) and rice (blue) DegP protease domains. DegP protease-like protease domains were aligned using ClustalW[Figure SF1. Multiple sequence alignment and phylogenetic analysis of  ClustalW program  ClustalW for reprClick here for fileArabidopsis and rice subtilisin-like proteins. Multiple sequence alignment of the subtilisin domain region of the annotated Arabidopsis and rice subtilisin protease-like proteins. The catalytic triad residues are indicated. Gene names correspond to those in Additional files Figure SF2. Multiple sequence alignment of Click here for fileArabidopsis and rice prolyl oligopeptidase -like proteins Multiple sequence alignment of the Prolyl oligopeptidase domain region of the annotated Arabidopsis and rice prolyl oligopeptidase-like proteins. The catalytic triad residues are indicated. Gene names correspond to those in Additional files Figure SF3. Multiple sequence alignment of Click here for fileArabidopsis and rice serine carboxypeptidase-like proteins A: Multiple sequence alignment of Arabidopsis and rice serine carboxypeptidase-like proteins. Multiple sequence alignment of the serine carboxypeptidase domain region of the annotated Arabidopsis and rice serine carboxypeptidase-like proteins. The catalytic triad residues are indicated. Gene names correspond to those in Additional files B: Phylogenetic analysis of Arabidopsis and rice serine carboxypeptidase-like proteins. Unrooted N-J tree computed from multiple sequence alignments of Arabidopsis (red) and rice (blue) serine carboxypeptidase domains. Serine carboxypeptidase-like protease domains were aligned using ClustalW[Figure SF4. Multiple sequence alignment and phylogenetic analysis of  ClustalW program  ClustalW for reprClick here for fileArabidopsis and rice serine beta-lactamase -like proteins. Pairwise sequence alignment of the serine beta-lactamase domain region of the annotated Arabidopsis and rice serine beta-lactamase-like proteins. The catalytic residues are indicated. Gene names correspond to those in Additional files Figure SF5. Pairwise sequence alignment of Click here for fileArabidopsis and rice serine Lon protease-like proteins. Multiple sequence alignment of the Lon_C protease domain region of the annotated Arabidopsis and rice Lon protease-like proteins. The catalytic dyad residues are indicated. Gene names correspond to those in Additional files Figure SF6. Multiple sequence alignment of Click here for fileArabidopsis and rice serine Type I SPase-like proteins. Unrooted N-J tree computed from multiple sequence alignments of Arabidopsis (red) and rice (blue) Type I SPase domains. Type I SPase domains were aligned using ClustalW[Figure SF7. Multiple sequence alignment of  ClustalW program  ClustalW for reprClick here for fileArabidopsis and rice serine S28 protease-like proteins. Multiple sequence alignment of the family S28 protease domain region of the annotated Arabidopsis and rice family S28 protease-like proteins. The catalytic triad residues are indicated. Gene names correspond to those in Additional files Figure SF8. Multiple sequence alignment of Click here for fileArabidopsis and rice Ctpase-like proteins. Multiple sequence alignment of the Ctpase domain region of the annotated Arabidopsis and rice Ctpase-like proteins. The catalytic dyad residues are indicated. Gene names correspond to those in Additional files Figure SF9. Multiple sequence alignment of Click here for fileArabidopsis and rice protease IV-like proteins. Multiple sequence alignment of the protease IV domain region of the annotated Arabidopsis and rice protease IV-like proteins. The active site serine residue is indicated. Gene names are derived from those in Additional files Figure SF10. Multiple sequence alignment of Click here for fileArabidopsis and rice rhomboid-like proteins. Multiple sequence alignment of the rhomboid protease domain region of the annotated Arabidopsis and rice rhomboid protease-like proteins. The catalytic triad residues are indicated. Gene names correspond to those in Additional files Figure SF11. Multiple sequence alignment of Click here for fileArabidopsis and rice nucleoporin autopeptidase-like proteins. Multiple sequence alignment of the nucleoporin autopeptidase domain region of the annotated Arabidopsis and rice nucleoporin autopeptidase-like proteins. The catalytic residues are indicated. Gene names correspond to those in Additional files Figure SF12. Multiple sequence alignment of Click here for file


Staphylococcus aureus was chosen for structure determination to elucidate its domain organization and confirm our earlier remote homology based prediction that it housed a nitrogen regulatory PII protein-like domain. SA1388 was predicted to contain a central PII-like domain and two flanking regions, which together belong to the NIF3-like protein family. Proteins like SA1388 remain a poorly studied group and their structural characterization could guide future investigations aimed at understanding their function.The protein encoded by the SA1388 gene from E. coli ybgI and the hypothetical protein SP1609 from Streptococcus pneumoniae. The openings on either side of the toroid are partially covered by trimeric "lids" formed by the PII domains. The junction of the two NIF3 domains has two zinc ions bound at what appears to be a histidine rich active site. A well-defined electron density corresponding to an endogenously bound ligand of unknown identity is observed in close proximity to the metal site.The structure of SA1388 has been solved to 2.0Å resolution by single wavelength anomalous dispersion phasing method using selenium anomalous signals. It reveals a canonical NIF3-like fold containing two domains with a PII-like domain inserted in the middle of the polypeptide. The N and C terminal halves of the NIF3-like domains are involved in dimerization, while the PII domain forms trimeric contacts with symmetry related monomers. Overall, the NIF3-like domains of SA1388 are organized as a hexameric toroid similar to its homologs, SA1388 is the third member of the NIF3-like family of proteins to be structurally characterized, the other two also being hypothetical proteins of unknown function. The structure of SA1388 confirms our earlier prediction that the inserted domain that separates the two NIF3 domains adopts a PII-like fold and reveals an overall capped toroidal arrangement for the protein hexamer. The six PII-like domains form two trimeric "lids" that cap the central cavity of the toroid on either side and provide only small openings to allow regulated entry of small molecules into the occluded chamber. The presence of the electron density of the bound ligand may provide important clues on the likely function of NIF3-like proteins. Despite the increased sophistication of annotation tools, a significant number of protein sequences emerging from genome sequencing efforts continue to remain in the realm of "hypothetical proteins" with little or no functional annotation associated with them. Ultimately, a definite functional annotation would require experimental characterization, which is often time consuming and expensive. Careful remote homology detection and manual analysis has in many occasions helped to glean useful structural and functional insights into these so-called "hypothetical proteins". Typically, such studies involve a combination of profile based methods like transitive PSI-BLAST , COMPASS2 secondary structural pattern, and function as trimers. While the nitrogen regulatory PII proteins that belong to the Group II of the superfamily have been studied extensively [One such study  had analensively , the funensively ,11, whilStaphylococcus aureus protein SA1388) for structure determination with two primary objectives: to structurally characterize both the Group III PII-like domain and NIF3 domains which may provide clues to their potential function that is otherwise unattainable from sequence information alone. Apart from providing confirmation of our prediction of the central P-II domain, the structure of SA1388 would also be important for functional analysis of the NIF3 domains, which were recently highlighted in the top 10 list of important structural targets due to their broad phylogenetic distribution, sequence conservation patterns with putative "active-site like" features and their uncharacterized role as a putative regulatory molecules of eukaryotic transcription [We chose the Group III proteins of the PII superfamily  refined to 2.0Å resolution consists of two subunits in the asymmetric unit. One subunit is traceable across the entire length of polypeptide from the N- to the C-terminus (residues 1 to 366) with the exception of a loop region containing 25 residues (168–193) in the central PII domain. In the other monomer, several more residues are disordered (139 – 193). A ribbon representation of one SA1388 monomers is shown in Figure The final model of SA1388  bulges out between the two NIF3 domains and is structured as a classical PII-like fold with a (β/α/β)e Figure , confirmE. coli ybgI  and SP1609 from Streptococcus pneumoniae . These two NIF3 homologs share sequence identities of 23% and 32%, respectively, with SA1388 sequence with the central PII-like domain (residues 128–235) excluded. A superimposition of the three structures is shown in Figure Structure comparison of the entire polypeptide of SA1388 as well as the NIF3 domains using DALI  clearly 2 secondary structural pattern described in SCOP as a ferredoxin-like fold [in vivo or in vitro.The PII-like domain of SA1388 is topologically identical to several PII proteins with characterized structures Figure , such asike fold . It rema2, or ~10% of the total surface area per monomer is buried in a largely hydrophobic (62%) interface  and consist primarily of five histidines, one glutamate and one aspartate. These residues are among the most conserved residues in the NIF3-like protein superfamily.We observe clear electron density for two tightly bound metal ions at the junction of the two NIF3 domains in each SA1388 monomer close to the protein surface that face inside the hexameric toroid Figure . The idelog ybgI , and is log ybgI , ribonuclog ybgI  and purplog ybgI . Some ofAdditionally, we observe a clear electron density for a ligand of unknown identity in close proximity to the two bound zinc atoms that would provide two additional metal ligands, one to each of the two metal ions to complete the octahedral metal center configuration Figure . By triaE. coli, T. thermophilus, Synecococcus and Herbaspirillum), GlnA (E. coli), glnK (E. coli), HisG (E. coli and M. tuberculosis), CutA1 , and a hypothetical protein from COG1993. Effectors of PII include transcription factors [Although homologs of both PII and NIF3 proteins are found ubiquitously in all three kingdoms of life, the function of SA1388 and its homologs that contain both PII-like and NIF3-like domains remain completely unknown. The fusion of the two proteins in one peptide chain indicates a functional coupling of these two proteins. The structural features revealed in the present study, such as the cage-like hexameric toroid structure with its NIF3 domains as walls and the two PII-like domain trimers as lids, the dinuclear metal site, and the intrinsically bound ligand, may provide certain clues of its potential function. The primary role of the nitrogen regulatory PII proteins is to integrate various intracellular carbon and nitrogen signals by regulating enzymes involved in nitrogen assimilation . PII exe factors ,11, sign factors , and met factors . Apart f factors ). It is E. coli and Methanococcus) have only the NIF3 domains with the PII-like domain being absent. Experiments on spermatogonia derived cell line GC-1 suggest that nif3L1 (Ngg1-interacting factor 3-like 1) gene, a homolog of sa1388, expresses copiously during embryonic development and participates in retinoic acid-primed neural differentiation by interacting with the transcriptional corepressor Trip15/CSN2 [E. coli, expression of NIF3 homolog ybgI increases dramatically upon genotoxic stress induced by DNA damage [Unlike PII domains, the NIF3-like domains have only recently begun to be structurally characterized, and their structure-function relationship remains sketchy. NIF3-like proteins are ubiquitously conserved from bacteria to higher eukaryotes . They hap15/CSN2 . Its prip15/CSN2 . Furtherp15/CSN2 . In E. cA damage . This obA damage -35. HoweA damage ).E. coli Ybgl proteins likely bind and perform some form of catalysis on small molecule ligands, possibly metabolic substrates, and whose function may be regulated by the PII-like domains. In an attempt to determine the chemical identity of the endogenous ligand, we have extracted the ligand from the purified protein by trichloroacetic acid (TCA)/acetone precipitation. The preliminary high-resolution mass spectroscopic analysis of the TCA/acetone extract suggested that the ligand might be a compound of novel chemical composition. Crystallization of the compound has not been successful probably due to its intrinsic structural flexibility, which may not be susceptible for crystallization. Alternative approaches, such as NMR spectroscopy, may be needed in order to determine the chemical formulas and three-dimensional structure of the endogenous ligand.Although the precise biochemical function of the NIF3-like proteins are not known, the presence of a binuclear metal center similar to those found in hemerythrins, ribonucleotide reductases and purple acid phosphatase, as well as the endogenously bound ligand in the current structure strongly suggest that SA1388 NIF3 domains and The recent upsurge in structures of a large number of hypothetical proteins by structural genomics efforts continue to provide important leads in the eventual determination of their function. This success has been largely due to either similarity of the overall structure, or local relatedness of catalytic residues to proteins of known function. Proteins with novel, previously unknown folds however are often limited in providing functional clues because of lack of similarity to any experimentally characterized proteins. Sometimes, the fortuitous observation of endogenously bound ligands offers important clues in this direction. The dinuclear metal center and the presence of a bound ligand at the active site of the NIF3 domain of SA1388 combined with the structural analysis of the two NIF3 homologs, ybgI and SA1609 should aid in the functional assignment of this widespread protein family.Staphylococcus aureus  and was cloned into a pProEX (Invitrogen) prokaryotic expression vector containing a trc promoter, 6xHis tag, and a tobacco etch virus (TEV) protease cleavage site. The resulting plasmid was transformed into the E. coli strain C41(DE3)  for protein expression. Expression was induced at OD600 of 0.4 and the cells were pelleted after overnight growth at 20°C. Protein was first purified with a nickel-nitrilotriacetic acid (NTA)-agarose column (Qiagen) followed by cleavage of the N-terminal His tag by TEV protease. The protein was further purified by anion exchange chromatography on a MonoQ column (Amersham Biosciences) using a linear gradient of salt  and finally by gel-filtration chromatography on a Superdex 200 column (Amersham Biosciences) that was pre-equilibrated with a buffer containing 50 mM Tris pH 7.8, 300 mM NaCl, 1 mM DTT. The selenomethionine-substituted SA1388 protein was expressed in the minimum medium supplemented with selenomethionine and other nutrients according to standard protocols [3 appeared within 3 days in the condition with 0.1 M Bis-Tris propane, pH 8.5, 0.2 M MgCl2 and 30% PEG 400 as the well solution. Crystals were directly frozen in liquid propane and stored in liquid nitrogen until data collection. All crystallographic data was processed using HKL2000 [The gene coding for SA1388 was PCR amplified from genomic DNA of rotocols  and puri HKL2000 2NYD).Structure of SA1388 was solved by single wavelength anomalous dispersion (SAD) method. Initial phases were obtained from a complete 2.3 Å anomalous dataset collected at the peak wavelength of selenium at Advanced Photon Source , beamline 19-BM (SBC-CAT). Phasing using SOLVE  assumingX-ray fluorescence scans were performed at the Advanced Photon Source (APS) beam line 19-BM at Argonne National Laboratory on a native SA1388 crystal. Metal analysis was also conducted on a Perkin Elmer Optima 3300 DV Inductively Coupled Plasma (ICP) atomic emission spectrometer calibrated with AccuStandard ICP Multi-Element solutions. The protein was first dialyzed against a buffer containing 50 mM Hepes, pH7.5 and 1 mM DTT, and then diluted to appropriate concentrations for ICP atomic emission spectra analysis. The dialysis buffer was used as blank.KSS performed data collection, structure determination, structure-function analysis, and drafting of the manuscript. XZ cloned, expressed, purified and crystallized both native and SeMET forms of the protein. LK and NVG provided significant bioinformatics input and target selection in the project. ML performed ICP atomic emission and ICP mass spectrometry analysis. HZ coordinated all the components of the project, critically edited the manuscript, and provided financial support. All authors have read and approved the final manuscript.