Regulation of the c-Abl (ABL1) tyrosine kinase is important because of its role in cellular signaling, and its relevance in the leukemiogenic counterpart (BCR-ABL). Both auto-inhibition and full activation of c-Abl are regulated by the interaction of the catalytic domain with the Src Homology 2 (SH2) domain. The mechanism by which this interaction enhances catalysis is not known. We combined computational simulations with mutagenesis and functional analysis to find that the SH2 domain conveys both local and global effects on the dynamics of the catalytic domain. Locally, it regulates the flexibility of the αC helix in a fashion reminiscent of cyclins in cyclin-dependent kinases, reorienting catalytically important motifs. At a more global level, SH2 binding redirects the hinge motion of the N and C lobes and changes the conformational equilibrium of the activation loop. The complex network of subtle structural shifts that link the SH2 domain with the activation loop and the active site may be partially conserved with other SH2-domain containing kinases and therefore offer additional parameters for the design of conformation-specific inhibitors. The Abl kinase is a key player in many crucial cellular processes. It is also an important anti-cancer drug target, because a mutation leading to the fusion protein Bcr-Abl is the main cause for chronic myeloid leukemia (CML). Abl inhibitors are currently the only pharmaceutical treatment for CML. There are two main difficulties associated with the development of kinase inhibitors: the high similarity between active sites of different kinases, which makes selectivity a challenge, and mutations leading to resistance, which make it mandatory to search for alternative drugs. One important factor controlling Abl is the interplay between the catalytic domain and an SH2 domain. We used computer simulations to understand how the interactions between the domains modify the dynamic of the kinase and detected both local and global effects. Based on our computer model, we suggested mutations that should alter the domain-domain interplay. Consequently, we tested the mutants experimentally and found that they support our hypothesis. We propose that our findings can be of help for the development of new classes of Abl inhibitors, which would modify the domain-domain interplay instead of interfering directly with the active site. The expression of the constitutively active BCR-ABL fusion tyrosine kinase is sufficient for the initiation and maintenance of chronic myelogenous leukemia (CML) in humans In the light of the above it is not surprising that the mechanisms regulating the activation and deactivation of both the wild type c-Abl and BCR-ABL tyrosine kinases have attracted a considerable interest In physiological conditions the catalytic activity of tyrosine kinases is tightly regulated through the interplay between various protein domains, phosphorylation events and associated conformational states of the catalytic domain (CD) In most non receptor-type tyrosine kinases, the catalytic domain is preceded by a Src homology 2 (SH2) domain While the molecular basis for the role of the SH2 and SH3 domains in Abl autoinhibition is well understood, the mechanism of their activating effect is less straightforward. Recent crystal structures and small angle X-ray scattering studies have revealed that the transition from the auto-inhibited to the fully activated form of Abl requires a complete reassembly of the complex formed by the CD, the SH2 and the SH3 domains, leading to migration of the SH2 domain from the C-lobe to the N-lobe of the CD (“top-hat” conformation)  [24]. ImThe SH3/linker region has also been shown to be involved in the regulation of Abl activation We used a multi-disciplinary approach combining elastic network models, extensive molecular dynamics simulations, free energy calculations and functional assays following mutagenesis to characterize the allosteric coupling of the CD of Abl with the SH2 domain as well as the modulation of the dynamic properties of the assembly by interactions in the “top-hat” conformation. Both the long atomistic simulations and the elastic network models indicate a significant change in the dynamics of key regulatory elements, providing a simple explanation of the mechanism of allosteric activation. Based on the computational results we designed a number of point mutants to validate the proposed model. These mutants were expressed in human cells and tested for kinase activity. We identified mutations, all distant from the active site, that were either activating the catalytic output of the kinase or were disrupting. Interestingly, we also identified a residue that when mutated lead to a decoupling of the activity of the CD from interaction with the SH2 domain. Collectively, the data suggested an effect of SH2 binding that results in changes of the properties of the αC helix, reminiscent of the effect that cyclins exert on cyclin-dependent kinases.To elucidate the mechanism by which the SH2 domain stimulates the catalytic activity of the Abl kinase we first characterized the dynamics of the CD alone and with the SH2 domain bound in the activating conformation using elastic network models. In the free CD, the two predominant modes emerging from the normal mode analysis (NMA) corresponded to the well-described hinge motion We analyzed the allosteric coupling of local conformational fluctuations along these normal modes The allosteric coupling analysis thus suggests that the SH2 domain acts primarily on the N-lobe loops and the A-loop. All these motifs are coupled among them through a dense network of allosteric interactions, which affect also the P-loop and the αC helix, two structural motifs that, together with the A-loop, participate actively in the catalytic process.We next carried out 1 µs long all-atom molecular dynamics (MD) simulations of the free CD and the activated SH2-CD complex in solution. The structure of the CD does not, in general, deviate much from the crystal structure . It is thus conceivable that the interaction with SH2 might play a role both in the inactive-to-active equilibrium and in the release of the products, which has been proposed to be the rate determining step of c-Abl-dependent phosphorylation.Next, we addressed the issue of how the SH2 domain affects global motions of the CD by using principal component analysis (PCA), which provides a description of the dominant motions of the CD during the simulations.The SH2 domain significantly restricts the movement along both EVs and leads to changes in the conformational equilibrium. The amplitude of the hinge motion along EV1 is shifted towards conformations in which the lobes close down over the active site . The effThe MD simulations indicate that the SH2 domain bound in the “top-hat” conformation exerts a double effect. On the one hand, it locally reduces the flexibility of a number of structural motifs of the CD, which participate in the catalytic process, such as the P-loop, the β3-αC loop and the αC helix, locking them in their active conformations. On the other hand, it modifies the collective motions of the CD, channeling energy away from unproductive twists and distortions into the catalytic hinge motion, and shifting the hinge motion towards closed conformations and the A-loop towards more “active-like” conformations.To investigate the effects of the SH2 domain on the A-loop conformation propensities we computed the inactive-to-active free energy landscape. To that aim we used a multiple-replica free energy algorithm  and a structure based hybrid force field. A similar computational strategy has been successfully used in the case of the CD of the highly homologous Src kinase to study the A-loop opening, where it was able to provide an accurate reconstruction of the free energy surface underlying the transition O-C≈6 kcal/mol). Moreover, the large free energy barrier separating the closed to the open A-loop state  disfavors the transition.In absence of SH2, the A-loop of the CD is mostly closed, as expected from its in-vitro low catalytic activity , left. TIn contrast, the effect of the SH2 regulatory domain is to shift the equilibrium towards an active-like conformation of the A-loop , right rBased on these computational results, we proposed a number of point mutations, both in the SH2 and in the catalytic domains, that we expect to modify the dynamic interaction between the domains by interrupting essential interactions or altering the flexibility of important motifs . We mainin vivo and in vitro. None of the candidate mutations have been described previously as clinically relevant and could in principle have either a moderate activating, neutral, or disruptive effect. The effect of the mutations on the catalytic activity of c-Abl was assessed both in the context of the SH2-CD module and in the isolated CD, in order to discern whether the mutation generally affects the fold or activation state of the kinase domain, or if it specifically targets the SH2-mediated regulation mechanism. The mutations were introduced in HA-tagged Abl SH2-CD or CD-only constructs which were transiently expressed in human embryonic kidney 293 (HEK293) cells, and the level of their in vivo activity was assessed by phospho-Y412-Abl and total phosphotyrosine immunoblotting of the crude lysates. The mutated proteins mostly accumulated to levels comparable to the wild-type constructs, indicating that they did not affect protein stability. The impact on c-Abl enzymatic activity using an in vitro assay with an optimal peptide substrate was assessed for selected mutations  severely decreases c-Abl activity, while introduction of additional flexibility (M297G) has a slightly activating effect and makes c-Abl activation less dependent on SH2 domain binding.A number of mutants , howeverThe pivotal role of the β3-αC loop is further confirmed by the effect of mutating E294 and V299 to prolines, which are the equivalent residues in c-Src, a protein closely related to c-Abl but not known to be activated by SH2. We would expect the E294P V299P double mutant to stiffen the β3-αC loop lever and, consequently, the αC helix. In turn, this should activate the CD and enhance the effect of the SH2 domain. The double mutant was indeed found to be markedly activating, both in the context of the catalytic domain alone as well as within the SH2-CD construct . The intLastly, we have investigated the effect of changes in the flexibility of the hinge region. In the MD simulations, Y339 in the hinge region showed the highest fluctuations. Mutation of the tyrosine to glycine, which should make the hinge even more flexible, has no effect on c-Abl kinase activity . In contin vitro measured c-Abl activity upon temperature increase  that are key to the interplay between catalytic and SH2 domain in different ways.The combination of structure-based normal mode analysis, unbiased MD simulations and free energy calculations with experiments Contrary to what could have been expected, the residues forming the hydrophobic spine are not directly affected by the formation of the activating complex. The picture that emerges from our studies is that the SH2 domain stabilizes and repositions the loops that form the binding site, which, in turn, interact with the surrounding loops, stabilizing the P-loop and the αC helix, redirecting the movement of the N-lobe and changing the inactive to active conformational equilibrium of the A-loop. The stabilization and repositioning of the αC helix also leads to an enhancement of the salt bridge between E305 and K290, which has been identified as one of the structural elements essential for kinase activation. The β3-αC loop preceding the αC helix is the key player in this complex mechanism, acting as a lever that transmits the effect from the SH2 domain-binding site towards the substrate binding residues and the catalytically important motifs. The effects of the M297G and E294P V299P mutants demonstrate the crucial role of the β3-αC loop. The observation that these mutations have a different impact on the CD and on the SH2-CD constructs shows that the β3-αC loop is involved in Abl activation by the SH2 domain. While the introduction of a glycine breaks the chain of transmission from the SH2 domain towards the active site and uncouples SH2 domain binding and kinase activation, the stiffening of the β3-αC lever by the introduction of two proline residues enhances the activating effect of the SH2 domain.The finding that at higher temperatures the unbound catalytic domain becomes less effective while the activity of the CD-SH2 construct increases indicates that the main role of the SH2 domain is to channel kinetic energy into directed, catalytically relevant motions. We propose that the SH2 domain modifies the so-called hinge motion, an opening and closing of the N-and C-lobes upon the active site. This is underlined by the fact that stiffening the hinge by the Y339P mutation decreases substantially the activity of the free CD, which cannot be rescued in the CD-SH2 construct. The hinge motion and the other changes in dynamics upon SH2 domain binding are involved in both the inactive-to-active conformational changes of the A-loop and the release of the products, the phosphorylated substrate and ADP. The latter has been proposed to be the rate-determining step of the phospho-transfer reaction in some kinases It is interesting to note that the effect on the β3-αC loop are reminiscent of the binding of cyclin to cyclin-dependent kinases Normal mode analyses (NMA) were applied to elastic network models of the CD alone and with the SH2 domain bound at the N-lobe in the activating conformation. The models were built from the crystal structure of the CD-SH2 complex .Allosteric couplings were calculated following the approach of Ref. The CD of c-Abl with the SH2 domain bound in the top-hat conformation  was solvated in tip3p water molecules The free energy calculations were performed with parallel tempering metadynamics (PT-metaD) For details on the set-up of the simulations and the coarse-grained model see Root mean square deviations (RMSD), root mean square fluctuations (RMSF), clustering, distances and principal component analysis were performed with GROMACS tools .All structural figures were created using PyMol Point mutations were obtained using the Quikchange Site-directed Mutagenesis kit (Stratagene) and pcDNA3.1-2×HA-TEV c-Abl CD and pcDNA3.1-2×HA-TEV c-Abl SH2-CD as templates Transfection of HEK 293 cells and immunoprecipitation of HA-tagged Abl protein was carried out as described previously M, each experiment was normalized for a reaction performed in the absence of the peptide, and the amount of kinase.The in vitro kinase assays were carried out as described previously Figure S1Global dynamics and allosteric coupling.  Schematic representation of the first and second normal modes of the elastic network model of the free CD (A) and the SH2-CD construct (B). Coloring by B-factors along the corresponding normal modes . Red arrows represent the direction and amplitude of motion of the individual residues along the normal modes. (C) Allosteric couplings in the SH2-CD complex. (D) Schematic representation of the first two eigenvectors from the principal component analysis of the MD simulation of the free CD. (E) The first two eigenvectors from the PCA of the MD simulation of the SH2-CD construct.(EPS)Click here for additional data file.Figure S2Flexibility of wild-type Abl. (A) Backbone root means square deviation (RMSD) of free CD (blue) and CD-SH2 complex (red), aligned on all backbone atoms. In orange, RMSD of the CD within the CD-SH2 complex, aligned on the backbone atoms of the CD. (B) Backbone root mean square fluctuations (RMSF) of the CD, unbound (blue) and in the CD-SH2 complex (red). For the analysis, only the last 750 ns of simulation were taken into account, in order to ensure full previous equilibration of the structures. Important structural motifs are marked in grey.  Abl in complex with an ATP-peptide conjugate (PDBid: 2G1T). Structures are colored by the RMSF from the MD simulations of the free CD (C) and the CD-SH2 complex (D). ATP is shown in a stick representation in grey and the pseudo-substrate in black.(EPS)Click here for additional data file.Figure S3Conformational plasticity and hinge motion of wt c-Abl.  Most representative structures of the CD in absence (A) and presence (B) of the SH2 domain. The representative structure of the largest cluster is shown in green and the one corresponding to the smallest cluster in blue. Upper panel: structures aligned on the C-lobe of the CD. Lower panel: detail of the αC helix, with the structures aligned on the N-lobe. (C) Distance between the Cα atoms of V299, at the N-terminus of the αC helix, and Y530, at the C-terminus of the activation loop during the simulations as a measure of the hinge motion. Top: Visualization of the corresponding residues in the CD. Bottom: distance in the free CD (in blue), and the SH2-CD (in red). The corresponding averages and standard variations are marked in grey, and the corresponding values are shown.(EPS)Click here for additional data file.Figure S4Flexibility of the all-atom versus the coarse-grained models. The average residue root mean square fluctuation (RMSF) for the CD in the open A-loop conformation is shown in the case of 100 ns of free molecular dynamics with and without SH2 in the top-hat position (solid red and blue lines respectively). The same quantities are shown with dashed lines in the case of the coarse-grained model.(TIF)Click here for additional data file.Figure S5Effect of mutations of residues in the SH2 domain on c-Abl activity. HEK293 cells were transfected with wild-type or mutant c-Abl constructs, and the lysates and immunoprecipitated proteins were analyzed by immunoblotting. E187K and N165A mutations are slightly disruptive to c-Abl activity as judged by total phos- photyrosine content in cell lysates (A). The same effect is seen when constructs were immunoprecipitated and subjected to in vitro kinase assays in the presence of an opti- mal Abl substrate peptide containing a single phosphorylation site (B). Activity of the mutants was normalized to SH2-CD wt. Error bars represent standard deviations from technical triplicates.(EPS)Click here for additional data file.Figure S6Effect of mutations of residue T291 on c-Abl activity. HEK293 cells were transfected with wild-type or mutant c-Abl constructs, and the lysates and immunoprecipitated proteins were analyzed by immunoblotting. Mutations in T291 are disruptive to c-Abl activity as judged by total phosphotyrosine content in cell lysates (A), the activity of the immunoprecipitated mutants in in vitro assays and their phos- phorylation on Y412 (B). Kinase activity was normalized to CD wt. Error bars represent standard deviations from technical triplicates.(EPS)Click here for additional data file.Figure S7Flexibility and structures of the M297G mutant and the E294P V299P double mutant from MD simulations. (A–C) Structures of CD (A) and SH2-CD (B) of the M297G mutant, colored by RMSF, and distance between E305(Cδ) and K290(Nζ) (C) in CD (blue lines) and SH2-CD (red lines). (D–F) Structures of CD (D) and SH2-CD (E) of the E294P V299P double mutant, colored by RMSF, and distance between E305(Cδ) and K290(Nζ) (F) in CD (blue lines) and SH2-CD (red lines). Average values of the E305-K290 distance, calculated for the last 750 ns of simulation time, are marked by cyan (CD) and orange (SH2-CD) lines and labelled with the corresponding values (in nm).(EPS)Click here for additional data file.Figure S8Conformational plasticity and flexibility of the M297G and E294P V299P mutants. (A–C) Conformation of the M297G CD. Most representative structures of the CD in absence (A) and presence (B) of the SH2 domain. The representative structure of the largest cluster is shown in green and the one corresponding to the small- est cluster in blue. Upper panel: structures aligned on the C-lobe of the CD. Lower panel: detail of the αC helix, with the structures aligned on the N-lobe. (C) Comparison of the P-loop and the β3-αC loop in the wt CD , the wt CD-SH2 (middle panel in red) and the M297G CD (lower panel in cyan).  Conformation of the E294P V299P mutant. Most representative structures of the CD in absence (D) and presence (E) of the SH2 domain.  Essential dynamics of the E294P V299P mutant compared to the wt. Projection of the trajectories of the wild type and E294P V299P mutant on eigenvectors 1 (hinge motion) and 2 (lobe twist) of the principal component analysis of the trajectory of the wt. (F) CD wt (blue dots) vs. CD E294P V299P (cyan dots). (G) SH2-CD wt (red dots) vs. SH2-CD E294P V299P (orange dots).(EPS)Click here for additional data file.Text S1Supplemental Methods. Table S1. Mutants tested for an effect on the activation of the CD and SH2-CD constructs.(PDF)Click here for additional data file.


Identification of ortholog groups is a crucial step in comparative analysis of multiple genomes. Although several computational methods have been developed to create ortholog groups, most of those methods do not evaluate orthology at the sub-gene level. In our method for domain-level ortholog clustering, DomClust, proteins are split into domains on the basis of alignment boundaries identified by all-against-all pairwise comparison, but it often fails to determine appropriate boundaries.We developed a method to improve domain-level ortholog classification using multiple alignment information. This method is based on a scoring scheme, the domain-specific sum-of-pairs (DSP) score, which evaluates ortholog clustering results at the domain level as the sum total of domain-level alignment scores. We developed a refinement pipeline to improve domain-level clustering, DomRefine, by optimizing the DSP score. We applied DomRefine to domain-level ortholog groups created by DomClust using a dataset obtained from the Microbial Genome Database for Comparative Analysis (MBGD), and evaluated the results using COG clusters and TIGRFAMs models as the reference data. Thus, we observed that the agreement between the resulting classification and the classifications in the reference databases is improved at almost every step in the refinement pipeline. Moreover, the refined classification showed better agreement than the classifications in the eggNOG databases when TIGRFAMs was used as the reference database.DomRefine is a useful tool for improving the quality of domain-level ortholog classification among microbial genomes. Combining with a rapid domain-level ortholog clustering method, such as DomClust, it can be used to create a high-quality ortholog database that can serve as a solid basis for various comparative genome analyses. Identification of orthologs constitutes the basis for comparative analysis of multiple genomes. It provides not only a foundation for inferring the evolutionary history of genes and genomes but also an important clue for inferring protein functions . OriginaOwing to rapid progress in sequencing technologies, an increasing number of genomes have been sequenced. In particular, accumulation of microbial genome data is remarkable ; severalSeveral previous studies have developed orthology inference algorithms and ortholog databases ,10. One Among numerous methods proposed to create ortholog groups, only a few methods consider orthology relationships at the sub-gene level. Figure The orthologous domains considered here are orthologous gene subsequences that have been stable (unsplit) during evolution after speciation from a common ancestor. To clarify the difference between orthologous domains and conventional homologous domains, let us consider the following evolutionary scenarios Figure B, C. In Note that the full length of a gene can be an orthologous domain. If the domain-reorganization event after speciation is either gene fusion or gene fission, the orthologous domain should correspond to the full length of a gene in at least one of the species Figure B. Thus, In practical applications, the determination of orthologous domains becomes more complicated because of several factors, including insertions/deletions of promiscuous domains and random disruption of coding sequences due to loss of function. These factors fragment orthologous domains into smaller pieces than expected as a unit of functional annotation. To avoid this over-splitting problem, the DomClust algorithm tries to split genes into the minimum number of domains required for ortholog clustering, i.e., a gene is split only when a different set of genes is putatively orthologous to each split segment with sufficiently large scores . MoreoveIn this study, we present a method for improving domain-level ortholog classification using multiple alignment information. We designed a scoring scheme to evaluate the inferred domain organization on the basis of multiple alignments and developed procedures to improve the inference by optimizing the score. The improvement procedures included the merge of adjacent domains to fix the over-splitting problem and determination of optimal domain boundaries. In addition, a phylogenetic tree was created for each cluster to check the cluster members in terms of orthology relation. To evaluate the improvements, we compared the obtained ortholog groups with the original ones by examining the agreement with COG and TIGRFAMs, which are the manually curated reference databases.merge and merge_divide_tree) were designed to solve the over-splitting problem; merge determined whether two adjacent clusters should be merged, whereas merge_divide_tree temporarily merged the adjacent clusters and then divided them into two groups (rather than split into two domains). The next two procedures (move_boundary and create_boundary) determined the optimized boundaries between the domains: the move_boundary procedure moved existing domain boundaries, whereas the create_boundary procedure introduced new boundaries. All the four procedures improved the domain organization on the basis of the maximization of the DSP score. In contrast, the last procedure (divide_tree) is a type of conventional tree-based approach for ortholog classification; it divided a cluster into subgroups along with the phylogenetic tree if the subgroups shared intraspecies paralogs.In this study, we assumed DomClust results as the input to our method, although any other domain-level clustering could have been applied. As illustrated in Figure merge procedure merged these clusters because of the increase in the DSP score after merge, which resulted from the gain of the SP score between the newly aligned residues in the merged alignment and the disappearance of gaps owing to inconsistent domain boundaries. Figure move_boundary procedure moved all the existing boundaries at the same time in the multiple alignment to the best position on the basis of the DSP score. The subsequent create_boundary procedure created new boundaries, and the creation of these boundaries increased the DSP score. As a result of these procedures, we obtained domain boundaries that perfectly matched the region detected by TIGRFAMs models  were clustered into ortholog groups by our method, and the results were compared with the manually curated COG clusters for evaluation. To test the utility of our method in a more practical situation, we also constructed a larger dataset (the FAMILY dataset including 309 organisms) by selecting a representative organism from each taxonomic family of the MBGD database. For each of the COG03 and FAMILY datasets, we first applied DomClust to classify genes into ortholog groups and then applied the DomRefine pipeline to improve the classification. For the FAMILY dataset, we compared our results with eggNOG, which was constructed by computationally extending COG. In the comparison with eggNOG, we extracted the common proteome between FAMILY and eggNOG (FAMILY210 dataset including 210 organisms).Table For more detail, we also examined the distribution of the cluster size (the number of proteins in each cluster) Figure . In gene10y = −1.499 log10x + 4.206, R2 = 0.90, Figure For the FAMILY dataset, the DomClust distribution follows a linear relationship in the log–log plot (logNCOG1to1) was then used as an indication for the agreement between two clustering results. Figure NCOG1to1 during the DomRefine procedures. We observed an increase in the agreement with COG during the merge and merge_divide_tree procedures . This is probably because increasing one-to-one relationships by moving a boundary requires exact matches of boundary positions; thus, divide_tree, which divides a cluster into subgroups to separate paralogs rather than modifying the domain organization. However, this result does not necessarily mean that divide_tree failed to improve ortholog classification, considering that a COG cluster often includes obvious outparalogs as members, resulting in a larger cluster than that produced by more rigorous ortholog grouping (see Discussion).To assess our refinement method, we examined whether our fully automated procedures could recover the manually curated COG database (COG02 including 43 organisms and COG03 including 66 organisms). To quantify the agreement of the clustering results between two methods (ours and COG) at the domain level, we first identified corresponding clusters as cluster pairs sharing at least one overlapping domain of the same protein and then extracted only those cluster pairs that had one-to-one correspondence (see Methods for details). The number of one-to-one corresponding cluster pairs against COG . This observation supports an assumption that the DSP score is able to quantify the quality of domain-level ortholog classification in terms of consistency using the COG database as a reference. We drew a LOWESS curve to reveal the details of the relationship between the score changes and Next, we examined the contribution of the DSP score to the refinement in the m Figure . We obsepositive 39 pairs To demonstrate the utility of our method in a more practical situation, we applied the method to the FAMILY dataset that covers the diversity of currently sequenced microbial genomes, in addition to the COG03 dataset. We here used the TIGRFAMs database instead of the COG database to evaluate the clustering result. TIGRFAMs is a database containing the profile hidden Markov models (HMMs) constructed from manually curated multiple alignments of functionally equivalent protein families   with “trNTIGR1to1) as a measure of consistency between two classifications. We examined the changes in NTIGR1to1 during the DomRefine procedure  to the COG03 and FAMILY datasets to classify genes and evaluated the resulting clusters using the TIGRFAMs database as a reference. As in the previous section, we considered the number of one-to-one corresponding cluster pairs against TIGRFAMs  exceeds the losses in total and in most steps that have sufficient numbers of modifications . For the COG03 dataset, NTIGR1to1 of the original COG classification was 1107, whereas for the FAMILY210 dataset, NTIGR1to1 of the eggNOG classification was 1149 , into two domains, it failed to split another plausible fused gene, ftu:FTT_0505 (Francisella tularensis) , NOG44136 (N = 7), and NOG145366 (N = 2), where N indicates the cluster sizes in the FAMILY210 dataset. eggNOG did not split the two plausible fused genes, ftu:FTT_0505 and nam:NAM_0533; it assigned ftu:FTT_0505 to NOG145366 and nam:NAMH_0533 to NOG12793. As a result, proteins with the same TIGRFAMs hits were separated into different clusters. In contrast, NOG12793 was the largest eggNOG cluster containing proteins with many different TIGRFAMs hits (97 families), indicating that it is too large in terms of grouping corresponding genes among organisms.On the basis of the resulting number of clusters for FAMILY210 (Table endA: tRNA intron endonuclease). Here genes of FAMILY210 were extracted to demonstrate the subset of the alignment . It is known that the two tandemly repeated domains, N-terminal repeat (NR) and C-terminal repeat (CR), have distinct functional roles and were suggested to have arisen by gene duplication and subfunctionalization [Nitrosopumilus maritimus, nmr:NMAR_0450 and nmr:NMAR_1039, which were assigned to the NR and CR clusters, respectively, were both located in the C-terminal half in the alignment. Another problem affecting the alignment was the presence of unconserved sequences in the N-terminal regions of eukaryotic genes, such as cdu:CD36_42500 (Candida dubliniensis). In domain inferences of DomClust and DomRefine, these regions are treated as C-terminal groups (colored in light blue). Influenced by such an unconserved region, regions such as nmr:NMAR_0450 are prevented from being aligned to the N-terminal region and are consequently aligned to the C-terminal region.Figure lization . Thus, imerge procedure of DomRefine , DomClust clusters became more consistent with both COG and TIGRFAMs after the  Figures  and 7, sdivide_tree procedure  among prokaryotic genomes . Therefoe Figure B, whereae Figure A. This ds Figure A, whereas Figure B. This wThe goal of this study was to construct a fully automated and reliable procedure to create ortholog database, a necessary resource in the era of huge amounts of genomic data. In this respect, the eggNOG database, which was constructed by computational extension of COG, is another ortholog database that covers the currently sequenced genomes and is periodically updated. However, eggNOG consists of two different types of ortholog groups, i.e., the extension of the original COGs and the remaining NOGs, because of the nature of its incremental updating procedure. COG-derived clusters tend to be larger, whereas the NOG clusters tend to be smaller  and discovered that our method showed better agreement than the COG/eggNOG classifications (Table NTIGR1to1 from the eggNOG classification to the DomClust classification using the FAMILY210 dataset (20%) was higher than that from the COG classification to the DomClust classification (12%) , i = 1, ..., Nseq , j = 1, …, Npos, where aij represents an amino acid or a gap, Nseq is the number of sequences, and Npos is the number of positions in the alignment. The positions of a domain on the amino acid sequences are also defined in the form of matrix D = (dij) of the same size of A, where dij = 1 if aij is within the domain or otherwise dij = 0. The DSP score of domain D in multiple alignment A is given byThe DSP score is calculated on the basis of multiple alignments. The score evaluates the consistency of domain-level ortholog clusters and multiple alignments. The basic idea is the sum-of-pairs score of a multiple alignment, which is a standard measure of evaluation of protein sequence alignment . The uniGopen is the gap-opening penalty. i-th sequence and i′-th sequence, where the open gaps are counted in the regions of dij=1 and di'j = 1, and the mismatches of the domain terminal positions are counted as open gaps. sdom is a function similar to a commonly used score matrix, but it returns a value depending on the domain as follows:where smat is a commonly used score matrix such as the BLOSUM score matrices, Gext is the gap extension penalty, and b(a) = 1 if a represents an amino acid or otherwise b(a) = 0. Therefore, a higher DSP score is obtained when the domain organization is such that sequence regions similar to each other  belong to the same domain and sequence regions dissimilar to each other  belong to different domains, because the DSP score counts similarity scores only between sequences belonging to the same domain. If the domain boundaries are not consistent with each other in the multiple alignment, they are penalized as external gaps, decreasing the score. Thus, an increase in the DSP score denotes that the domain boundaries are more consistent with each other in multiple alignment and/or the sequences belonging to the same domain produce a higher sum-of-pairs score. To normalize the DSP score with respect to the number of sequences and sequence lengths, we divide the DSP scores or the differences in the DSP scores by Nseq and Naa, where Naa is the total number of amino acids included in the alignment.where merge procedure, all the split proteins in the dataset are re-examined in multiple alignments. Consider a pair of clusters that share at least one common protein whose sub-sequences are members of each cluster. We define two clusters as adjacent if they have a shared protein whose sub-sequences in each cluster are adjacent to each other in the shared protein sequence. To determine whether a pair of adjacent clusters should be merged, the DSP scores are evaluated before and after the merge. First, the score is calculated for each of the clusters before the merge. Then, the clusters are merged by canceling the split between the clusters. The clusters are to be merged under the condition of the normalized score change S and S′ are the scores before and after the merge, respectively, and Sδ is a threshold for the merge. Following the examination of adjacent cluster pairs, all the pairs to be merged are merged at once.In the merge_divide_tree procedure temporarily merges a pair of adjacent clusters and then divides them into two groups as a split of a phylogenetic tree. Because this procedure is preceded by the merge procedure, we assume that clusters that should be merged are already merged.The merge_divide_tree procedure adopts the modified classification only when the resulting subgroups are consistent with the gene phylogeny, i.e., when they correspond to a split of the gene tree, as well as when the resulting DSP score becomes higher than before.A motivating example of this procedure is as follows: suppose there are two domains A and B. Some proteins have both domains (domain organization A + B) and the others have only domain A (domain organization A). In this case, we may want to classify these proteins into two groups corresponding to the two domain organizations, A + B and A, instead of the original domain-level classification, A and B. The More precisely, this procedure re-defines the two groups on a phylogenetic tree as follows. If a root of the tree is determined, two subgroups are produced. The initial domain patterns are compared between the newly defined subgroups, and the difference is quantified as follows:G1 and G2 represent initial clusters, t1 and t2 represent newly defined subgroups, g12 is the set of genes in both G1 and G2, g1 is the set of genes in G1 but not in G2, and g2 is the set of genes in G2 but not in G1. We calculated tdiff for all candidate roots and selected the root showing the largest tdiff. If several candidate roots show the same value of tdiff, the root with the longest edge among them is selected. Finally, the DSP score change was calculated comparing the original and resulting states, and the modification was executed only when the score increases.where move_boundary procedure moves the set of domain boundaries between two adjacent clusters at the same time, keeping them in the same column on the multiple alignment. By moving the position from the N terminus to the C terminus on the multiple alignment, the position showing the highest score is selected. If the best score is higher than the score of the initial state, the move of the boundaries is retained.The create_boundary procedure creates a new boundary on candidate sequences, which are not split into domains in the initial state. Following the examination of all the protein sequences without splits, if the set of newly introduced splits increases the DSP score, boundary creation is applied.The divide_tree procedure checks whether the resulting clusters contain paralogous genes using a species overlap criterion that is used in DomClust as well as several tree-based ortholog classification methods. For this purpose, using FastTree, we created phylogenetic trees on the basis of multiple alignments produced by Clustal Omega. Although the obtained tree is unrooted, the root is placed on one of the edges so that the height of the resulting rooted tree is minimized. Division of a cluster into subgroups is determined by a species overlap rule as follows: t1 and t2 represent candidate subgroups of the phylogenetic tree, Rsp is a threshold, and Ssp(ti) represents the set of species included in ti.The ao (member overlap for merging adjacent clusters) of 0.8, ai  of 0.95, V  of 0.6, and C (cutoff score for domain split) of 80. For the execution of the DomRefine pipeline, the following parameters were set: Gopen of 10, Gext of 0.5, Sδ of −0.05, and Rsp of 0.5, and BLOSUM45 was used as the score matrix smat. In the tests to recover COG classification by DomClust, an additional parameter was used to specify a condition that at least three phylogenetically distinct organisms must be included in each cluster, as described previously [The 2002 version of the COG database (COG02) contains genes from 43 species in 3307 clusters. We excluded ortholog groups comprising genes of fewer than three phylogenetically distinct organisms, retaining 3192 clusters, as described previously . Using tThe FAMILY dataset was created using the MBGD database . Using NCi and a DomClust cluster Dj, whereas no overlapping fragments are observed between Ci and Dj for any Ci and Dj is called a one-to-one relationship. When we have two clustering results, we can evaluate the consistency between them using the number of one-to-one relationships between them. To evaluate clustering results showing moderate agreement with the reference classification more appropriately than counting the number of one-to-one relationships, the agreement of clustering results was quantified as follows. The overlap ratio of fragment c∈Ci and fragment d∈Dj is calculated as rover = |c ∩ d|/max. The mean overlap ratio rover for the overlapping fragments.If overlapping fragments are observed between a COG cluster auto option. FastTree [http://search.cpan.org/dist/GD/). The tool colors the amino acid residues according to the conservation rate pcons in the multiple alignment: red for pcons ≥ 70%, yellow for 70% > pcons ≥ 50%, and cyan for 50% > pcons ≥ 30%. The scatter plot was created using R (http://www.r-project.org/). A significance test of the results obtained by the binomial test was performed using the binom.test function of R considering gains and losses as successes and failures in trials, respectively. TIGRFAMs release 13.0 [http://mbgd.genome.ad.jp/domrefine/.The core part of the pipeline that calculates the DSP score was implemented in the C language. Other parts of the pipeline are implemented in the Perl language. The programs were executed on Linux. DomClust  was usedFastTree  was usedase 13.0  was usedase 13.0  was usedDSP score: Domain-specific sum-of-pairs score; MBGD: Microbial Genome Database for Comparative Analysis; COGs: Clusters of Orthologous Groups; NOGs: Non-supervised orthologous groups; HGT: Horizontal gene transfer.The authors declare that they have no competing interests.HC designed the study, performed the analysis, and drafted the manuscript. IU conceived of the study, participated in its design, and helped to draft the manuscript. All authors read and approved the final manuscript.Supplementary figures and tables.Click here for file


Coxiella burnetii seropositive dairy herds, to describe the geographical distribution, and to identify risk factors. Using the CHEKIT Q-fever Antibody ELISA Test Kit (IDEXX), the study revealed a prevalence of 79.2% seropositive herds, 18.3% seronegative herds, and 2.5% serointermediate herds based on the instructions provided by the manufacturer. Multifactorial logistic regression showed statistically significant associations (P < 0.01) between C. burnetii seropositivity and increasing herd size (OR = 1.02 per cow increment) and increasing regional average number of cattle per dairy herd . Herds >150 cows had 17.9 times higher odds of testing positive compared to herds <80 cows. The regional average number of cattle herds per square kilometer was borderline significantly related to the occurrence of seropositive dairy herds (P = 0.06). The results indicate an increased prevalence of seropositive dairy herds since the previous survey in 2008 and an adverse impact of increasing herd size and cattle density on the risk of seropositivity.A study based on bulk tank milk samples from 120 randomly selected dairy cattle herds was conducted to estimate the prevalence of  Coxiella burnetii, an obligate intracellular bacterium and a zoonotic agent that may cause Q fever in animals and humans, occurs in cattle almost worldwide ]. Th]. ThCoxiwas 59%  based oin 2010 . Publicin 2010 , 65% inin 2010 , 67% inC. burnetii antibodies in BTM samples. The sample size was calculated using the formula n = Z2pq/l2 with an assumed prevalence p = 0.50 and an allowable error on the estimate of l = 0.10 at the 95% confidence level. Although we had a prior knowledge of p = 0.59 [[C. burnetii using the commercially available CHEKIT Q-fever Antibody ELISA test  based on C. burnetii inactivated phase 1 and 2 antigens following manufacturer’s instructions. The optical density (OD) of each sample was corrected by subtracting the OD of the negative control. The results were expressed as sample-to-positive values and estimated as S/P = [(OD sample – OD negative control)/(OD positive control – OD negative control) X 100]. According to the manufacturer, S/P ≥ 40%, S/P < 30% and results in the interval 30% ≤ S/P < 40% were considered as positive, negative and intermediate respectively. However, for the purpose of risk factor analysis in logistic regression we dichotomized the test results as positive for samples with S/P ≥ 40% and as not positive for samples with S/P < 40%. Supplementary herd information for the year 2012 was extracted from the Danish Cattle Database.In a cross sectional designed survey we randomly selected 120 dairy herds to be tested for the presence of  = 0.59  we used2) test was used to compare the prevalence found in this study with the prevalence in 2008. Association between herd antibody status and herd size, dominant milk breed type, animal purchase, bulk tank milk somatic cell count, average fat and protein percentage, average milk delivery to a dairy plant per cow, herd type , regional number of cattle herds per km2  and regional average number of cattle per dairy herd, and regional average number of cattle per cattle herd  were tested by univariable logistic regression followed by multivariable logistic regression with backward elimination of non-significant variables. Statistical significance of the covariates was assessed using the likelihood ratio test based on P ≤ 0.05. Collinearity among the selected variables was assessed, and variables with correlation coefficients |ρ| ≤ 0.5 were considered for inclusion in the final model. The values of Hosmer-Lemeshow goodness of fit test were used to validate the final model.The data were analyzed in SAS. The prevalence of positive, negative and intermediate results with confidence interval was estimated using the Proc SURVEYFREQ command. The chi-square  estimating Kappa (κ) for the agreement between the test results and McNemar’s test.P < 0.01) than the estimate in the study in 2008 [[Descriptive analysis showed that an array of the S/P values of the BTM samples ranged from 1 to 293  in both approaches. Regional number of cattle herds per km2  was borderline significant (P = 0.06). The final model from multivariable analysis included increasing herd size and increasing regional average number of cattle per dairy herd as continuous scaled variables significantly associated with C. burnetii seropositivity (Table P = 0.74). The regional number of cattle herds per km2  and the distribution of the sampled herds is presented in Figure Herd size, average milk delivery per cow to dairy plant, and regional average number of animals per dairy herd and regional average number of cattle per cattle herd  were analyzed as continuous variables and as categorized into three groups. The variables were found significant in univariable analyses (2 = 0.96). When categorizing the 29 duplicate tests as positive or negative, there was full agreement between the test results (κ =1.00 and P = 1). This shows a high precision of the laboratory procedure. This result was expected because the ELISA plates used for the two tests were from the same lot.Comparison of duplicate test results of 29 BTM samples in two separate ELISA plates showed a high correlation between the two test results (rC. burnetii seropositive Danish dairy herds in 2012 shows an increase compared to the prevalence of 59% in 2008. This corresponds well to an increasing trend in results from other European countries as reviewed above and in reviewed literature in [[The prevalence of 79% ture in . AlthouThe cattle herd density is highest in the south western and in the north western areas of Denmark. These are also the areas where most of the sampled herds are located. However, there is no clear clustering of herd status compared to density. Garcia-Seco et al.  did notLike in our study, Ryan et al.  and McCC. burnetii seropositive dairy herds in Denmark. However, as the sample of 120 herds may be slightly too small for more detailed cluster analysis, we have only used simple mapping of the study herds compared to the herd density (Figure The study is based on a random sample of herds, and the results are considered valid for the current prevalence of It is concluded that the prevalence of seropositive dairy herds has increased since the previous survey in 2008, and that there is an adverse impact of increasing herd size and of the regional average dairy herd size on the risk of a dairy herd being seropositive.The authors declare that they have no competing interests.Together we designed the study, analysed the data and wrote the manuscript. Both authors read and approved the final manuscript.


Rhodopseudomonas palustris strain 42OL was isolated in 1973 from a sugar refinery waste treatment pond. The strain has been prevalently used for hydrogen production processes using a wide variety of waste-derived substrates, and cultured both indoors and outdoors, either freely suspended or immobilized. R. palustris 42OL was suitable for many other applications and capable of growing in very different culturing conditions, revealing a wide metabolic versatility. The analysis of the genome sequence allowed to identify the metabolic pathways for hydrogen and poly-β-hydroxy-butyrate production, and confirmed the ability of using a wide range of organic acids as substrates. Rhodopseudomonas palustris is a PNSB belonging to the class Alphaproteobacteria. According to Imhoff et al. [Alphaproteobacteria and Betaproteobacteria, containing photosynthethic pigments and able to carry out anoxygenic photosynthesis.f et al. , the terR. palustris have been isolated from a variety of different environments, from eutrophic lagoons to moist soils, from freshwater ponds to marine coastal sediments [R. palustris throughout a variety of habitats is due to its extreme metabolic versatility, with all modes of metabolism represented ; moreover, the organism is a facultative anaerobe [Strains of ediments –4. The vanaerobe .R. palustris is considered a model organism for studying biological hydrogen production, due to its capacity of efficiently producing hydrogen during organic wastes degradation [All PNSBs are characterized by the ability of carrying out anoxygenic photosynthesis; in the presence of oxygen, photosynthesis is inhibited and a number of PNSBs are able to carry out respiration . Under aradation .R. palustris 42OL has been used previously for hydrogen production processes under various conditions [R. palustris 42OL was also used as a biosorbent for metal removal from wastewaters [c2 were studied [nditions –22, i.e.nditions . Its bionditions . The accnditions , 11, 25.tewaters , 27. Fur studied –30. The  studied .The long history and the versatility of this particular strain render it a very good candidate for further investigating the basis of its ability to acclimate to very different culturing conditions.R. palustris 42OL was isolated in 1973 from a catch-basin collecting the effluents of a sugar refinery waste treatment pond, in Castiglion Fiorentino (AR), Italy. The enrichment was carried out aimed at selecting waste degrading phototrophs. The isolated microorganism has been stored since 1973 at CSMA Collection (WDCM number 147) under the collection name CSMA73/42, growing anaerobically on solid RPN medium [-1 as the carbon source and supplemented with 0.4 g L-1 of yeast extract. The general features of the isolate are reported in Table N medium  with malR. palustris. Phylogenetic analysis performed subsequently (unpublished results) by comparing 16S rRNA gene sequences revealed that the isolate might be indeed affiliated to the species R. palustris. With the present work, a further phylogenetic analysis was conducted and, as shown in the phylogenetic tree in Fig. The isolate 42OL was firstly assigned morphologically to R. palustris 42OL is a Gram-negative rod shaped bacterium, of 0.6–1.2 μm  and non-fixing (NH4+ supplied) conditions. A significantly lower amount of protein was produced in presence of ethanol, while the nitrogen source did not have any effect. However, both nitrogen and carbon sources significantly influenced the amino acid composition of the protein biomass [The first characterization of  biomass .The carbon metabolism of the strain was investigated in terms of substrates that could be used for growth and hydrogen production. Short chain fatty acids such as acetate, pyruvate, lactate, malate and succinate were found to be photodissimilated by the strain with substrate conversion efficiency of 40, 52, 61, 56, and 67 %, respectively . ButyratR. palustris 42OL in terms of hydrogen production and acetylene reduction with different atmosphere composition and at different pH values. The optimal conditions for hydrogen production were under Argon gas as the atmosphere, for early logarithmic stage cells, at pH 6.8. The authors also demonstrated the presence of a hydrogenase enzyme, recycling the hydrogen produced during late logarithmic and light-limited stage.Vincenzini et al.  characteEvidences of its suitability for the treatment of wastes combined with hydrogen production were reported , using wR. palustris 42OL could accumulate up to 18 % w/w of PHB on cell dry weight. R. palustris 42OL accumulates PHB in large amorphous granules, as shown in Fig. PHB is synthetized as a reservoir for reducing equivalents by the strain, in this way competing with hydrogen production , especiaAnother relevant characteristic of this strain is the possibility of cultivation outdoors, under light/dark cycles both for biomass , 38 and LCZM00000000).The organism was selected for genome sequencing on the basis of its metabolic versatility and biotechnological relevance, as witnessed by its long history and by the diversity of applications. Project information is available from the Genomes OnLine Database , under tR. palustris 42OL (CSMA73/42) was maintained anaerobically on solid RPN medium [-1 as the carbon source and supplemented with 0.4 g L-1 of yeast extract. For the extraction of genomic DNA a single colony of cells grown on agar plate was harvested and cultured anaerobically on the same liquid medium in 20 mL sealed glass tubes, at room temperature with a light irradiance of 80 μmol of photons m-2 s-1. Cultures were then transferred into 100 ml round bottles and the headspace was exchanged with Argon gas for anaerobiosis. Cells were harvested at an OD660 = 0.5, in mid-logarithmic phase, pelleted and stored at −20 °C. DNA was isolated from the cells using a CTAB bacterial genomic DNA isolation method, and checked on agarose gel. The genomic DNA purity was assessed by spectrophotometric measurements [N medium  with malurements .The draft genome sequence was generated using the Illumina technology. A Nextera XT DNA library was constructed and sequenced using Illumina MiSeq platform which generated 23,625,870 reads. After trimming, a total of 7,574,912 paired end reads were obtained and assembled into 308 high quality contigs (larger than 5419 bp each) using Abyss 1.0.0 software present on the Galaxy Orione server [Genes were identified using the prokaryotic genome annotation software Prokka 1.4.0  Galaxy .Table 3GR. palustris 42OL resulted to be 5,128,858 bp in length with a GC content of about 65.74 % , genes involved in carbon fixation (RubisCO), the complete tricarboxylic acid cycle, the glyoxylate shunt, a Embden-Meyerhof pathway, and a pentose phosphate pathway. Genes coding for the synthesis of glycogen and poly-β-hydroxyalkanoates as carbon storage polymers were also found, as well as genes related to the photosynthetic apparatus, similarly to all the other R. palustris strains so far sequenced.The genome of R. palustris 42OL was analyzed in terms of synteny with other strains sequenced. The genome was found to be highly syntenic with those of other strains of R. palustris (data not shown). Exclusive reactions were then mapped on KEGG with respect to other R. palustris strains sequenced so far  by using DuctApe v 0.17.2 software [Data obtained are reported in Table TIE1. The total number of reactions ranged between 2442 and 3012, respectively for strains BisA53 and CGA009. Strain Haa2 resulted to have the highest number of unique reactions.The genome of software . Data obR. palustris strain 42OL isolated from a wastewater pond of a sugar refinery in 1973. Along the last four decades, this strain has been successfully used in a wide number of applications, from hydrogen production on wastewaters (its major application) to PHB production. The present genome analysis supported those findings.In this study, we characterized the genome of


Transducer of Cdc42-dependent actin assembly protein 1 (TOCA1) is an effector of the Rho family small G protein Cdc42. It contains a membrane-deforming F-BAR domain as well as a Src homology 3 (SH3) domain and a G protein-binding homology region 1 (HR1) domain. TOCA1 binding to Cdc42 leads to actin rearrangements, which are thought to be involved in processes such as endocytosis, filopodia formation, and cell migration. We have solved the structure of the HR1 domain of TOCA1, providing the first structural data for this protein. We have found that the TOCA1 HR1, like the closely related CIP4 HR1, has interesting structural features that are not observed in other HR1 domains. We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP. TOCA1 binds Cdc42 with micromolar affinity, in contrast to the nanomolar affinity of the N-WASP G protein-binding region for Cdc42. NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization. This suggests that TOCA1 binding to Cdc42 is an early step in the Cdc42-dependent pathways that govern actin dynamics, and the differential binding affinities of the effectors facilitate a handover from TOCA1 to N-WASP, which can then drive recruitment of the actin-modifying machinery. The Ras superfamily of small GTPases comprises over 150 members that regulate a multitude of cellular processes in eukaryotes. The superfamily can be divided into five families based on structural and functional similarities: Ras, Rho, Rab, Arf, and Ran. All members share a well defined core structure of ∼20 kDa known as the G domain, which is responsible for guanine nucleotide binding  P2) , 19–22.  binding , 23 or b binding . Followi binding . The imp studies , 26–32, es 26–28–, 33, 34,es 26–28–, neuritees 26–28–, transcres 26–28–, and intes 26–28–. A role es 26–28–, 39.TOCA1 comprises an N-terminal F-BAR domain, a central homology region 1 (HR1) domain, and a C-terminal SH3 domain. The F-BAR domain is a known dimerization, membrane-binding, and membrane-deforming module , 40, 41 Other HR1 domains studied so far, including those from the PRK family, have been found to bind their cognate Rho family G protein-binding partner with high specificity and affinities in the nanomolar range –45. The –The interactions of TOCA1 and N-WASP with Cdc42 as well as with each other have raised questions as to whether the two Cdc42 effectors can interact with a single molecule of Cdc42 simultaneously. There is some evidence for a ternary complex between Cdc42, N-WASP, and TOCA1 , but theHere, we present the solution NMR structure of the HR1 domain of TOCA1, providing the first structural data for this protein. We also present data pertaining to binding of the TOCA HR1 domain to Cdc42, which is the first biophysical description of an HR1 domain binding this particular Rho family small G protein. Finally, we investigate the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and N-WASP, contributing to our understanding of G protein-effector interactions as well as the roles of Cdc42, N-WASP, and TOCA1 in the pathways that govern actin dynamics.Xenopus tropicalis TOCA1 HR1 domain  were amplified from cDNA (TOCA1 accession number NM_001005148) and cloned into pGEX-6P-1  or pGEX-HisP (Xenopus laevis FBP17 HR1 domain (residues 385–486) from IMAGE clone 5514481, and the X. tropicalis N-WASP G protein-binding domain (GBD) (residues 197–255) from IMAGE clone 5379332, and all were cloned into pGEX-6P-1. The resulting constructs express the proteins as N-terminal GST fusions with a 3C protease-cleavable tag, with pGEX-HisP expressing an additional C-terminal His6 tag. Human Cdc42Δ7Q61L and full-length Cdc42 were cloned into pGEX-2T  and pGEX-6P-1, respectively. A C-terminally extended construct of TOCA1 comprising residues 330–545 was cloned into pMAT10-P.X. tropicalis TOCA1, TOCA1 F-BAR (residues 1–287), and TOCA1 ΔSH3 (residues 1–480) were PCR-amplified from a cDNA clone (IMAGE 5157175) and cloned into pET-His6-SNAP using FseI and AscI sites that had been incorporated into the primers to create His-SNAP-TOCA1 proteins.The GEX-HisP . The HR1E. coli BL21 cells (Invitrogen). Stationary cultures were diluted 1:10 and grown at 37 °C until an A600 of ∼0.8 was reached and then induced with 0.1 mm isopropyl-β-d-thiogalactopyranoside for 20 h at 20 °C. The GST-N-WASP GBD construct was expressed in E. coli BL21-CodonPlus®-RIL (Agilent Technologies). The proteins were purified using glutathione-agarose beads (Sigma) and eluted from the beads by cleavage of the GST tag with 3C protease  or thrombin  prior to gel filtration on a 16/60 S75 column . His-MBP-HR1-SH3 was purified using nickel-nitrilotriacetic acid-agarose beads (Life Technologies) prior to cleavage with 3C protease and gel filtration. Full-length TOCA1, TOCA1 F-BAR, and TOCA1 ΔSH3 were expressed from pET-His6-SNAP in BL21 pLysS, grown at 37 °C until an A600 of ∼0.6 was reached, and induced with 0.3 mm isopropyl-β-d-thiogalactopyranoside overnight at 19 °C. Proteins were coupled to nickel-nitrilotriacetic acid-agarose (Qiagen), eluted using increasing concentrations of imidazole, and further purified by gel filtration using a 16/60 S200 column . All protein concentrations were determined by amino acid analysis .GST fusion proteins (HR1 domains and Cdc42) were expressed in 3H]GTP using [8-3H]GTP (PerkinElmer Life Sciences), as described previously  as described previously . For scieviously . The pro3H]GTP. Binding curves were fitted using a direct binding isotherm to obtain dK values and their curve-fitting errors for the G protein-effector interactions  recorded on a Bruker DRX500 and a 13C-separated NOESY (100-ms mixing time) recorded on an Avance AV600. NMR data were processed using AZARA  and analyzed using ANALYSIS  and δ(15N) are the chemical shift changes for the 1H and 15N dimensions, respectively. Residues that had disappeared were assigned a δ value larger than the maximum calculated δ for the data set, and residues that were too overlapped to be reliably assigned in the complex spectra were assigned δ = 0. The residues that had shifted more than the mean chemical shift change across the spectrum were classed as significant and were filtered for solvent accessibility using NACCESS  as low as 26 nm , suggesting that the interaction is predominantly entropically driven. Other G protein-HR1 domain interactions have also failed to show heat changes in our hands.3H]GTP·Cdc42 was preformed, and the effect of increasing concentrations of untagged TOCA1 HR1 domain was examined. Competition of GST-ACK GBD bound to [3H]GTP·Cdc42 by free ACK GBD was used as a control and to establish the value of background counts when Cdc42 is fully displaced. The data were fitted to a binding isotherm describing competition , in agreement with the low affinity observed in the direct binding experiments.The affinity was therefore determined using competition SPAs. A complex of a GST fusion of the GBD of ACK, which binds with a high affinity to Cdc42 , with rapetition . Free ACof 23 nm . The TOC23 nm . Thus, the C-terminal region of Cdc42 is not required for maximal binding of TOCA1 HR1.The Cdc42 construct used in the binding assays has seven residues deleted from the C terminus to facilitate purification. These residues are not generally required for G protein-effector interactions, including the interaction between RhoA and the PRK1 HR1a domain . In contin vitro translated human TOCA1 fragments had suggested that residues N-terminal to the HR1 domain may be required to stabilize the HR1 domain structure (e.g. the BAR domain of Arfaptin2 binds to Rac1 and Arl1) (A) were therefore assessed for binding to full-length Cdc42 by direct SPA. The isolated F-BAR domain showed no binding to full-length Cdc42 (B). Full-length TOCA1 and ΔSH3 TOCA1 bound with micromolar affinity (B), in a similar manner to the isolated HR1 domain (A). The HR1-SH3 protein could not be purified to homogeneity as a fusion protein, so it was assayed in competition assays after cleavage of the His tag. This construct competed with GST-ACK GBD to give a similar affinity to the HR1 domain alone . Taken together, these data suggest that the TOCA1 HR1 domain is sufficient for maximal binding and that this binding is of a relatively low affinity compared with many other Cdc42·effector complexes.Another possible explanation for the low affinities observed was that the HR1 domain alone is not sufficient for maximal binding of the TOCA proteins to Cdc42 and that the other domains are required. Indeed, GST pull-downs performed with tructure . Furthernd Arl1) , while and Arl1) . TOCA1 dnd Arl1) A were thth Cdc42 B. Full-laffinity B, in a s1 domain A. The HRm, respectively) (D and E), suggesting that low affinity interactions with Cdc42 are a common feature within the TOCA family.The low affinity of the TOCA1 HR1-Cdc42 interaction raised the question of whether the other known Cdc42-binding TOCA family proteins, FBP17  and CIP4ctively) , D and E15N-separated NOESY and 13C-separated NOESY experiments. There were 1,845 unambiguous NOEs and 757 ambiguous NOEs after eight iterations. 100 structures were calculated in the final iteration; the 50 lowest energy structures were water-refined; and of these, the 35 lowest energy structures were analyzed. Because the TOCA1 HR1 domain was sufficient for maximal Cdc42-binding, we used this construct for structural studies. Initial experiments were performed with TOCA1 residues 324–426, but we observed that the N terminus was cleaved during purification to yield a new N terminus at residue 330 (data not shown). We therefore engineered a construct comprising residues 330–426 to produce the minimal, stable HR1 domain. Backbone and side chain resonances were assigned as described . 2,778 nA. The two α-helices of the HR1 domain interact to form an anti-parallel coiled-coil with a slight left-handed twist, reminiscent of the HR1 domains of CIP4 (2KE4) and PRK1 (1CXZ and 1URF). A sequence alignment illustrating the secondary structure elements of the TOCA1 and CIP4 HR1 domains and the HR1a and HR1b domains from PRK1 is shown in Fig. 3B.The structure closest to the mean is shown in Fig. 3 of CIP4   but does not form an α-helix. It instead forms a series of turns, defined by NOE restraints observed between residues separated by one  or two (residues 337–340) residues in the sequence and the φ and ψ angles, assessed using Stride . Interestingly, side chains of residues within the loop region point back toward helix 1; for example, there are numerous distinct NOEs between the side chains of Asn-380 and Met-383 of the loop region and Tyr-377 and Val-376 of helix 1 (D). The backbone NH and CHα groups of Gly-384 and Asp-385 also show NOEs with the side chain of Tyr-377.The two α-helices of TOCA1 HR1 are separated by a long loop of 10 residues (residues 380–389) that contains two short 3 helix 1 D. The baTOCA1-Cdc42 interface was investigated using NMR spectroscopy. A series of 15N HSQC experiments was recorded on 15N-labeled TOCA1 HR1 domain in the presence of increasing concentrations of unlabeled Cdc42Δ7Q61L·GMPPNP to map the Cdc42-binding surface. A comparison of the 15N HSQC spectra of free HR1 and HR1 in the presence of excess Cdc42 shows that although some peaks were shifted, several were much broader in the complex, and a considerable subset had disappeared (A). This behavior cannot be explained by the increase in molecular mass (from 12 to 33 kDa) when Cdc42 binds and is more likely to be due to conformational exchange. This leads to broadening of the peaks so that they are not detectable. Overall chemical shift perturbations (CSPs) were calculated for each residue, whereas those that had disappeared were assigned a shift change of 0.2 (B). A peak that disappeared or had a CSP above the mean CSP for the spectrum was considered to be significantly affected.The HR1appeared A. This be of 0.2 B. A peak15N HSQC shift mapping experiments report on changes to amide groups, which are mainly inaccessible because they are buried inside the helices and are involved in hydrogen bonds. Therefore, 13C HSQC and methyl-selective SOFAST-HMQC . The changes were localized to one end of the coiled-coil, and the binding site appeared to include residues from both α-helices and the loop region that joins them. Residues outside of this region were not significantly affected, indicating that there was no widespread conformational change. The residues in the interhelical loop and helix 1 that contact each other (D) show shift changes in their backbone NH and side chains in the presence of Cdc42. For example, the side chain of Asn-380 and the backbones of Val-376 and Tyr-377 were significantly affected but are all buried in the free TOCA1 HR1 structure, indicating that local conformational changes in the loop may facilitate complex formation. The chemical shift mapping data indicate that the G protein-binding region of the TOCA1 HR1 domain is broadly similar to that of the CIP4 and PRK1 HR1 domains (B and 4D).TOCA1 residues whose signals were affected by Cdc42 binding were mapped onto the structure of TOCA1 HR1 C. The chch other D show sh domains B and 4D.15N and 13C NMR experiments were also recorded on 15N-Cdc42Δ7Q61L·GMPPNP or 15N/13C -Cdc42Δ7Q61L·GMPPNP in the presence of unlabeled HR1 domain. The overall CSP was calculated for each residue. As was the case when labeled HR1 was observed, several peaks were shifted in the complex, but many disappeared, indicating exchange on an unfavorable, millisecond time scale (A). Detailed side chain data could not be obtained for all residues due to spectral overlap, but constant time 13C HSQC and methyl-selective SOFAST-HMQC experiments provided further information on certain well resolved side chains .The corresponding me scale A. DetailB and C) are invisible in NMR spectra recorded on free Cdc42 due to conformational exchange. These switch regions become visible in Cdc42 and other small G protein·effector complexes (C)As many of the peaks disappeared, the mean chemical shift change was relatively low, so a threshold of the mean plus one S.D. value was used to define a significant CSP. Residues that disappeared were also classed as significantly affected. Parts of the switch regions , B and Complexes , 61, 66 omplexes C8 shows TOCA1 complex was not amenable to full structural analysis due to the weak interaction and the extensive exchange broadening seen in the NMR experiments. HADDOCK  in Fig. 6B.The orientation of the HR1 domain with respect to Cdc42 cannot be definitively concluded in the absence of unambiguous distance restraints; hence, HADDOCK produced a set of models in which the HR1 domain contacts the same surface on Cdc42 but is in various orientations with respect to Cdc42. The cluster with the lowest root mean square deviation from the lowest energy structure is assumed to be the best model. By these criteria, in the best model, the HR1 domain is in a similar orientation to the HR1a domain of PRK1 bound to RhoA and the HR1b domain bound to Rac1. A representative model from this cluster is shown in Fig. 6tructure  . Some of these can be rationalized; for example, Thr-24Cdc42, Leu-160Cdc42, and Lys-163Cdc42 all pack behind switch I and are likely to be affected by conformational changes within the switch, while Glu-95Cdc42 and Lys-96Cdc42 are in the helix behind switch II. Other residues that are affected in the Cdc42·TOCA1 complex but that do not correspond to contact residues of RhoA or Rac1  include Gln-2Cdc42, Lys-16Cdc42, Thr-52Cdc42, and Arg-68Cdc42. Lys-16Cdc42 is unlikely to be a contact residue because it is involved in nucleotide binding, but the others may represent specific Cdc42-TOCA1 contacts. In the model, these side chains are involved in direct contacts .A sequence alignment of RhoA, Cdc42, and Rac1 is shown in Fig. 6TOCA1 complex and the structure of Cdc42 in complex with the GBD of the N-WASP homologue, WASP (1CEE), shows that the HR1 and GBD binding sites only partly overlap, and, therefore, a ternary complex remained possible (A). Interestingly, the presence of the TOCA1 HR1 would not prevent the core CRIB of WASP from binding to Cdc42, although the regions C-terminal to the CRIB that are required for high affinity binding of WASP (A).From the known interactions and effects of the proteins in biological systems, it has been suggested that TOCA1 and N-WASP could bind Cdc42 simultaneously . Studiesue, WASP  . The dK that was determined (37 nm) is consistent with the previously reported affinity . Unlabeled HR1TOCA1 was then added to the Cdc42·N-WASP complex, and no changes were seen, suggesting that the N-WASP GBD was not displaced even in the presence of a 5-fold excess of HR1TOCA1. These experiments were recorded at sufficiently high protein concentrations  to be far in excess of the dK values of the individual interactions . A comparison of the HSQC experiments recorded on 15N-Cdc42 alone, in the presence of TOCA1 HR1, N-WASP GBD, or both, shows that the spectra in the presence of N-WASP and in the presence of both N-WASP and TOCA1 HR1 are identical (C).An N-WASP GBD construct was produced, and its affinity for Cdc42 was measured by competition SPA B. The Kdaffinity . Unlabelng HSQCs C. Unlabedentical C.15N-TOCA1 HR1 was monitored in the presence of unlabeled Cdc42Δ7Q61L·GMPPNP (1:1) before and after the addition of 0.25 and 1.0 eq of unlabeled N-WASP GBD. The spectrum when N-WASP and TOCA1 were equimolar was identical to that of the free HR1 domain, whereas the spectrum in the presence of 0.25 eq of N-WASP was intermediate between the TOCA1 HR1 free and complex spectra (D). When in fast exchange, the NMR signal represents a population-weighted average between free and bound states, so the intermediate spectrum indicates that the population comprises a mixture of free and bound HR1 domain. Hence, a third, intermediate state that includes all three proteins is unlikely. Again, the experiments were recorded on protein samples far in excess of the individual dK values (600 μm each protein). These data indicate that the HR1 domain is displaced from Cdc42 by N-WASP and that a ternary complex comprising TOCA1 HR1, N-WASP GBD, and Cdc42 is not formed. Taken together, the data in C and D, indicate unidirectional competition for Cdc42 binding in which the N-WASP GBD displaces TOCA1 HR1 but not vice versa.Furthermore,  spectra D. When iXenopus extracts into which exogenous TOCA1 HR1 domain or N-WASP GBD was added, to assess their effects on actin polymerization. Actin polymerization in all cases was initiated by the addition of PIP2-containing liposomes. Actin polymerization triggered by the addition of PIP2-containing liposomes has previously been shown to depend on TOCA1 and N-WASP . This is over 100 times lower than that of the N-WASP GBD (dK = 37 nm) and considerably lower than other known G protein-HR1 domain interactions. The polybasic tract within the C-terminal region of Cdc42 does not appear to be required for binding to TOCA1, which is in contrast to the interaction between Rac1 and the HR1b domain of PRK1 but more similar to the PRK1 HR1a-RhoA interaction. A single binding interface on both the HR1 domain and Cdc42 can be concluded from the data presented here. Furthermore, the interfaces are comparable with those of other G protein-HR1 interactions . TOCA1 and CIP4 both bind weakly to Cdc42, whereas the HR1a domain of PRK1 binds tightly to RhoA and Rac1, and the HR1b domain binds to Rac1. The structural features shared by TOCA1 and CIP4 may therefore be related to Cdc42 binding specificity and the low affinities. In free TOCA1, the side chains of the interhelical region make extensive contacts with residues in helix 1. Many of these residues are significantly affected in the presence of Cdc42, so it is likely that the conformation of this loop is altered in the Cdc42 complex. These observations therefore provide a molecular mechanism whereby mutation of Met383-Gly384-Asp385 to Ile383-Ser384-Thr385 abolishes TOCA1 binding to Cdc42 . Phe-56Cdc42, which is a Trp in both Rac1 and RhoA (C), is thought to pack behind switch I when Cdc42 interacts with ACK, maintaining the switch in a binding-competent orientation  may contact HR1TOCA1 directly (D). Gln-2Cdc42, which has also been identified as a contact residue in the Cdc42·ACK complex , suggesting a direct electrostatic contact between switch II of Cdc42 and helix 2 of the HR1 domain. The equivalent Arg in Rac1 and RhoA is pointing away from the HR1 domains of PRK1. The importance of this residue in the Cdc42-TOCA1 interaction remains unclear, although its mutation reduces binding to RhoGAP, suggesting that it can be involved in Cdc42 interactions  and Rac binders being more rigid (mT of the TOCA1 HR1 domain is 61.9 °C (data not shown), which is the highest mT that we have measured for an HR1 domain thus far. As such, the ability of the TOCA1 HR1 domain to bind to Cdc42 (a close relative of Rac1 rather than RhoA) fits this trend. An investigation into the local motions, particularly in the G protein-binding regions, may offer further insight into the differential specificities and affinities of G protein-HR1 domain interactions.We have previously postulated that the inherent flexibility of HR1 domains contributes to their ability to bind to different Rho family G proteins, with Rho-binding HR1 domains displaying increased flexibility, reflected in their lower melting temperatures     suggests tubules , and TOCbules (2 . Furtherbules (2 . Once at(2.5 μm) ) and tha2 to the basic region just N-terminal to the GBD further favors the active conformation (It has been postulated that WASP and N-WASP exist in equilibrium between folded (inactive) and unfolded (active) forms, and the affinity of Cdc42 for the unfolded WASP proteins is significantly enhanced . The unformation . A substormation . Our dat2 via its F-BAR domain, where the local increase in concentration favors F-BAR-mediated dimerization of TOCA1. Cdc42 is activated in response to co-incident signals and can then bind to TOCA1, further stabilizing TOCA1 at the membrane. TOCA1 can then recruit N-WASP Pt N-WASP  via an it N-WASP . The rect N-WASP . WIP inht N-WASP . It may 2, activated Cdc42 and TOCA1, and oligomeric activation implemented by TOCA1 would lead to full activation of N-WASP and downstream actin polymerization.In a cellular context, full-length TOCA1 and N-WASP are likely to have similar affinities for active Cdc42, but in the unfolded, active conformation, the affinity of N-WASP for Cdc42 dramatically increases. Our binding data suggest that TOCA1 HR1 binding is not allosterically regulated, and our NMR data, along with the high stability of TOCA1 HR1, suggest that there is no widespread conformational change in the presence of Cdc42. As full-length TOCA1 and the isolated HR1 domain bind Cdc42 with similar affinities, the N-WASP-Cdc42 interaction will be favored because the N-WASP GBD can easily outcompete the TOCA1 HR1 for Cdc42. A combination of allosteric activation by PIPTOCA1 interaction prior to downstream activation of N-WASP.In such an array of molecules localized to a discrete region of the membrane, it is plausible that WASP could bind to a second Cdc42 molecule rather than displacing TOCA1 from its cognate Cdc42. Our NMR and affinity data, however, are consistent with displacement of the TOCA1 HR1 by the N-WASP GBD. Furthermore, TOCA1 is required for Cdc42-mediated activation of N-WASP·WIP , implyinIn light of this, we favor an “effector handover” scheme whereby TOCA1 interacts with Cdc42 prior to N-WASP activation, after which N-WASP displaces TOCA1 from its bound Cdc42 in order to be fully activated rather than binding a second Cdc42 molecule. Potentially, the TOCA1-Cdc42 interaction functions to position N-WASP and Cdc42 such that they are poised to interact with high affinity. The concomitant release of TOCA1 from Cdc42 while still bound to N-WASP presumably enhances the ability of TOCA1 to further activate N-WASP·WIP-induced actin polymerization. There is an advantage to such an effector handover, in that N-WASP would only be robustly recruited when F-BAR domains are already present. Hence, actin polymerization cannot occur until F-BAR domains are poised for membrane distortion.TOCA1 complex indicates a mechanism by which such a handover could take place (Our model of the Cdc42·HR1ke place  because ke place  are conske place . The regke place , would tin vitro and that the interaction is of micromolar affinity, lower than that of other G protein-HR1 domain interactions. The analogous HR1 domains from other TOCA1 family members, FBP17 and CIP4, also exhibit micromolar affinity for Cdc42. A role for the TOCA1-, FBP17-, and CIP4-Cdc42 interactions in the recruitment of these proteins to the membrane therefore appears unlikely. Instead, our findings agree with earlier suggestions that the F-BAR domain is responsible for membrane recruitment (In conclusion, the data presented here show that the TOCA1 HR1 domain is sufficient for Cdc42 binding ruitment , 33. Theruitment  and withruitment .TOCA1 complex. The displacement of the TOCA1 HR1 domain from Cdc42 by N-WASP may represent a unidirectional step in the pathway of Cdc42·N-WASP·TOCA1-dependent actin assembly.It is clear from the data presented here that TOCA1 and N-WASP do not bind Cdc42 simultaneously and that N-WASP is likely to outcompete TOCA1 for Cdc42 binding. We therefore postulate an effector handover mechanism based on current evidence surrounding WASP/N-WASP activation and our model of the Cdc42·HR1J. R. W. generated constructs and proteins, set up NMR experiments, analyzed NMR data, and performed binding experiments; D. N. set up NMR experiments; H. M. F. generated longer TOCA clones and proteins; J. L. G. supervised the pyrene actin assays; D. O. supervised the protein binding assays; and H. R. M. performed NMR experiments and analyzed NMR data. J. R. W., D. O., and H. R. M. wrote the paper with input from all authors.


A putative globin-coupled sensor from Methylacidiphilum infernorum, HGbRL, contains an N-terminal globin domain whose open and closed structures reveal an untypical dimeric architecture. Helices E and F fuse into an elongated helix, resulting in a novel site-swapped globin fold made up of helices A–E, hence the distal site, from one subunit and helices F–H, the proximal site, from another. The open structure possesses a large cavity binding an imidazole molecule, while the closed structure forms a unique Lys–His hexacoordinated species, with the first turn of helix E unravelling to allow Lys52(E10) to bind to the haem. Ligand binding induces reorganization of loop CE, which is stabilized in the closed form, and helix E, triggering a large conformational movement in the open form. These provide a mechanical insight into how a signal may be relayed between the globin domain and the C-terminal domain of HGbRL, a Roadblock/LC7 domain. Comparison with HGbI, a closely related globin, further underlines the high degree of structural versatility that the globin fold is capable of, enabling it to perform a diversity of functions.Globins are haem-binding proteins with a conserved fold made up of  Methylacidiphilum infernorum, isolated from the Hell’s Gate (Tikitere) geothermal area in New Zealand, is an aerobic methanotrophic bacterium growing optimally at 60 °C and pH 2.02 and an unusually low autoxidation rate2 or acetateThe bacterium 2, CO and NO, ligands as large as econazole or even a phospholipid have been reported in the structures of yeast flavohaemoglobinRalstonia eutropha flavohaemoglobin (FHP)67Escherichia coli flavohaemoglobin (HMP), interestingly, the sixth coordination site is physically occupied by Leu57(E11)The globin fold, having one of the most versatile architecture, can bind a variety of different ligands in the distal site. Besides small ligands such as Oby Leu57E11.Bacillus subtilis aerotaxis, the C-terminal domain is a methyl-accepting chemotaxis proteinB. subtilis, on the other hand, comprises a C-terminal STAS domain and forms a gigantic stressosome with RsbS and RsbT, which plays a role in the bacterium’s general stress responseB. subtilis RsbR represents an extreme example of a globin that has evolved to lose its haem, the RsbR homologues from Saprospira grandis still retain their haem and are able to bind O2Globins have also been found fusing to the N termini of a variety of proteins, forming multi-domain proteins which are widely distributed in prokaryotes and eukaryotes alike. In flavohaemoglobins, which function in cellular responses to nitrosative stress, the globin domain is fused to a ferredoxin reductase-like module consisting of a FAD- and an NADH-binding domainM. infernorum is a putative globin-coupled sensor of 274 residues, consisting of an N-terminal globin domain and a C-terminal Roadblock/LC7 domain. It is unique to the genus Methylacidiphilum, and presently is found only in two other closely related bacteria, M. kamchatkenseM. fumariolicumN-HGbRL, is 38% identical to HGbI, with both belonging to the same group of bacterial globins that lack helix D and conserve both Tyr(B10) and Gln(E7) (6Vitreoscilla stercoraria haemoglobin (VsHb)N-HGbRL, which has been solved in both the open and closed forms with several novel features.HGbRL from  GlnE7) . The str . The stN-HGbRL was in the ferric state, whose spectrum revealed a predominantly hexacoordinated form with a Soret band at 412 nm and a visible peak at 534 nm , which also coordinates a water molecule, W1  except for Tyr28(B10) and Lys52(E10). The A-ring propionate of the haem also hydrogen-bonds to the side chains of Arg80 and Tyr84, and the D-ring propionate to Lys43 N and the Tyr28-bound water W1  to close in to bind to the haem at the sixth coordination site and to a water molecule, W2 c quinol dehydrogenasec-550 from Paracoccus versutus, whose haem-coordinating Met100 was replaced by lysineN-HGbRL is hence the first globin structure with Lys–His hexacoordination.Further optimization subsequently yielded crystals of the closed form, also in the same space group cule, W2 . Tyr28 and considerably better at helices F–H (rmsd 0.46 Å), with the distal site undergoing some conformational changes. A ~ 25° bending of helix E results in a large shift of subunits B* between the two forms , creatin tunnels . In the  tunnels . Loop CE tunnels . The sid tunnels . In the  tunnels .N-HGbRL may sense a signal. When a signalling ligand binds the haem in the closed form, it dislodges Lys52(E10) and pushes helix E outwards, inducing part of loop CE to fold into the extra turn of helix E of the open form  in the closed form, but only slight movements of its helix E and a sideways rotation of Leu57(E11) are already sufficient to open up its distal site to bind a molecule of azide, thiocyanate or imidazole19VsHb’s Leu57(E11) is, in HMP it may require a larger outward movement of helix E for ligand binding.The huge pocket of N-HGbRL and these multi-domain globins probably constitute a subgroup of novel bacterial globins that share distinct features such as an open and a closed form, a huge distal pocket, haem hexacoordination or shielding, and, upon ligand binding, reorganization of loop CE and helix E which may possibly lead to a large movement of a neighbouring domain. A signature of this subgroup may be haem hexacoordination or shielding with a residue at site E10 or E11, instead of E7, such as Lys52(E10) in N-HGbRL and Leu57(E11) in HMP. Haem hexacoordination normally involves the invariant His(E7), and only recently has an E11 residue been reported, i.e. His66(E11) in Geobacter sulfurreducens globin-coupled sensor (GsGCS)N-HGbRL has been solved in both forms, more structures of globins in either form are needed for further studies.N-HGbRL and HGbIN-HGbRL  of HGbI, for instance, is structurally aligned not to N-HGbRL’s corresponding Gln49(E7) but to Lys52(E10), and HGbI’s Val57(E14) is aligned to N-HGbRL’s Trp59(E17), which may have a role as a pivot for the shift –Trp100(G14) has also been observed to shift N-HGbRL’s helix B by about half a helical turn –Ala70(E28) corresponding to HGbI’s loop EF, are also employed for the formation and accommodation of the fused helices E and F. The conversion of HGbI’s Ser51(E8) to Gly50(E8) has not only added flexibility for helix E’s unwinding, but also avoided clashes with helix B. Substituting HGbI’s Gly64 and Gly70 with Asn63(E21) and Ala69(E27), in turn, increases the propensity for helix formation in loop EF, while replacing HGbI’s conserved Pro73 with Asp72(E30) also contributes to helix forming by providing a free backbone N . Although spectrally ferrous N-HGbRL also binds O2  to move in. On the other hand, the hexacoordinated spectra of HGbIHGbI also has a smaller distal pocket which is connected to the solvent through several tunnels opening . In HGbIbinds O2 , it is uN-HGbRL between its open and closed forms, by the same token, is also expected to trigger changes in HGbRL’s C-terminal Roadblock/LC7 domain. Roadblock/LC7 proteins form one of the light chains of dynein in eukaryotes, a large multisubunit complex that transports cellular cargo along mircrotubules. In bacteria, they constitute an ancient protein superfamily which may function in NTPase regulationMyxococcus xanthus, MglB, is the cognate GTPase-activating protein (GAP) of the GTPase MglA, and they are involved in regulating the dynamic switching of cell polarity in bacterial motility34M. infernorum genome also encodes a pair of genes similar to the MglB–MglA operon, but whose functions are still unknown — a single-domain Roadblock/LC7 protein (GenBank: ACD84470) 34% identical to the C-terminal domain of HGbRL, and an MglA-like GTPase (GenBank: ACD84469) encoded 183 bp upstream.As observed in the reorientation of FHP’s NADH domainMethylacidiphilum, and could have evolved from the duplication and subsequent fusion of the genes of HGbI and the Roadblock/LC7 protein. With both domains having their own carbon copies in the genome — hence able to relieve themselves of the same functions — HGbRL was therefore free to have developed a new role of its own. Its globin domain, upon binding a yet to be identified ligand, is likely to mechanistically relay the signal to the Roadblock/LC7 domain through the former’s conformational changes. The downstream pathway might involve cell motility as mediated by the Roadblock/LC7 domain, but M. infernorum has been reported as a non-motile rodHGbRL is unique to the genus N-HGbRL  was amplified by PCR using primers containing the NdeI and BamHI restriction sites, with a His-tag sequence inserted before the stop codon. The PCR product was cloned into the pET-3a expression vector (Novagen), and the resulting vector was transformed into Rosetta 2(DE3)pLysS (Novagen). The cells were grown in LB medium containing 100 μg/ml ampicillin and 34 μg/ml chloramphenicol. Expression was induced with 50 μM isopropyl-β-D-thiogalactopyranoside, supplemented with 100 mg/l FeSO4•7H2O and 17 mg/l δ-aminolevulinic acid. After overnight incubation at 37 °C, the cells were harvested by centrifugation and resuspended in 50 mM Tris-HCl, 200 mM NaCl, pH 8.0, sonicated and centrifuged to remove cell debris. The supernatant was then applied to a Ni2+-charged HiTrap IMAC HP column , and N-HGbRL was eluted with a gradient of up to 1.0 M imidazole. Fractions containing the protein were pooled and further purified by size-exclusion chromatography on a Superdex 200 column .The gene encoding N-HGbRL was measured in 50 mM Tris-HCl, pH 8.0 at room temperature. The deoxy form was obtained by adding freshly prepared sodium dithionite to a final concentration of 100 mg/ml, and the absorption spectra were measured every ten minutes for an hour.Extensive buffer exchange was carried out to remove traces of imidazole. The absorption spectrum for the met form of N-HGbRL was concentrated to 10–20 mg/ml prior to crystallization. Using the sitting-drop vapour diffusion technique, the imidazole-bound crystals were formed at 20 °C in 3.5 M 1,6-hexanediol, 0.1 M sodium citrate pH 5.6, and cryoprotected with 12% glycerol. Crystals of the hexacoordinated closed form, meanwhile, were formed at 4 °C on a big salt crystal in 50% 2-methyl-2,4-pentanediol, 0.1 M Tris-HCl pH 8.0, 0.2 M (NH4)2HPO4. Diffraction data for both types of crystals, flash-cooled at 100 K, were collected on a Rigaku MicroMax-007 HF X-ray generator (λ = 1.5418 Å) equipped with an R-AXIS IV++ area detector, and processed with XDSC2 but with different cell parameters.http://www.pymol.org).The HGbI structureAccession codes: Atomic coordinates have been deposited with the Protein Data Bank under accession codes 3WFW for the hexacoordinated closed form, and 3WFX for the imidazole-bound open form. How to cite this article: Teh, A.-H. et al. Open and Lys–His Hexacoordinated Closed Structures of a Globin with Swapped Proximal and Distal Sites. Sci. Rep. 5, 11407; doi: 10.1038/srep11407 (2015).