Escherichia coli and Bacillus subtilis, respectively. The third one (PolC + DnaE1), found in Clostridia, represents a novel, so far experimentally uncharacterized, set.The analysis of ∼2000 bacterial genomes revealed that they all, without a single exception, encode one or more DNA polymerase III α-subunit (PolIIIα) homologs. Classified into C-family of DNA polymerases they come in two major forms, PolC and DnaE, related by ancient duplication. While PolC represents an evolutionary compact group, DnaE can be further subdivided into at least three groups (DnaE1-3). We performed an extensive analysis of various sequence, structure and surface properties of all four polymerase groups. Our analysis suggests a specific evolutionary pathway leading to PolC and DnaE from the last common ancestor and reveals important differences between extant polymerase groups. Among them, DnaE1 and PolC show the highest conservation of the analyzed properties. DnaE3 polymerases apparently represent an ‘impaired’ version of DnaE1. Nonessential DnaE2 polymerases, typical for oxygen-using bacteria with large GC-rich genomes, have a number of features in common with DnaE3 polymerases. The analysis of polymerase distribution in genomes revealed three major combinations: DnaE1 either alone or accompanied by one or more DnaE2s, PolC + DnaE3 and PolC + DnaE1. The first two combinations are present in  DNA polymerase III is a tripartite protein machine responsible for replication of bacterial genome . It consEscherichia coli. PolC is present in low-GC Gram-positive bacteria such as Bacillus subtilis. The two different PolIIIα forms are thought to be the result of an ancient gene duplication event predating the radiation of Gram-positive and Gram-negative bacteria  produces opposite effects  identified in a much larger sample (close to 2000) of complete bacterial genomes. We took advantage of the available 3D structures of PolIIIα representatives and surveyed various sequence, structure and surface properties as well as their differences within and between distinct groups of C-family polymerases. Among other things, the results enabled us to suggest a specific evolutionary pathway leading to the emergence of DnaE and PolC from the common ancestor. We also surveyed the combinations of PolIIIα homologs found in genomes in an attempt to get more clues about functional properties of different polymerase groups and a deeper insight into the evolution of bacterial replication systems. As a result, in addition to two typical replication systems represented correspondingly by Supplementary Table S1) were obtained from NCBI (ftp://ftp.ncbi.nlm.nih.gov/genomes/Bacteria/). The associated data on bacterial taxonomic classification, various physiological properties, metabolic features and habitats were obtained from the Integrated Microbial Genomes database (http://img.jgi.doe.gov/). C-family DNA polymerases (DNA PolIIIαs and their homologs) were identified by performing protein sequence searches with PSI-BLAST (E. coli PolIII α-subunit) of C-family representatives as search probes. To make sure that no unannotated sequences were missed, representatives of C-family were also used to search the collection of genomic sequences using TBLASTN . Results of both types of searches were combined. If a polymerase sequence contained an intein, it was excised before further analysis. A number of cyanobacterial polymerase sequences are split by an intein  program . The tre program  with the program , run wit program  as imple program  and iTOL program .Conserved structural-functional domains in polymerase sequences were identified using three different approaches designed to maximize the sensitivity of domain detection.E. coli DnaE and B. subtilis PolC sequence regions corresponding to structural-functional domains defined based on DnaE and PolC crystal structures . Matches with E-value ≤ 1e–03 were considered significant.Next, for each of the PolIIIα domains, the sequences identified in the PSI-BLAST searches and filtered to 90% sequence identity were used to construct domain-specific Hidden Markov Models (HMMs). HMMs constructed from MAFFT alignments using the HMMer software suite  were app2 and Exo domains. At the same time, for a number of polymerase sequences, no conserved domains with statistically significant values were identified in the N- and C-terminal regions, where at least OB domain and CTD (in the case of DnaE) or NTD and OB domain (for PolC) could be expected. To further test the domain composition of these terminal regions, HHsearch HHsearch , a more HHsearch . For eveHHsearch  against HHsearch . To avoiFor full-length sequences of each polymerase group, separate multiple sequence alignments were generated. The alignments were inspected visually and adjusted manually if necessary. For each analyzed motif, relative positions from multiple sequence alignments for each group were extracted. In cases where multiple nonconsecutive positions were analyzed , concatenated alignments for each group were made. The WebLogo  represenThermus aquaticus DnaE1 (PDB ID: 3E0D) and Geobacillus kaustophilus PolC (PDB ID: 3F2B) with bound DNA were used as templates to generate models for DnaE and PolC polymerases accordingly. Sequence-structure alignments were generated using HHsearch. The 3D structural models were constructed with Modeller  in 1877 completely sequenced bacterial genomes. Once the polymerases  were compiled, we selected a representative nonredundant set of bacterial genomes as described in ‘Materials and Methods’ section. This mostly removed closely related strains of the same species that otherwise might have strongly biased the data. All the subsequent analyses were performed using the representative set of 945 bacterial genomes coding for 1590 putative C-family DNA polymerases (the redundancy of both genomes and polymerases was reduced approximately twice). Detailed information about each of the representative polymerases is presented in 2 domains. We then used this region to analyze the evolutionary relationship between C-family polymerases. Phylogenetic analysis revealed that the two major forms are well-separated (100% bootstrap support) and yet show a different degree of diversity (B. subtilis PolIIIα subunits (PolC), represent a single, evolutionary compact, group. In contrast, DnaE-type polymerases are significantly more diverse. The most distinct among DnaE-type polymerases is the DnaE2 group (90% bootstrap support). One of the best characterized members of this group is M. tuberculosis DnaE2, a nonessential error-prone DNA polymerase  of C-family polymerases has been noticed some time ago . We usediversity . PolC polymerase ,35. Overnclature , we labenclature  to the DTo maximize phylogenetic tree resolution, a minimal number of strongly diverged sequences were removed from the analysis. All of the omitted sequences, except three, were present as additional copies in respective genomes, suggesting that they are not the primary replicative polymerases. They were labeled DnaEX, while the three essential polymerases were assigned to the specific polymerase group based on the sequence similarity alone.E. coli). Consistent with an earlier study  of DnaE2. Although in general C-family polymerases are rarely encoded in plasmids (only 3% in our set), of those that are, about two-thirds are DnaE2s. This observation suggests that horizontal gene transfer may be an important route of the DnaE2 dispersal within bacterial genomes. However, despite the inferred ‘mobility’ of DnaE2 polymerases, they are extremely rarely found in PolC genomes  combinations? Based on the results of the analysis presented in er study , we findrmotogae . These m genomes . This maTo better understand differences between distinct polymerase groups, we compiled their domain architectures . To this end, for every polymerase sequence we asked which of the conserved domains revealed by DnaE and PolC structures  as well Supplementary Table S2) revealed that each polymerase group has a typical architecture. At the same time, we observed a significant variability within each group. Most often, new variants are associated with the loss of one or more domains, but there are also cases of the domain gain. The most conserved subset of structural-functional domains includes the combination of PHP, Pol3 and (HhH)2 domains. For the sequence to be annotated as a putative C-family DNA polymerase, we required that it should have the intact region harboring the polymerase active site. Therefore, the conservation of the polymerase core (Pol3) is expected. Somewhat surprisingly, PHP and (HhH)2 domains are also nearly universally present, suggesting their important structural and/or functional role.The survey  domain, Pol3 and the (HhH)2 motif (Thermotogae) were found exclusively in Clostridia and Negativicutes, two classes of Firmicutes bacteria. Firmicutes belonging to these two classes all have PolC paired with DnaE1 and not with DnaE3 (Lactococcus genus.The most common architecture of PolC (86%) is identical to that of )2 motif . For a s)2 motif . Thus, ith DnaE3 . An examE. coli PolIIIα subunit and includes PHP, Pol3, (HhH)2, OB and CTD domains. The PolIII proofreading exonuclease activity in E. coli is supplied by the separate ε-subunit complexed with α-subunit . This difference indicates that the incorporation of the exonuclease domain into PolC and DnaE1 are unrelated evolutionary events. Other variants display differences in the region, C-terminal to the (HhH)2 motif. In some of these cases, the CTD could not be identified with statistically significant values within the unassigned C-terminal region. Since CTD, similarly to NTD of PolC, is poorly conserved, at least a number of CTDs may have escaped identification. One of the minor variants features domain expansion (duplicated OB domain) in the otherwise canonical architecture. Another variant is truncated right after the OB domain, thus excluding any possibility of the CTD presence. Also, a second DnaE1 copy from δ-proteobacterium Desulfococcus oleovorans  has CTD replaced with a small domain related to the restriction alleviation protein Lar (PFAM family: PF14354), a predicted rubredoxin-like zinc binding domain.The most typical architecture of the DnaE1 group (91%) is represented by the Tenericutes all lack CTD, and only 40% of them have an identifiable OB domain. Thermotogae sequences, which show the strongest divergence within the DnaE3 group, all lack recognizable OB and CTD domains.The dominating architecture in the DnaE3 group is the same as in the case of DnaE1, but less typical. Only 60% of DnaE3s, compared with 91% of DnaE1s, have this architecture, and a larger fraction of DnaE3s lacks one or more domains at the C-terminus. As many as 16% are truncated after the OB domain (do not have CTD), and additional 4% lack both OB and CTD domains. There is even a variant with the excised OB domain but with CTD present. DnaE3s in At the level of domain architecture, DnaE2 polymerases differ from both DnaE1 and DnaE3 in that not a single DnaE2 has the CTD. Although some have protruding C-terminal tails, disorder predictions suggest that they are mostly unstructured. Several DnaE2 sequences lack not only CTD but also the OB domain.Yersinia pestis and Salmonella enterica, OB and CTD domains are replaced with the archaeal-type uracil DNA glycosylase domain (Of those DnaEs that were not assigned to one of the above groups (DnaEX), most have architectures already found in DnaE1-3 groups. In an unusual variant, found as an additional polymerase in some strains of e domain , a membe2 conserved core, the OB domain is also nearly universally present (detected in at least 97% of polymerases). However, its position in relation to the conserved core is completely different: in DnaE, it is C-terminal, while in PolC, it is N-terminal. This observation inevitably raises a question: do OB domains of both PolC and DnaE derive from the ancestral C-family polymerase or had they been acquired independently by DnaE and PolC lineages? If the first scenario were true, DnaE and PolC OB domains would be expected to be closest to each other. Conversely, the second scenario would imply that DnaE and PolC OB domains should be more similar to their respective parental OB domain families than to each other. To distinguish between the two alternatives, we collected a large number of diverse homologs of both DnaE and PolC OB domains  and clustered them according to all-against-all sequence similarities. Clustering revealed that DnaE OB domains are connected to a considerable number of OB domain families, while PolC OB domains are almost exclusively linked to OB domains of DnaE (In addition to the PHP-Pol3-(HhH) of DnaE . These r2 regions . Similar, but less severe reduction of the PHP domain can be seen in DnaE3s of most Tenericutes and at least some Firmicutes. In addition to the degraded PHP domain, Thermotogae DnaE3 polymerases also feature a strongly reduced ‘thumb’ . Their ‘thumb’ lacks a helix-loop-helix motif, corresponding to T. aquaticus DnaE1 residues 513–552, that provides additional contacts with the DNA duplex  harbors the binding site of the proofreading ε-subunit  were experimentally shown to be important for maintaining the interaction with α-subunit . The corresponding surface region in DnaE2 polymerases is not conserved at all, suggesting that they do not bind ε-subunit or at least not in the same way as E. coli DnaE1.It has been known for some time that the PHP domain of -subunit . Recentle linker . Our anae linker . Conserve linker . Residuetructure . At leas-subunit ,70. εHis2]. As a simple initial test, we computed theoretical isoelectric points (pI) for each protein. Despite simplicity of the approach, the pI calculation results revealed striking parallels with the results of sequence length analysis. Both the average pI values and the pI distribution for DnaE1 and PolC are almost identical  revealed that extreme pI values tend to coincide with major structural deviations. For example, DnaE3 polymerases of Thermotogae and Tenericutes featuring significant loss of structural elements have the highest pI values . In Supplementary Figure S3). DnaE1 polymerases with high pI values are almost exclusively found in insect symbionts, while PolCs are mostly found in mycoplasmas and phytoplasmas. In contrast, high pI values in DnaE3 and DnaE2 groups are not specifically associated with small genome size or low GC content.Owing to different functional roles, different polymerase groups might be expected to have distinct electrostatic properties. To make a proper comparison between polymerase groups, similarly to the length analysis, we only used the common conserved core  that2+-dependent proofreading exonuclease activity in at least some C-family polymerases . It turned out that DnaE1 sequences of three major classes of Proteobacteria  all have extensive substitutions in the PHP active site. Notably, not a single DnaE1 in these bacteria possesses all nine (or even eight) residues from the consensus pattern. This finding is consistent with previous studies suggesting that at least some of Proteobacteria might have an inactivated PHP exonuclease active site  appear to form a distinct clade in the phylogenetic analysis. On the other hand, none of the DnaE3 subgroups showed any conservation, suggesting that the defective PHP metal binding site is a hallmark of DnaE3 polymerases.Since polymerase groups, in particular DnaE1, span a variety of bacterial lineages, we also looked at the PHP metal binding site in more detail, taking into account bacterial taxonomy , indeed have a strongly conserved putative ε-binding site . Nonetheless, DnaE1s of other bacteria with the perfectly conserved PHP active site  still retain considerable surface conservation . In other words, the presence of the intact PHP metal binding site does not seem to preclude the ε-subunit binding. Apparently, the differences between DnaE groups are more relevant. For example, DnaE-type polymerases in Firmicutes, partitioned into DnaE1 and DnaE3 groups . These DnaE1 polymerases have both the intact PHP metal binding site and a strongly conserved putative ε-binding surface patch. In contrast, DnaE3s have both the disrupted metal binding site and almost no conservation of the PHP surface.Potentially, DnaE polymerases could have two different types of exonuclease activity, one due to the bound ε-subunit and the second due to the exonuclease activity of the PHP domain. An interesting question is whether the two exonuclease activities are mutually exclusive? Recently, it was suggested that they likely might be . In such3 groups , show thBacterial replicative DNA polymerases interact with the DNA sliding clamp to achieve high speed and processivity. The interaction is mediated by a short sequence motif within PolIIIα subunit. The consensus β-clamp binding motif has been identified previously as the pentapeptide QL[S/D]LF  in whichSupplementary Figure S8). For example, DnaE1 polymerases in Deferribacteres (five sequences) and a small fraction of Bacteroidetes (five sequences) do not have a recognizable β-clamp binding motif at all. In these bacteria, DnaE1 appears to be the primary replicative polymerase, raising a question of how the replication processivity is achieved in those cases. Notably, DnaE1 sequences that co-occur with PolC in Clostridia (Firmicutes) have a motif typical to other DnaE1 sequences and not to the ‘weak’ motif of DnaE3  . A small fraction of DnaE3 sequences (14%), most of them in bacteria of the order Bacillales, also have motifs similar to those of DnaE1 or PolC . B. subtilis is one of these bacteria and its DnaE3 has a relatively ‘strong’ motif (QMGLF).Like in the case of PHP metal binding site, a more detailed look at taxonomy-based β-clamp binding motifs revealed significant variation (α-proteobacteria. A similar consensus motif (xLPLF) is typical for γ-proteobacteria, but β-proteobacteria, although taxonomically close to γ-proteobacteria, seem to have a different and even ‘weaker’ consensus motif, xxxLL. Some Actinobacteria seem to have a nontypical QLPLx motif, which in almost half of the cases can be extended to the hexameric QLPLxL motif, similar to the consensus motif of Hda, DnaA-related protein  is predominantly found in  protein ,79.Supplementary Figure S9A). The motif is part of the region, predicted to be intrinsically unstructured . High conservation combined with the lack of a defined structure typically is the signature of a protein–protein interaction motif, but in this case its specific function remains to be established. This motif is found in the majority of DnaE2s of Proteobacteria (except most of δ-proteobacteria), part of Actinobacteria (including M. tuberculosis), a few Bacteroidetes and some minor phyla. Interestingly, all of these DnaE2 sequences clustered together in the phylogenetic tree , despite the omission of the motif during the tree construction.In addition to the known functional sites, we also searched for any other conserved motifs that stand out in at least one of the groups. One such motif is associated with ∼77% of DnaE2 sequences. This motif, noticed previously , featureOur analyses presented above indicate that there are significant differences between distinct groups of C-family polymerases. In addition, both the number and the type of polymerases encoded in genomes vary considerably. It might be expected that different polymerase combinations may represent different functional capabilities pertaining to DNA replication such as speed and mutation bias and/or dealing with oxidative stress exerted onto DNA. Therefore, we asked whether polymerase combinations correlate with global characteristics of bacterial species such as the genome size, genome GC content and the use of oxygen.First, we looked at the genome size. We divided genomes into several bins according to their size and asked which fraction of genomes in each bin has a particular polymerase combination. The results revealed several clear trends A and B. Candidatus Zinderia insecticola, a member of β-proteobacteria  and microaerophiles , while the second group consisted of anaerobes. Genomes with DnaE1 as a sole C-family polymerase are common for both groups . HoweverSupplementary Figures S3 and S10). The strongest correlation (Spearman’s rank correlation coefficient ρ = 0.58) was observed between the length of the DnaE2 polymerase core 2) and GC content of the genome, while no correlation was found with the genome size . Moreover, no significant correlation with either GC content or genome size was observed for DnaE1, DnaE3 or PolC polymerases. The variation in sequence length of DnaE2 polymerases is mainly due to additional structural elements or deletions in the PHP and ‘thumb’ domains. Functional importance of these differences is not obvious, but the correlation with GC content provides another hint for the possible involvement of DnaE2 in shaping genomic GC content.Taken together, these results show that specific polymerase sets encoded in a given genome strongly correlate with genome size, GC content and oxygen requirement. In particular, it appears that the presence of DnaE2 together with DnaE1 is linked to bacteria featuring large GC-rich genomes and living in aerobic environments. However, this does not necessarily imply the causal relationship as genome size and GC content are also correlated to each other . DnaE3 is never found alone in any genome , indicating that it is specialized to complement the PolC function. DnaE2 is also never found alone consistent with its role of a nonessential polymerase, not involved in bulk DNA replication   . These dd on SSB , supportd on SSB .E. coli DnaE1 and the ε-subunit has been determined, revealing the exact interaction site between these two subunits , implying that these polymerases most probably do not bind an ε-subunit, at least not in a similar way as E. coli DnaE1. However, separate  exonuclease might be not the only means to harbor exonuclease activity by C-family polymerases. At least in some polymerase α-subunits, the PHP domain has been found to be associated with a Zn2+-dependent proofreading activity (Proteobacteria) show highly conserved metal binding site . In contrast, DnaE3 polymerases show no conservation of corresponding positions whatsoever. Most of DnaE2 polymerases except a subgroup of Actinobacteria also lack the intact metal binding site. Therefore, our results suggest that DnaE3 and most of DnaE2 polymerases are devoid of proofreading activity. This is the actual case for B. subtilis DnaE3, for which no exonuclease activity could be detected  have both PolC and DnaE1 polymerases, some have only a single DnaE1. Furthermore, all PolC lacking the integral exonuclease are only found in clostridial genomes. Alternatively, it may be that PolC and DnaE1 work together at the replication fork and that the replication process is even more complex. Experimental approaches are clearly needed to gain knowledge on how the PolC + DnaE1 replication system really functions.Summing up all the observations about different polymerase groups, three distinct replication systems corresponding to DnaE1, PolC + DnaE3 and PolC + DnaE1 polymerase sets could be outlined . The firy itself . In B. s primers . The PolFirmicutes Clostridia would be placed at the root of bacterial evolution. This is supported by the observation that two different replication systems are found in Clostridia: PolC + DnaE1 and a single DnaE1. Deep branching of Firmicutes  hypotheostridia ,87. The bacteria ,88. If tThere have been previous reports suggesting the involvement of PolIIIα subunits in shaping global properties of the genome such as the GC content ,90. To fSupplementary Data are available at NAR Online.Research Council of Lithuania [MIP-079/2011]. Funding for open access charge: European Community’s Seventh Framework Programme [FP7-REGPOT-2009-1] project ‘MoBiLi’.Conflict of interest statement. None declared.


Agrobacterium tumefaciens is a typical T4SS. It facilitates the bacteria to translocate the VirD2-T-DNA complex to the host cell cytoplasm. In addition to protein-DNA complexes, the VirB/D4 system is also involved in the translocation of several effector proteins, including VirE2, VirE3 and VirF into the host cell cytoplasm. These effector proteins aid in the proper integration of the translocated DNA into the host genome. The VirD2-binding protein (VBP) is a key cytoplasmic protein that recruits the VirD2–T-DNA complex to the VirD4-coupling protein (VirD4 CP) of the VirB/D4 T4SS apparatus. Here, we report the crystal structure and associated functional studies of the C-terminal domain of VBP. This domain mainly consists of α-helices, and the two monomers of the asymmetric unit form a tight dimer. The structural analysis of this domain confirms the presence of a HEPN (higher eukaryotes and prokaryotes nucleotide-binding) fold. Biophysical studies show that VBP is a dimer in solution and that the HEPN domain is the dimerization domain. Based on structural and mutagenesis analyses, we show that substitution of key residues at the interface disrupts the dimerization of both the HEPN domain and full-length VBP. In addition, pull-down analyses show that only dimeric VBP can interact with VirD2 and VirD4 CP. Finally, we show that only Agrobacterium harboring dimeric full-length VBP can induce tumors in plants. This study sheds light on the structural basis of the substrate recruiting function of VBP in the T4SS pathway of A. tumefaciens and in other pathogenic bacteria employing similar systems.The Type IV Secretion System (T4SS) is the only bacterial secretion system known to translocate both DNA and protein substrates. The VirB/D4 system from  Agrobacterium tumefaciens causes crown gall disease (tumors) in agriculturally important plant species. It initiates infection through its Ti plasmid, which integrates a portion of its own DNA (T-DNA) into that of the host genome. The T-DNA is bound to VirD2 relaxase, and this complex is required for the efficient translocation and integration of the T-DNA into the plant genome for tumor formation. Two additional proteins, among others, are also required for Agrobacterium tumorigenesis: VirD4-coupling protein (CP) and VirD2-binding protein (VBP). VBP is responsible for recruiting VirD2–T-DNA to VirD4 CP to help localize T-DNA to the Type IV Secretion System apparatus for transfer. However, it is still unclear how VBP recruits the complex to VirD4 CP. Here, we report the crystal structure and associated functional studies of the C-terminal domain of VBP. We show that the C-terminal domain is the dimerization domain of VBP and only dimeric VBP is functional and essential for the induction of tumor in plants. This study enhances the understanding of the role of VBP in recruiting VirD2–T-DNA in A. tumefaciens prior to its transfer into the host plant. This mode of action can be extended to other pathogenic bacteria employing similar secretion systems. The Type IV Secretion System (T4SS) has an unmatched versatility among the seven different secretion systems known in bacteria. T4SS can translocate not only proteins but also DNA to phylogenetically diverse taxa, including many bacterial species and various eukaryotic cells, as well as import and export DNA from the extracellular milieu. The T4SS shares a common ancestry with bacterial conjugation systems Agrobacterium tumefaciens is a typical example of a T4SS, which serves as both a conjugation and an effector translocation system. Conjugation systems within the T4SS facilitate the translocation of protein-DNA complexes from the bacterium into the cytoplasm of the host cell. For instance, A. tumefaciens translocates to the host cell a segment of the Ti (tumor inducing) plasmid between the right and left borders (T-DNA) in complex with a cytoplasmic relaxase protein (VirD2) A. tumefaciens VirB/D4 system, is an inner membrane protein with a large cytoplasmic domain essential for the transfer of both the T-DNA strand and VirE2 to host cells The VirB/D4 secretion system of et al. reported the existence of a subset of proteins defined as ‘recruiting proteins’ that are involved in bringing the nucleoprotein substrate complex formed in the bacterial cytoplasm to the VirD4 CP. The VirD2-binding protein (VBP) was subsequently identified as a key protein belonging to this subset and was shown to recruit the VirD2–T-DNA complex to the VirD4 CP The VirD2–T-DNA complex forms in the bacterial cytoplasm, but there is no evidence to suggest that VirD4 CP can recruit the bulky substrate complex to the T4SS apparatus A. tumefaciens is a gram-negative bacteria that causes crown gall disease (tumor formation) in over 140 species of dicots Agrobacterium to infiltrate and infect other organisms to create transgenic plants. Thus, given the importance of this bacterium in both crop infection and as a research tool, we sought to better understand how VBP recruits the protein-DNA complex to VirD4 CP by exploring the structure of VBP.Agrobacterium harboring dimeric VBP can interact with the key proteins to induce tumor formation in plants. These studies broaden our understanding about the role of VBP in the VirD2–T-DNA transfer from A. tumefaciens to the plant cell.Here we report the crystal structure of the C-terminal domain of VBP along with its functional studies. We show that the two monomers of the asymmetric unit form a tight anti-parallel dimer, with the dimerization confirmed by solution studies. Moreover, we demonstrate that this C-terminal domain is the dimerization domain of full-length VBP. Pull-down analyses and plant virulence assays showed that only 2 . The dimer is held together by tight interactions between α1 and α2 helices from both monomers  domain at the C-terminus. The NT (nucleotidyltransferase) domain is implicated in nucleotidyl transfer function, whereas the HEPN domain is implicated in nucleotide bindingStaphylococcus aureus (PDB code: 1KNY) with an rmsd of 2.9 Å for the 104 Cα atoms. Although VBP has very low sequence identity (14 to 16%) with its structural homologs, it might have similar nucleotide binding and transfer function.PSI–BLAST searches of the non-redundant protein database using the full length VBP (gi|159141484) resulted in several hits of nucleotidyltransferase proteins from The crystal structure of the HEPN domain shows the presence of a tight dimer in the asymmetric unit. The molecular mass based on the sequence of VBP is 37.5 kDa (including the 6His tag). However, size-exclusion chromatography showed that VBP elutes as a single peak at an elution volume corresponding to an apparent molecular mass of 75 kDa . FurtherWe examined the role of the HEPN domain in VBP oligomerization by generating individual constructs for the N-terminal (NT) and C-terminal (HEPN) domains of VBP. The size-exclusion chromatography of the purified proteins showed that the NT domain elutes as a single peak at an elution volume corresponding to an apparent molecular mass of 16.8 kDa (NT as monomer), whereas the HEPN domain elutes as a single peak at an elution volume corresponding to an apparent molecular mass of 37 kDa (HEPN as a dimer) . These rThe structural analysis indicated that the HEPN dimer is held together by contacts throughout helices α1 and α2; in particular, residues Asp173, Lys184 and Asn186 of the HEPN domain play important roles in maintaining the dimer. Asn186 is located at the edge of a loop connecting the α1 and α2 helices of the dimer interface . This reNext, we verified the role of these key residues using full-length VBP. Single point substitutions of Asp173Asn, Lys184Asp or Asn186Asp in full-length VBP caused the dimer to break and the protein to elute as a single peak at an elution volume corresponding to an apparent molecular mass of 37.5 kDa (monomeric VBP). Further AUC analyses of full-length VBP bearing one of these point substitutions showed that the protein sediments as a single species at an apparent molecular mass corresponding to monomeric VBP (37.5 kDa) . The cirin vitro pull-down assay with recombinant proteins (6His-VBP and MBP-VirD2), we showed that wild-type VBP binds to VirD2, whereas VBP with one of these aforementioned single point substitutions—Asp173Asn, Lys184Asp or Asn186Asp (which results in monomeric VBP)—does not bind VirD2 ). WT His-VBP was able to pull down VirD2 and VirD4 CP, whereas substituted VBP could not pull down either of these proteins. These results indicate that the dimeric nature of VBP is important for its interaction with VirD2 and VirD4 CP (VBP is the key recruiting protein that binds to the VirD2–T-DNA complex and brings it into contact with VirD4 CP for subsequent translocation to the host cell nd VirD2 . To confVirD4 CP .vbp KO strain of A. tumefaciens) was complemented with pQH300 plasmid harboring substituted constructs for a functional vbp gene, a gene expressing the HEPN domain, or a gene expressing the NT domain. We found that null mutants transformed with a plasmid expressing VBP were able to cause a tumor-like phenotype in the wild-type plants , whereas VBP with the three point substitutions described above does not bind to AMPPNP  and pRSET  vectors, respectively. N-terminal nucleotidyltransferase (NT) domain and C-terminal HEPN domain constructs were created using specific primers that amplify these regions and were cloned into pGEX-6p1 . Site-specific mutations in vbp were introduced by overlapping PCR, as described previously virF cassette cloned from pTiBo542 (GenBank: DQ058764.1) was inserted into the SphI–ApaI site on pCB301 virF coding sequence was substituted with a multiple cloning site, resulting in pQH300.The strains and plasmids used are given in vbp was transformed into E. coli BL21 (DE3) cells and was grown in LB broth at 37°C overnight. The overnight culture was transferred into 1 L of LB broth and the protein expression was induced at an absorbance of 0.6 with 350 µM IPTG for 20 h at 20°C.Cells were harvested and lysed in lysis buffer . Cell lysates were centrifuged and the supernatants transferred to affinity columns containing Ni-NTA agarose , pre-equilibrated with the lysis buffer. The 6His-VBP bound to Ni-NTA was eluted with 400 mM imidazole following three wash steps to remove non-specific proteins. The eluted protein was purified through size-exclusion chromatography using a HiLoad 16/12075 Superdex75 gel filtration column  containing buffer . The GST fusion proteins (GST-HEPN and GST-NTD) were expressed as described above using M9 media The plasmid pET32a-2 cold stream.Initial crystallization conditions were identified by hanging drop vapor diffusion method using an index screen . Diffraction-quality crystals were obtained by equilibrating l.0 µl drop of protein (4 mg/ml) in 30 mM CHES, pH 9.0, 200 mM NaCl and 5% glycerol mixed with 1.0 µl of reservoir solution (8% (w/v) PEG 3350, 2% v/v tacsimate, 5% v/v 2-proponal, and 0.1 M imidazole) suspended over 1 ml of reservoir solution. Crystals grew in 1–3 days at 16°C. For data collection, 15% glycerol was added as a cryo-protectant and the crystals were flash-cooled in an N12121 space group. There were two monomers in the asymmetric unit corresponding to Vm = 2.49 Å3 Da−1 with a solvent content of 50.6%. The position of the selenium atoms were determined using the program Phenix-Autosol free = 0.28) for reflections I>σ (I) to 2.7 Å resolution  under accession code 4NQF.A complete single wavelength anomalous diffraction (SAD) solution . The modThe oligomeric state of full-length VBP, HEPN and NTD domain of VBP and their mutants was investigated by monitoring the sedimentation properties of each protein in sedimentation velocity experiments. For these experiments, 400 µl of samples at 1 mg/ml in appropriate buffer were used, with the experiments carried out in duplicate. The sedimentation velocity profiles were collected by monitoring the absorbance at 280 nm. The samples were centrifuged at 40,000 rpm at 24°C in a Beckman Optima XL-I centrifuge fitted with a four-hole AN-60 rotor and double-sector aluminum centerpieces and equipped with absorbance optics. Eighty scans were collected and analyzed using the Sedfit program A. tumefaciens crude extracts, E. coli strain BL21 was used to produce 6His-VBP/substituted 6His-VBP as described above.6His-VBP/substituted 6His-VBP bound to Ni-NTA metal affinity resin was incubated with freshly prepared A. tumefaciens (vbp KO strain) crude extracts . After incubation at 4°C for 1 h, the resin was washed four times. The bound proteins were eluted with 250 mM imidazole. The eluted proteins were resolved on 12.5% SDS-PAGE. For western blot analysis, the proteins were transferred to a PVDF membrane. VirD2 and VirD4 CP in the eluent were detected by western blot using anti-VirD2 (1∶5000) and anti-VirD4 (1∶4000) antibodies. The signal was detected using the Super Signal WestPico Chemiluminescent substrate  under the conditions recommended by the manufacturer.MBP-VirD2 bound to amylose resin  was incubated with purified 6His-VBP, with or without substitution at Asp173Asn/Lys184Asp/Asn186Asp. The beads were washed several times before resolving on 12.5% SDS-PAGE. For western blot analysis, the proteins were transferred to a PVDF membrane. 6His-VBP was detected by the addition of diluted anti-His antibody . The signal was detected using the Super Signal WestPico Chemiluminescent substrate  under the conditions recommended by the manufacturer. For pull down assay from 6His-VBP, with or without substitution of key residues, were purified in gel filtration buffer containing 30 mM Tris-HCl, pH 8.0 and 200 mM NaCl. ITC experiments were carried out using a VP-ITC calorimeter  at 25°C using 0.01 mM VBP protein in the sample cell and 0.25 mM AMP-PNP in the injecting syringe. All samples were thoroughly degassed and then centrifuged to remove precipitates. With the exception of the first injection, 10 µl volumes per injection were used for different experiments. Consecutive injections were separated by 5 min to allow the peak to return to baseline levels. ITC data were analyzed with a single-site binding model using Origin 7.0  software.Far UV spectra (260–190 nm) of VBP, HEPN, NTD domains and their mutants were measured using a Jasco J-810 spectropolarimeter  in phosphate buffer (pH 7.5) at room temperature using a 0.1-cm path length-stoppered cuvette. Six scans were recorded, averaged and then baseline-subtracted.A. tumefaciens strains were grown in MG/L liquid medium overnight at 28°C supplemented with appropriate antibiotics. The bacterial cells were collected by centrifugation and re-suspended in a buffer solution consisting of 10 mM MgCl2 and 10 mM MES, pH 5.5. Cell concentrations were adjusted to OD600 = 0.1. The leaves of Kalanchoe plants were wounded with a hypodermic needle and 5 µl of bacterial cell suspension was inoculated onto each wound area. The tumors were photographed at different time points after inoculation.Figure S1Electron density map and topology diagram of HEPN domain. (A) A sample 2Fo-Fc electron density map (contoured at 1 σ) of HEPN domain of VBP. (B) Topology diagram of the HEPN domain of VBP.(TIF)Click here for additional data file.Figure S2Comparison of the gel filtration elution profiles of NT domain and HEPN domain of VBP. NT domain elutes as a single peak (in green) at an elution volume corresponding to an apparent molecular mass of 17 kDa while HEPN domain elutes as a single peak (in brown) at an elution volume corresponding to a molecular mass of 37 kDa. The molecular mass standard is shown in red.(TIF)Click here for additional data file.Figure S3Substitution of key residues disrupts dimerization in HEPN domain. (A) Analytical ultra-centrifugation profile of Asp173Asn substituted HEPN domain of VBP. (B) Analytical ultra-centrifugation profile of Lys184Asp substituted HEPN domain of VBP.(TIF)Click here for additional data file.Figure S4Comparison of the gel filtration elution profiles of HEPN and substituted HEPN domains of VBP. HEPN domain elutes as a single peak (in brown) at an elution volume corresponding to an apparent molecular mass of 37 kDa while HEPN domain with substitution Asp173Asn (in black)/Lys184Asp (in green)/Asn186Asp (in blue) elutes as a single peak at an elution volume corresponding to a molecular mass of 18.5 kDa. The molecular weight standard is shown in red.(TIF)Click here for additional data file.Figure S5Substitution of key residues disrupts dimerization in VBP. (A) Analytical ultra-centrifugation profile of VBP Asp173Asn. (B) Analytical ultra-centrifugation profile of VBP Lys184Asp.(TIF)Click here for additional data file.Figure S6Comparison of the gel filtration elution profiles VBP and substituted VBP. VBP domain elutes as a single peak (in green) at an elution volume corresponding to an apparent molecular mass of 75 kDa while VBP Asp173Asn (in brown)/Lys184Asp /Asn186Asp (in blue) elutes as a single peak at an elution volume corresponding to a molecular mass of 37.5 kDa. The peak (green) at 670 kDa corresponds to highly aggregated VBP that elutes in the void. The molecular weight standard is shown in red.(TIF)Click here for additional data file.Figure S7CD spectroscopy of HEPN domain and VBP. (A) HEPN domain and HEPN domain with substitution Asp173Asn/Lys184Asp/Asn186Asp have identical CD spectra. The graph is color coded. (B) VBP and VBP with substitution Asp173Asn/Lys184Asp/Asn186Asp have identical CD spectra. The graph is color coded.(TIF)Click here for additional data file.Figure S8VBP is a functional dimer. The effect of VBP mutations on tumorigenesis. A. tumefaciens strains were grown in MG/L medium at 28°C overnight. The cell density was adjusted to 108 cells/ml. The wounds on the Kalanchoe leaf were inoculated with this cell suspension (5 µl) of A.tumefaciens WT strain or GMV123 strain complemented with plasmid expressing VBP WT, VBP N186D, NT domain or HEPN domain. Only wounds inoculated with A.tumefaciens WT strain or GMV123 strain complemented with plasmid expressing VBP WT showed tumor, other wounds showed no tumor clearly indicating that only Agrobacterium harboring full length VBP can induce tumor. The tumors shown here were photographed at 35 days to show the growth of tumors over period of time. The particular mutants are labeled at the respective scars.(TIF)Click here for additional data file.Figure S9Interaction of substituted VBP with AMPPNP  by isothermal titration calorimetry (ITC). Representative ITC profiles are shown. The upper part of each panel shows the thermogram  after baseline correction and the bottom part of each panel shows the binding isotherm . (A) Calorimetric titration for VBP K184D. (B) Calorimetric titration for VBP D173N.(TIF)Click here for additional data file.Table S1Structural homologs of HEPN domain as predicted by DALI search.(DOCX)Click here for additional data file.Table S2Thermodynamic parameters for VBP interaction with AMPPNP obtained by ITC.(DOCX)Click here for additional data file.Table S3Strains and plasmids used in this study.(DOCX)Click here for additional data file.


Airway symptoms are common among workers exposed to flour dust. However, no such evaluation had been done in Kinshasa.To study the prevalence of airway symptoms, sensitization profile and risk factors of allergic disease in Congolese workers exposed to flour dust.A cross-sectional study was performed among 263 workers directly exposed to flour , 278 indirectly exposed to wheat flour and 268 controls. Indirect exposure was defined as administrative workers of the bakeries. Individual rhinitis and asthma symptoms were asked for, and skin prick tests (SPT), peak nasal inspiratory flow and pulmonary function performed.Workers that were directly exposed to flour dust showed a prevalence of rhinitis (57.4%), rhinoconjonctivitis (43.5%), and nocturnal cough (11.0%) significantly higher than the indirectly exposed  and controls , all p<0.05. 36.8% of flour dust directly exposed workers showed positive SPT to at least one allergens, that was significantly lower than 44.2% of controls. DPT and cockroaches were the most prevalent allergens in all groups. Storage mite was more prevalent among workers directly exposed, while pollen mix, sunflower pollen and crab were more prevalent in controls. Within the directly exposed group, sensitization to manioc flour was higher among individuals exposed to manioc and/or maize flour than those exposed to wheat flour. Of individuals reporting rhinitis, 37.0% showed positive SPT results. In multivariate analysis, direct exposure to flour, neighborhood of flour mill and mice in the house significantly increased the risk of having airway symptoms. Mice in the house increased the risk of sensitization. However, cooking with electricity was negatively associated with both airway symptoms and sensitization.Although sensitization was lower in the directly exposed group, this study showed that flour dust constitutes an increased risk for having airway symptoms compared to controls. Flour dust control measures is needed at workplace.


MutS family proteins are widely distributed in almost all organisms from bacteria to human and play central roles in various DNA transactions such as DNA mismatch repair and recombinational events. The small MutS-related (Smr) domain was originally found in the C-terminal domain of an antirecombination protein, MutS2, a member of the MutS family. MutS2 is thought to suppress homologous recombination by endonucleolytic resolution of early intermediates in the process. The endonuclease activity of MutS2 is derived from the Smr domain. Interestingly, sequences homologous to the Smr domain are abundant in a variety of proteins other than MutS2 and can be classified into 3 subfamilies. Recently, the tertiary structures and endonuclease activities of all 3 Smr subfamilies were reported. In this paper, we review the biochemical characteristics and structures of the Smr domains as well as cellular functions of the Smr-containing proteins. MutSr strain , 13, it bination , 11. Thitructure , 11, 14,Sgmt-MutS, a MutS homologue present in Sarcophyton glaucum mitochondria. Sgmt-MutS contains an HNH endonuclease domain in its C-terminal region [The endonuclease domain is located in the C-terminal region of MutS2, which is called the small MutS-related (Smr) domain . While tl region . Althougl region –20. In tFirmicutes, Deinococcus-Thermus, Bacteroidetes, Deltaproteobacteria, and Epsilonproteobacteria phyla of bacteria and plants. Plant MutS2 is believed to be derived from the genomes of incorporated cyanobacteria [E. coli YdaL and E. coli YfcN. Proteins belonging to this subfamily exist in both prokaryotes and eukaryotes. In general, MutS2 and the stand-alone type Smr domains do not coexist within the same organism, except for a few species [Smr domains can be categorized roughly into 3 subfamilies on the basis of the domain architecture of the proteins in which the Smr domains are included . The firbacteria . In thisbacteria . We refe species , 11. We α/β-sandwich structure with a βαβαββ fold consisting of a four-stranded β-sheet stacked against 2 α-helices , has also been determined, and it shows the same overall structure as the human N4BP C-terminal domain : 2.2 Å; sequence identity: 27%) [E. coli YdaL, was reported [ E. coli YdaL is also similar to those of the C-terminal domains of human N4BP2 and T. thermophilus MutS2. Thus, all 3 Smr subfamilies share a common overall structure as expected from their sequence similarity. As pointed out by Gui et al. [The solution structure of a family 2 Smr domain, namely, the C-terminal domain of human N4BP2 has been solved (PDB ID: 2D9I (unpublished), and 2VKC ). The ov-helices . As showty: 27%) . Althougreported . The ovei et al. , the lar T. thermophilus MutS2 Smr domains shows homology to those of the catalytic domain of E. coli RNase E (2BX2)  and the N-terminal DNA-binding domain of bovine DNase I (2DNJ)   [Bacillus stearothermophilus IF3C, a translational initiation factor (1TIG) [ B. stearothermophilus ribosomal S8 protein (1SEI) [ In addition to reported : E. coliy (1DCJ)  (Z-scorer (1TIG)  (Z-scoren (1SEI)  (Z-scoren (1SEI)   that spatially corresponds to Asp-303 of E. coli RNase E  were surveyed in detail by using NMR measurement and site-directed mutagenesis . The resγ (that comprises MSH4 and MSH5) [T. thermophilus MutS2 is as tight as that of the intact MutS2 [T. thermophilus MutS2 showed specificity to the Holliday junction though its Kd value (260 nM) was significantly higher than that of the N-terminal domain (60 nM) [E. coli YdaL domain showed a significant binding preference for branched DNA structures, including bubble DNA structure and Holliday junctions [It has been reported that MutS2 preferably binds to branched DNA structures, such as Holliday junctions, D-loops, and pseudo-Y structures , 11, 14.nd MSH5) , 35, whiunctions . Substraγ (MSH4/MSH5) [T. thermophilus MutS2, which are located between the N-terminal and the Smr domains, are responsible for the dimerization of the Smr domain [E. coli YdaL, were found to be enhanced by the presence of the N-terminal 1–38 residues that are not included in Smr core domain [ E. coli YdaL. It has been known that E. coli RNase E also functions in a dimeric form [E. coli RNase E catalytic domain are quite dissimilar to each other. The quaternary structure is likely to be closely correlated to the substrate specificity of these widely distributed folds of proteins.Generally, branched DNA-recognizing proteins, with a few exceptions, are dimeric or tetrameric molecules, because they need to hold multiple “arms” of the substrate. For instance, T7 endonuclease I , T4 endoH4/MSH5) , eukaryoH4/MSH5) , and arcH4/MSH5)  functionH4/MSH5)  and RuvCH4/MSH5)  are knowr domain , 18. Conr domain . Recentle domain . Those Nric form  and thatAs mentioned above, a variety of Smr-containing proteins are distributed across a wide range of organisms. The biochemical characterization of Smr domains would improve the understanding of their cellular functions. Helicobacter pylori MutS2 recognizes DNA containing 8-oxoguanine, a major DNA lesion caused by oxidative stress, and deletion of mutS2 gene results in an accumulation of 8-oxoguanine in the cell [ The family 1 Smr-containing protein MutS2 has been implicated to participate not only in the suppression of homologous recombination but also in the protection of cells from oxidative DNA damages , 43. HelArabidopsis thaliana GUN1 was identified as the key component in the plastid-to-nucleus retrograde signaling pathways that couple nuclear gene expressions and chloroplast functions [ Arabidopsis thaliana has at least 7 GUN1 paralogues that contain both the PPR motif and the Smr domain [Family 2 Smr-containing proteins are extreme diverse in their domain architecture. Among these, the plant GUN1 and the mammalian N4BP2 are relatively well characterized. unctions . GUN1, aunctions , 46, as unctions . It was unctions . Furtherunctions . In addir domain . One of r domain , 49. Anor domain , where tMammalian N4BP2 was originally identified as a protein that specifically interacts with the E3 ubiquitin ligase NEDD4 . SubsequLactobacillus casei phage ϕFSW repressor can be classified as a family 3 Smr domain [ϕFSW repressor protein has no N-terminal stretch, and its molecular and cellular function may be distinct from those of other family 3 Smr domains. To date, there are no reports about the cellular functions of other stand-alone type Smr domains. r domain . It woulϕFSW repressor protein is reminiscent of those of GUN1 and N4BP2. Although an endonuclease activity hardly seems to be correlated with the regulation of transcription, it has been shown that human NM23-H2 is a transcriptional regulator with DNA-cleaving activity [ϕFSW repressor protein, GUN1, and N4BP2 have multiple cellular functions and the endonuclease activity is not correlated with the regulation of transcription.The transcription regulatory role of the activity , 58. Furactivity . NER is activity , 61. Howactivity . Thus, iactivity , 62. TheThe sequences homologous to the Smr domains of MutS2 proteins are conserved in almost all organisms except for archaea. Smr domains are classified into 3 subfamilies on the basis of the domain architecture of the proteins in which Smr domains are present. Three-dimensional structures of Smr domains revealed that all 3 subfamilies share a common overall structure despite the local differences in loop regions. Consistent with this, all 3 subfamilies showed endonuclease activity and specificity for branched DNA structures. Immediate identification of the catalytic residues is required to study the reaction mechanism of this endonuclease. Since the relationship between the cellular and molecular functions of the majority of the family 2 or 3 Smr domain-containing proteins is still unknown, detailed characterization of these Smr domains may lead to the discovery of a novel biological phenomenon. For this purpose, an unavoidable task in the future will be to identify the most preferable substrate of the endonuclease or DNA-binding activity.


The availability of over 3000 published genome sequences has enabled the use of comparative genomic approaches to drive the biological function discovery process. Classically, one used to link gene with function by genetic or biochemical approaches, a lengthy process that often took years. Phylogenetic distribution profiles, physical clustering, gene fusion, co-expression profiles, structural information and other genomic or post-genomic derived associations can be now used to make very strong functional hypotheses. Here, we illustrate this shift with the analysis of the DUF71/COG2102 family, a subgroup of the PP-loop ATPase family.The DUF71 family contains at least two subfamilies, one of which was predicted to be the missing diphthine-ammonia ligase (EC 6.3.1.14), Dph6. This enzyme catalyzes the last ATP-dependent step in the synthesis of diphthamide, a complex modification of Elongation Factor 2 that can be ADP-ribosylated by bacterial toxins. Dph6 orthologs are found in nearly all sequenced Archaea and Eucarya, as expected from the distribution of the diphthamide modification. The DUF71 family appears to have originated in the Archaea/Eucarya ancestor and to have been subsequently horizontally transferred to Bacteria. Bacterial DUF71 members likely acquired a different function because the diphthamide modification is absent in this Domain of Life. In-depth investigations suggest that some archaeal and bacterial DUF71 proteins participate in B12 salvage.This detailed analysis of the DUF71 family members provides an example of the power of integrated data-miming for solving important “missing genes” or “missing function” cases and illustrates the danger of functional annotation of protein families by homology alone.This article was reviewed by Arcady Mushegian, Michael Galperin and L. Aravind. This inference does not imply, however, that no HGT occurred in these Domains. Indeed, some incongruence between the DUF71 phylogeny and the reference phylogeny of organisms [Thermofilum pendens DUF71 that robustly groups with Methanomicrobia (Euryarchaeota) and not with other Thermoproteales  = 1). Moreover it recovered the monophyly of most eukaryotic and archaeal major lineages  are strongly clustered on the chromosome with vitamin B12 salvage genes. More precisely 75/102 are adjacent to vitamin B12 transporter genes (such as the BtuCDF genes) [cbiB genes encoding adenosylcobinamide-phosphate synthetase, an enzyme shared by the de novo and salvage pathways [Because diphthamide is a modification specific to the archaeal and eukaryotic EF-2 proteins and bacteria lack all known diphthamide biosynthesis genes, we propose that cluster 1 in our phylogeny corresponds to s Figure . This fus Figure  are invoF genes)  and 18/1pathways   harbored both families  in Dph6 proteins is replaced by a ENGEF/YH188  motif in the DUF71-B12 proteins  or belong to the C-terminal conserved sequence motif (EGGE/D-X-E188) such as G182, G184, G185, E186, F187  and post-genomic evidence  is so strong that it could be used as an example where the functional annotation of a protein of unknown function could be derived from comparative genomic alone. On the other hand our analyses show that a subgroup of the DUF71 family is most certainly involved in a metabolic pathway unrelated to diphthamide synthesis and that transferring functional annotations from homology scores alone would be inappropriate in this case. We believe that this integrated functional annotation approach will play an important role in future pipelines for annotation of protein families.The author(s) declare that they have no competing interests.VdC-L conducted the comparative genomic analysis and made the functional predictions. CB-A performed the phylogenetic analysis. FF, LT and JFH did the structural analysis. All authors participated in writing/reviewing the manuscript. All authors read and approved the final manuscript.Reviewer number 1: Arcady MushegianStowers Institute for Medical Research, 1000 E 50thStreet, Kansas City, Missouri 64110The study by de Crecy-Lagard and co-authors pinpoints the DUF71/COG2102 asthe most likely archaeal/eukaryotic ATP-dependent diphthine-ammonia ligase,the so far unaccounted-for enzyme in the pathway of diphtamide biosynthesis, which pathway is responsible for the formation of unique derivative of the conserved histidine within the translation elongation factor 2. A distinct subfamily of this protein family appears to play another role in bacteria and a subset of archaea, most likely in the salvage of an intermediate of cobalamine biosynthesis. The evidence presented in the paper consists of genome context information, sequence-structure prediction and the data from yeast concerning gene expression and chemical-genomics profiling. Taken together, the evidence seems compelling to me. The data from yeast represent partial functional validation of predictions made for prokaryotes. I would recommend only to tone down the suggestion that all this is a “novel paradigm” in analysis of gene function: researchers have been inferring gene functions from phenotypes, as well as from directly detected changes in genotype, for a long, long time, and the current study is a logical extension of these approaches. What is different in the last 15 years is that we can compare these properties across many species with completely sequenced genomes; but even this is a logical extension of the previous work  - it was not any prescription of a previous scientific paradigm that constrained the work, but rather the lack of the data.The references to a “novel paragdim” were eliminated in the abstract and the introduction as suggested.Response: Reviewer number 2: Michael GalperinNCBI, NLM, NIH Computational Biology Branch, 8600 Rockville Pike MSC 6075, Building 38A, Room 6N601, Bethesda, MD 20894-6075The paper by de Crecy-Lagard and colleagues is a fine example of using comparative genomics to patch the remaining holes in the metabolic pathways. The key conclusion of this work, prediction of the participation of the members of the DUF71/COG2102 family in diphtamide biosynthesis in archaea and eukaryotes and in B12 metabolism in some bacteria and archaea, is extremely convincing and hardly even needs an experimental verification. The second conclusion, that ammonia used in the diphthine ammonia lyase-catalyzed reaction in different organisms could use generated by two different enzymes, asparagine synthetase and the RidA domain, also sounds convincing. However, proving beyond reasonable doubt that DUF71/COG2102 family members with their ATP-pyrophosphatase activity comprise the key part of diphthine ammonia lyase does not prove that they are the only subunits of this enzyme. Even if the proposed reaction scheme  as against a ligase. The conclusions are definitive and the article makes a useful contribution to the understanding of protein modification and cofactor biosynthesis. This said, there are certain issues with the current form of the article that authors necessarily need to address in their revision: 1) (pg 8) The authors state that the MJ0570-like enzymes have a HUP domain followed by a distinct C-terminal domain. They do not explain the meaning of this properly nor cite the reference of the paper (PMID: 12012333) pertaining to the HUP domains where this family was identified as a PP-loop ATPase, along with the observations .” A reference to the PP-loop ATPase family was added in the abstract as requested. A reference to the same work was added when talking about the RidA fusion. For the phylogenetic distribution the results presented here are a bit different from the previous study because many more genome are available after 10 years and we show that the family is also bacterial.2) The authors persistently refer to the domain as DUF71. This name is no longer current in Pfam and it has long been recognized as mentioned in the reference noted above that these proteins are not “domains of unknown function” but PP-loop ATPases. The domain is correctly termed ATP_bind_4 (PF01902) in Pfam. This Pfam (not the misleading DUF71) name and Pfam number should be indicated with just a statement in the introduction that it was formerly DUF71.This domain is currently called “Domain of unknown function DUF71, ATP-binding domain” in the InterPro database (IPR002761) even if it is called ATP_bind_4 (PF01902) in Pfam. It is much shorter to use (as well as easier for the reader to follow) the DUF71 abbreviation rather than the ATP_bind_4 abbreviation. We therefore prefer to keep DUF71. We however introduced a statement giving the different names of this domain in the InterPro, Pfam and COG databases at the end of the introduction.3) The authors apparently have a misapprehension regarding the Methanohalophilus mahii protein both in the text and the domain architecture rendered in the figure. First, these proteins have two N-terminal domains fused tothe MJ0570-like module: namely aN-terminal class-II glutamineamidotransferase  and second PP-loop ATPase domain thereafter (i.e. one related to asparagine synthetase). This GAT domain as in the case of other PP-loop enzymes could supply ammonia by cleaving it off glutamine. But this does not explain which PP-loop domain utilizes it. In the case of the Asn-synthetase it is used by the cognate PP-loop domain. In this case the presence of two PP-loop domains suggests that it is either utilized by both for different reactions or else the second domain does not receive the NH3 from this GAT. This also leads to the question what reaction is the Asn synthetase like PP-loop domain catalyzing? Quality of written English: Acceptable3 moiety to both the DUF71 and the Asn_Synthase_B_C enzymes.”The source of the confusion came from the fact that the Asn Synthase domain (AsnB) contains two domains the GAT-II domain and the Asn_Synthase_B_C PP-Loop ATPase domain. Both the figure and the text were modified to avoid the confusion. Based on the reviewer’s comments the sentence discussing the potential role of the AsnB domain was modified as follows: “This domain organization strongly suggests that in this subset of enzymes, the hydrolysis of glutamine catalyzed by the fused GAT-II domain could provide the NH4) Based on phyletic complementarity the authors suggest that bacterial CbiZ might be displaced by the bacterial MJ0570-like enzymes. This seems unusual - Why utilize a PP-loop ATPase for the reverse reaction, i.e. amidohydrolase? Typically there is little overlap between the families involved in amidohydrolase as opposed to ATP-dependent ligase activity. Of the almost 12 distinct major inventions of amidoligase activity, hardly any representatives of these superfamilies have been reused as amidohydrolases. So do the authors note anything special in the case of the bacterial representatives that might support such a functional shift?This hypothesis is derived from phylogenetic distribution and it is not unprecedented that ligases and hydrolases are found in the same family (see example in PMID:12359880). However, we agree that this hypothesis derives mainly from phylogenetic patterns analysisand beyond the differences in the predicted substrate binding pocket found in the DUF71-B12 family we did not identify specify changes that could point to a shift to hydrolase, hence our caution in our prediction as stated in the text.Quality of written English: AcceptableTable S1. Genbank RefSeq identities and corresponding organisms for all proteins used in the phylogenies.Click here for file Table S2. GO Term Enrichment Spell analysis (http://imperio.princeton.edu:3000/yeast) with YLR143w as input.Click here for fileFigure S1. Top 10 interactors with YLR143W by homozygous co-sensitivity in S. cerevisiae (from the Yeast fitness database http://fitdb.stanford.edu/fitdb.cgi?query=YLR143W). Figure S2 Multiple sequence alignment of selected Dph6 family and DUF71-B12 family sequences generated using the Multialin platform  Strictly conserved residues between the two families are in red. Residues conserved only in the Dph6 family are boxed in green. Residues found around the phosphate group of ATP are noted by red arrows. Secondary structural elements, yellow rectangles for α-helix and cyan arrows for β-strand, shown above the alignment, are from the crystal structure of P. furiosus_Dph6 (PF0828) (PDB id: 3RK1). Figure S3 Bayesian tree of archaeal and eukaryotic Dph6 sequences. The scale bar represents the average number of substitutions per site. Number at nodes represent posterior probabilities. For clarity only values greater than 0.85 are indicated. Figure S4 (Top) Sequence logo derived from 95 Dph6 sequences extracted from Diphthamide subsystem in SEED. The E188 reside (PF0828 numbering) is located at position 10 in the logo. (Bottom) Sequence logo derived of the corresponding region derived from 102 DUF71-B12 sequences extracted from the DUF71-B12 subsystem in SEED. Both logos were made at http://weblogo.berkeley.edu/logo.cgi based on clustalw derived alignments. Click here for file


Kinases are often aberrantly activated in various tumor types by overexpression, point mutations, chromosomal translocations and other mechanisms. Therefore, small molecule inhibitors that block the enzymatic activity of kinases have been developed for different targets and are successfully used for the treatment of cancer patients. A major drawback of these molecularly targeted therapies is the frequent development of therapy resistance, often caused by point mutations in the target protein . This cThe hallmark of chronic myeloid leukemia (CML) is the expression of the oncogenic kinase Bcr-Abl, which arises from the Philadelphia chromosome translocation . Bcr-AblRecently, the myristate binding pocket in the C-terminal lobe of the Abl kinase domain was identified as a site for allosteric regulation of Bcr-Abl activity . A smallIn addition, Bcr-Abl as well as its proto-oncogenic constituent c-Abl (ABL1) have a Src homology 2 (SH2) domain N-terminal to their tyrosine kinase domains - a conserved feature among the class of cytoplasmic tyrosine kinases. The analysis of active Abl conformations lead to the finding that SH2 domains are positive allosteric effectors in cytoplasmic tyrosine kinases via the formation of an intramolecular interface of the SH2 domain with the kinase domain . The SH2The exquisite affinity and specificity of monobodies exemplifies its general utility as target validation tools for preclinical studies. On the other hand, the necessity for intracellular delivery that may only be achieved using lentiviral gene transfer or fusion of the monobody to membrane-permeable peptides will likely limit or preclude the use of monobodies as drug-like molecules in clinical applications.In contrast, small-molecule protein-protein interaction inhibitors have been developed for a number of protein targets, breaking with the dogma that protein-protein interfaces are ‘undruggable’. However, interfering with intramolecular domain interactions (like the SH2-kinase interface in Bcr-Abl) might require very high local concentrations of inhibitor that may be difficult to achieve using intracellular delivery of proteins or peptides. Therefore, we believe that the development of a small-molecule inhibitor of the SH2-kinase domain interface that may be used in combination with approved Bcr-Abl tyrosine kinase inhibitors could be feasible.


Protein-domains play an important role in mediating protein-protein interactions. Furthermore, the same domain-pairs mediate different interactions in different contexts and in various organisms, and therefore domain-pairs are considered as the building blocks of interactome networks. Here we extend these principles to the host-virus interface and find the domain-pairs that potentially mediate human-herpesvirus interactions. Notably, we find that the same domain-pairs used by other organisms for mediating their interactions underlie statistically significant fractions of human-virus protein inter-interaction networks. Our analysis shows that viral domains tend to interact with human domains that are hubs in the human domain-domain interaction network. This may enable the virus to easily interfere with a variety of mechanisms and processes involving various and different human proteins carrying the relevant hub domain. Comparative genomics analysis provides hints at a molecular mechanism by which the virus acquired some of its interacting domains from its human host. Protein-protein interactions (PPIs) play a key role in the dialogue between viruses and their hosts. Datasets of host-virus protein interactions based on large- and small-scale studies are available for a growing number of viruses, particularly herpesviruses e.g:Escherichia coli to human, can be attributed to the experimentally-determined interacting domain-pairs. Furthermore, we showed that different organisms use the same interacting domain-pairs, and suggested that there is a limited repertoire of domain-pairs that underlie interactome networks, and that DDIs can be considered as the building blocks of the interactome.It is well established that many of the PPIs are achieved through domain-domain interactions DDIs, . In receHere we take this approach one step further and examine whether conserved DDIs also mediate host-virus interaction networks (in particular human-herpesvirus interactions). We point to the domain-pairs that may mediate these interactions and provide a dataset of viral and human domains and domain-pairs involved in herpesvirus infection. We find that indeed, statistically significant fractions of the human-virus interactions can be attributed to the intra-species conserved DDIs. Analyzing this dataset enables us to further investigate evolutionary and molecular aspects of both the domains (human and virus domains) involved in the interactions, and also the human-virus domains-pairs. We show that viral domains tend to interact with human hub domains and find hints at a molecular mechanism by which the virus may have acquired interacting domains from its human host.This study focuses on three herpesviruses: Herpes Simplex Virus type I (HSV), Epstein-Barr Virus (EBV) and Kaposi Sarcoma-associated Human Virus (KSHV). For each virus we compiled a dataset of intra-viral and human-virus protein interactions, as detailed in et al.We next asked whether the human-virus PPIs could be attributed to the interacting domain-pairs. If two interacting proteins contained domains documented as interacting, where one domain resides in one protein and the other domain resides in the interacting partner, the interaction of these proteins was attributed to this/these domain-pair/s. For EBV we also used information on the protein interacting regions reported by Calderwood For each of the viruses included in the study we found a statistically significant fraction of both the intra-viral and human-virus interactions that could be attributed to the interacting domain-pairs . To avoiSince human-virus interactions involve human domains that are also used for mediating interactions in the human cellular interaction network, it is possible that viral and human domains compete for interaction with the same human domains. For example, the Kaposi sarcoma's Cyclin homolog . To assess the statistical significance of this phenomenon we generated the human DDI network based on human single-domain protein complexes, where there is no doubt as to the domains mediating the interactions. In this network each node is a domain and edges connect between nodes of interacting domains. The degree of a node is the number of domains it interacts with. We compared the degrees of the viral-interacting human domains  to the degrees of all other human domains . We found that human domains involved in interactions with viral domains have statistically significantly higher degrees in comparison to human domains not involved in interactions with viral domains . These hcodon usage database−4). Furthermore, for several interacting domains we found that their sub-domain sequence organization is consistent with that of the human gene. For example, the viral Interleukin-10 homolog protein (Uniprot accession P03180) interacts with the human protein Interleukin-10 receptor subunit alpha  and consequently may control the inflammatory response, affect the antigen presentation by the cells and consequently may enable the virus to escape from the host immune detection We have shown that human-virus protein interactions can be attributed to specific domain-pairs. As it is known that viruses can acquire proteins from their host, it is interesting to find out whether viral domains involved in interactions are more similar to human domains than other viral domains that were not found in the analysis as involved in interactions. It was recently reported that the general similarity in GC content between virus and host is indicative of viral adaptation towards the host Figure S1Distributions of fractions of PPIs attributed to DDIs in the random networks. The fractions in the actual networks are written in the upper right box in each graph.(TIF)Click here for additional data file.Dataset S1The domain-pairs that potentially mediate the human-virus and intra-virus PPIs.(XLS)Click here for additional data file.