Vinculin is an important protein for the linkage between adhesion molecules and the actin cytoskeleton. The activation mechanism of vinculin is still controversial. In order to provide useful information for a better understanding of its activation, we analyze the motion mode of vinculin with elastic network model in this work. The results show that, to some extent, the five domains will present structural rigidity in the motion process. The differences between the structure fluctuations of these domains are significant. When vinculin interacted with other partners, the central long alpha-helix of the first domain becomes bent. This bending deformation can weaken the interaction between the first domain and the tail domain. This motion mode of the first domain is in good agreement with the information extracted from some realistic complex structures. With the aid of the anisotropy elastic network mode, we analyze the motion directions of these domains. The fourth domain has a rotational motion. This rotation is favorable for the releasing of the tail domain from the pincer-like clamp, which is formed by the first and the third domain. All these motion modes are an inherent feature of the structure, and these modes mainly depend on the topology character of the structure. As a cytoskeletal protein, vinculin is associated with cell-cell and cell-matrix junctions through the link between the integrin adhesion molecules and the actin cytoskeleton . VinculiVinculin contains 1066 amino acids with a 117-kDa molecular weight. As shown in Many interaction sites for numerous binding partners have been mapped onto all these regions . The Vh In its inactivated state, vinculin holds a “closed” and auto-inhibited conformation. In this inactivated conformation, these binding sites are all blocked by the intra-molecular interaction between the vinculin head and the tail domain. Once vinculin becomes activated, the interaction between Vh and Vt domain will be severed, and all the binding sites will be exposed.Vinculin activation needs the release of the Vt domain from the binding interaction between the D1 and D3 domains with Vt. However, the detailed structural mechanism of this activation process is still under dispute ,13. In sFor the activation mechanism of vinculin, experimental research is very challenging. The activation process of vinculin involves a large-scale conformational change, which is related to its biological function ,16. ThusFrom another perspective, it is very time-consuming to study the conformational change with the molecular dynamic simulation for such a big protein, vinculin .Therefore, it is valuable to do a motion mode analysis for the whole protein with some simple computational models . In thisFrom the crystal structures, the GNM method can directly estimate the amplitude of the conformation transition. The directional information of this transition can be obtained from the analysis with ANM.The ENM and normal-mode analysis have been successfully used in biological physics. Such as: the difficult molecular replacement problem , the preAt atomic level, the calculation technique of the low frequency motion modes for a large biological molecule has been developed, and some meaningful works have been carried out ,31.The details of the calculation process of ENM have been described elsewhere ,32,33, sIn the Gaussian network model, the three dimensional structure of protein is described as an elastic network with C-alpha atoms as nodes. These nodes are connected by harmonic springs when two nodes are within a certain cutoff distance (7.3 Å is used in this work). For all springs, the force constant is taken as identical. When we include all contacting residues, the Hamiltonian of the protein system can be defined as follow:γ is the harmonic force constant. We use N to represent the residues’ number, {ΔR} is for the 3N-dimensional column vector for the fluctuation vectors ΔR1,ΔR2, ...ΔRN of these nodes. The superscript T denotes the transposition of a matrix. E is a 3 × 3 identity matrix. Symbol ⊗ represents the direct product. Γ is a N × N symmetric Kirchhoff matrix in which the elements are defined aswhere Rij represents the distance between the ith and jth nodes and rc is the cutoff distance.where The mean-square fluctuations of each node and the cross-correlation fluctuations between different nodes are in proportion to the diagonal and off-diagonal elements of the pseudo inverse of the Kirchhoff matrix. The inverse matrix of the Kirchhoff matrix can be decomposed as:ui (1 ≤ i ≤ N). Λ is a diagonal matrix with the eigenvalues of Kirchhoff matrix– λi as its elements. The cross-correlation fluctuations between the ith and jth nodes can be calculated bywhere U is an orthogonal matrix with eigenvectors of Γ as its columns vectors kB is Boltzmann constant, T is absolute temperature.where i = j, we can get the mean-square fluctuations of the ith residue. In the kth mode, the mean-square fluctuation of the ith residue can be calculated withWhen We do the normalization calculation for the cross-correlation with the follow equation:With GNM model, for the fluctuations of nodes, we can only get the information about the amplitudes but no information for the directions. To including the directional information in the analysis process, an anisotropic elastic network model (ANM) is used. In this model, the motion mode of a protein can be determined by the Hessian matrix H.where the element hij is a submatrix with order 3 × 3. The detailed calculation of hij is:For the elements of hij, its analytical expression can be written as follows,γ and R are the same as in The meanings of The slow and long wavelength collective modes represent the functionally relevant motions of the protein. With the slowest mode from GNM, the protein structure can be divided into some dynamic domains, and the hinge sites for the domain motions also can be identified –36. In FThe whole structure of vinculin is composed of alpha-helix bundles, which has a bigger rigidity than other types of structural elements (such as beta-sheet and loop). Thus, these helixes are distinctly shown as a unit in the figure, especially for the D1 and D2 domain. Each helix corresponds to a line segment. These line segments are separated by the local minima points between them.For the flexible linker between D4 and Vt domain, it also holds a drastic change of fluctuation. This flexible linker is a little far from the main-body of vinculin, so it will have a small influence on the motion tendency of the whole protein. As a disordered part, this linker is lost in the protein’s structural data. Thus, we did not include this linker in the analysis process. For residues in the tail domain, the residue index is re-indexed simultaneously. The aim of this re-indexing is to repair the index gap due to the lost part.D1 domain contains many important binding sites for different binding partners to interact with vinculin. The binding with talin is thought to have an important function for integrin activation and focal adhesion assembly. For the D1 domain, there are three zones that have a low fluctuation. These zones correspond to the link between H (helix) 2–H3, H4–H5 and H6–H7, marked with ovals as shown in For a rigid body, if one end maintains a stationary state and the other has a motion tendency, then this rigid body may have a rotational motion, especially when there is an angle between the motion direction with the axis of the rigid body. As shown in We calculated the cross-correlations between the fluctuations of C-alpha atoms. The modes with low frequency correspond to functional slow motions, and modes with high frequency correspond to localized fast motions. To improve the signal-to-noise ratio, only low frequency modes are used in the calculation.Concretely, the first 40 modes were used in this work. In For this cross-correlation map, it is obvious that there are four orange-red portions anti-diagonally. These four orange-red portions correspond to the five distinct domains. The boundary between D3 and D4 is not as clear as other boundaries, such as between D1 and D2. For D1, D2 and D3, all three domains are composed of two four-helix bundles. From the cross-correlations map, we can see that these three domains are obviously divided into two parts. These parts correspond to the two helix bundles in each domain, especially for D2 domain. This means that the local structures of these two bundles are more stable than that of the central helix. In other words, the conformation change of these three domains can be shown mainly as the structural change of their central helix, such as bending.Along the main diagonal section, there are two obvious bright areas. One corresponds to the interactions between the first helix bundle of D1 and Vt. The other represents the interactions between the second bundle of D1 and D3 domain.As for the D4 and Vt domain, the interactions between them are mainly negatively correlated. But there are still some sites, shown as small bright points in this figure, with positive correlation between them. The correlation between the residue’s fluctuations is dependent on the interaction between them. A strong attraction will lead to a positive correlation motion. This phenomenon means that these two domains (D4 and Vt) will have a tendency to move in an uncorrelated manner, but there are still some weak interactions between some specific regions. The correlation between D3 and Vt is negative. When D4 is removed from the main-body of the molecular structure, the weak interactions between D4 and Vt is helpful for the release of Vt from the crab pincer, which is formed by D1 and D3.In the analysis process for the large-scale motions of nodes, the GNM model can only provide the magnitudes of the displacements departing from their equilibrium positions. In order to get the directions of these displacements, ANM is used to analyze the motion mode of this structure.The first slowest mode of ANM results mainly corresponds to a rotational motion of D4. In The second slowest mode from ANM also can be described as a rotational motion involving two domains D2 and D1. For D1, such a rotational motion is also helpful for the release of Vt.When vinculin interacts with different partners, it undergoes a conformational change. This structural change is partners specific. Thus, the information for the real motion of vinculin will be stored in the complex structure, then we can extract this information from the protein structural data.At present, the available vinculin complex structures in PDB are mainly focused on the D1 domain. There are two potential binding regions for this domain. One is the groove formed with H1, H2, H3 and H4, shown in Correspondingly, the complexes that vinculin D1 interacted with different partners can be classified into two groups. For the first group, vinculin D1 domain interacted with other proteins only at one binding region—the groove from H1–H4  has a rotation due to the attraction from VBs. At the opposite end, the flexible link between H6–H7 has a local conformation change. As there is no binding partner, this change is only due to the flexibility of the linker.For 2IBF, there are two binding regions. The conformation change of the groove formed with H1–H4 is similar to that of 1RKC. In the other groove, there is also a rotation around the linker between H4–H5, H6–H7. The deformation of the first groove in 2IBF is bigger than that of the 1RKC. This is due to the fact that there are more binding regions in 2IBF than in 1RKC. More binding regions will lead to more interactions between D1 and other partners. Furthermore, more interactions will bring about a bigger conformational change. For the same reason, the conformational change of the linker between D1 and D2 in 2IBF is bigger than that of 1RKC.In these two situations, the angle of the bent H4 of D1 is opposite to the Vt domain. The position of the middle part of H4 helix remains unchanged, but the two terminals of this helix depart from their equilibrium position and move away from Vt domain. As mentioned above, in conjunction with the interaction between different partners with D1, the conformation of D1 will change greatly. The bending of H4 will weaken the interaction between D1 and Vt. This conformation change is helpful for the release of Vt. Thus, we can say that the binding of D1 with other partners, regardless of whether it interacts at one or two regions, will help D1 to move away from the Vt. Further, through the weakening of the pincer-like structure, this departure will assist the release of Vt.The motion modes are inherently dependent on the topology character of the structure. From the analyses mentioned above, we have attained an outline of the motion modes of vinculin. First, the fourth domain rotates around the terminal which is situated away from the tail domain. With this rotation, the pincer-like binding will be weakened creating a passageway for Vt. These factors combine to release the tail domain from the pincer-like binding of D1 and D3. Second, when D1 domain binds with other partners, the longest helix in the first domain becomes bent. This bending is also helpful for the release of the tail domain. Briefly, the rotation of D4 and the bending of H4 of D1 all assist the activation of vinculin. Detailed structural mechanisms of vinculin activation remain unsolved by this experiment. However, this useful information about motion modes, achieved from ENM, improves the understanding of the activation process for vinculin.


In the accompanying papers we have shown that sequence errors of public databases and confusion of paralogs and epaktologs (proteins that are related only through the independent acquisition of the same domain types) significantly distort the picture that emerges from comparison of the domain architecture (DA) of multidomain Metazoan proteins since they introduce a strong bias in favor of terminal over internal DA change. The issue of whether terminal or internal DA changes occur with greater probability has very important implications for the DA evolution of multidomain proteins since gene fusion can add domains only at terminal positions, whereas domain-shuffling is capable of inserting domains both at internal and terminal positions. As a corollary, overestimation of terminal DA changes may be misinterpreted as evidence for a dominant role of gene fusion in DA evolution. In this manuscript we show that in several recent studies of DA evolution of Metazoa the authors used databases that are significantly contaminated with incomplete, abnormal and mispredicted sequences  and/or the authors failed to separate paralogs and epaktologs, explaining why these studies concluded that the major mechanism for gains of new domains in metazoan proteins is gene fusion. In contrast with the latter conclusion, our studies on high quality orthologous and paralogous Swiss-Prot sequences confirm that shuffling of mobile domains had a major role in the evolution of multidomain proteins of Metazoa and especially those formed in early vertebrates. All domain-types may be duplicated/deleted by this mechanism in both prokaryotes and eukaryotes but the rate of duplication/deletion may be significantly increased by intronic recombination, explaining why mobile modules (which frequently participate in exon-shuffling) are also prone to undergo tandem duplication .It is widely accepted that fusion of neighboring genes is another major mechanism for DA change in both prokaryotes and eukaryotes. The most plausible pathway for gene fusion in animals is through co-transcription and alternative splicing of neighboring genes, followed by fixation of genomic changes that favor fusion over separate transcription of the constituent genes. This view is supported by the observation that transcripts frequently span two adjacent, tandem genes ,3. TypicIn the case of gene fusion, the DA of the resulting chimeric gene is dictated by the relative position of the neighboring genes in the species where gene fusion occurs in as much as the upstream gene provides the N-terminal domain(s), whereas the downstream gene provides the C-terminal domain(s) of the chimeric protein. As a corollary, the degree of freedom with which genes (domains) may be combined by gene fusion depends on the rate of genomic rearrangements during evolution.It must be emphasized that fusion of genes  leads to an N-terminal DA change from the perspective of the downstream gene  and C-terminal change from the perspective of the upstream gene , therefore the frequencies of N-terminal and C-terminal DA changes due to gene fusion are expected to be similar.cis regulatory regions of the downstream gene  than to gain both of these (as in gene fission). Since the balance of gene fusion/fission is thus tilted in favor of fusion, it is frequently assumed that gene fusion is the main driver of the evolution of more complex multidomain proteins.Most studies agree that the process when a gene encoding a multidomain protein is ‘split’ in a way such that its constituent domains are expressed separately (AB → A + B) is much rarer than gene fusion. ,6. A plaWe wish to emphasize that fission of genes (resulting in DA change of the type AB → A + B) involves an N-terminal change from the perspective of one of the resulting genes  and C-terminal change from the perspective of the other gene , therefore the frequencies of N-terminal and C-terminal DA changes due to gene fission are also expected to be similar. It should be noted, however, that the term gene fission is sometimes  used inIn principle, changing signals for translation initiation and termination may favor loss over gain of terminal domains, since the use of a novel translation initiation site upstream of the original site (converting 5'-untranslated region to translated region) or conversion of the original stop codon to a sense codon (converting the 3'-untranslated region to translated region) is unlikely to result in the gain of a new domain since the 5'- and 3'-untranslated regions were not selected to encode folded domains. Conversely, the gain of a novel translation initiation site downstream of the original one may lead to the loss of an N-terminal domain  or the gain of a novel translation termination site upstream of the original one may lead to the loss of C-terminal domains of a protein . As a corollary, changing signals for translation initation and termination would favor loss over gain of terminal domains. As to the relative probabilities of loss of N-terminal or C-terminal domains: we must take into account the fact that nonsense-mediated decay (NMD) may detect premature stop codons  and may It is generally agreed that shuffling of symmetrical class 1-1 domains (domains flanked by phase 1 introns) by intronic recombination contributed significantly to the evolution of multidomain proteins of Metazoa but it is also clear that intronic recombination is not an absolute prerequisite of domain-shuffling –14.Analysis of a large number of cases where the evolutionary history of the DA change involving class 1-1 domains could be reliably reconstructed revealed that—as a rule—exons/exons-sets encoding class 1-1 domains are inserted in pre-existing phase 1 introns of the recipient gene . The resThe same genomic features that are essential for exon-shuffling  also facilitate the loss of domains acquired by exon-shuffling through fixation of exon skipping .A survey of the various genetic mechanisms that may change the DA of proteins thus suggests that unequal crossing-over, gene-fusion and domain-shuffling are the ‘creative’ mechanisms that may increase the complexity of the DA of multidomain proteins. Conversely, the DA complexity of multidomain proteins may be decreased by unequal crossing-over, gene fission, point mutations that change the boundaries of the open reading frame and fixation of exon-skipping.It must be emphasized that a major difference between gene fusion and domain-shuffling is that the former may alter DAs only at the termini  whereas exon-shuffling does not have this requirement: it may add domains both internally  or at the termini . As a corollary, the relative frequency of DA change in internal positions versus N-terminal and C-terminal positions may be used to assess the relative contribution of gene fusion and domain-shuffling to DA evolution.For example, if we assume that gene fusion is the dominant mechanism responsible for DA change we expect that the rates of DA change at the N-terminal and C-terminal ends of proteins significantly exceeds that observed at internal positions. Conversely, if we assume that domain-shuffling was the dominant mechanism responsible for DA change we expect that the rates of DA change at N-terminal, C-terminal and internal positions are roughly similar. It should be noted, however, that this analysis would be meaningless if we disregard the fact that in the case of one-domain ↔ two domain transitions (type 1 transitions that account for the majority of DA changes), domain architecture change can only be classified as terminal .1.2.Domains of multidomains proteins are frequently labeled with epithets to express some aspects of their role in the evolution of different domain architectures. The most frequently used terms are ‘versatile domains’, ‘promiscuous domains’, ‘mobile domains’ and sometimes these terms are used as if they were synonyms .The generally accepted definition of domain promiscuity/versatility is that domains are promiscuous/versatile if they are present in many different domain architectures –20. Typii.e., moved from one local environment (within a gene) to a new local environment (of another gene). Accordingly, the mobility of a domain is related to but not equivalent with its versatility/promiscuity: mobile domains are necessarily versatile, but versatile domains are not necessarily mobile. For example, domain A is involved in a mobility event if it is inserted into a new environment of a recipient gene  but the XY and Z domains of the recipient gene are not since they did not move from their original environment: the mobility score of domain A is increased by one count, but the mobility scores of X, Y and Z are unaffected. The situation is quite different for versatility/promiscuity scores: the DA change of XYZ → XYAZ will equally affect the versatility/promiscuity score of all four domains involved. This point may be illustrated by the cases of the trypsin domains of regulatory proteases such as TPA_HUMAN, THRB_HUMAN or NTR_HUMAN: these vertebrate-specific multidomain architectures arose by shuffling of mobile domains  whereas, in these DA changes, their common trypsin domain served as recipients for the mobile domains.In contrast with this, the term ‘domain mobility’ is intended to reflect the frequency with which a domain is shuffled, Despite the practical problems associated with genome-scale reconstruction of such events some prototypical studies may serve to illustrate the importance of the distinction of versatility and mobility of domains. Analysis of the evolutionary history of proteases of the blood coagulation and fibrinolytic cascade revealed that during evolution of these paralogous proteins of the trypsin-family a variety of mobile modules  were inserted into the ‘recipient’ genes ,21. In tIn our definition domain-shuffling (domain mobility) is restricted to cases where the partners involved in DA changes are non-equivalent; the domain is moved from one genome location (a donor gene) to a recipient gene. This definition excludes gene fusion as a mobility event since both partners can be considered donor and acceptor. Conversely, domain insertion  is a mobility event; since the roles of the recipient gene and donor genes are non-equivalent and can be clearly distinguished.1.3.As emphasized in an accompanying paper , reliable analysis of DA evolution of multidomain proteins requires that the protein sequences compared are valid, correct and complete. It must be pointed out that many authors realized that gene annotation errors may cause some problems in the analysis of DA of proteins but most studies implicitly assumed that such errors may be neglected in genome-scale analyses and that they occur at random thus they do not obscure the general tendencies of DA evolution. We have shown in an accompanying paper that neither of these assumptions is justified . First, in the case of most Metazoan species the contribution of gene prediction errors to domain architecture differences of orthologous and paralogous proteins is comparable/greater than those of true gene rearrangements. Second, the accuracy of gene prediction itself has a strong positional bias, in as much as it is most reliable for internal exons and least reliable for N-terminal exons , thus, eAs discussed above, neither unequal crossing-over, nor gene fusion nor domain-shuffling are expected to favor N-terminal versus C-terminal DA change, thus we have no obvious genetic explanation for the observation in genome-scale studies that DA changes are preferred at the N-terminal end . It seemOur findings thus cautioned that earlier proteome-scale studies that neglected the contribution of sequence errors may have led to erroneous conclusions about the evolution of novel domain architectures of multidomain proteins. Our observation on high quality Swiss-Prot sequences, that the contribution of internal DA alterations increased in vertebrates , is consistent with our suggestions that exon-shuffling played a major role in shaping the DA of multidomain proteins unique to vertebrates .1.4.We have demonstrated that contamination of protein families with epaktologs (proteins that are related only through the independent acquisition of the same domain types) increases the apparent rate of DA change and introduces a strong bias in DA differences in as much as it increases the proportion of terminal over internal DA differences . These findings cautioned that earlier studies based on analysis of datasets of protein families that were contaminated with epaktologs may have led to some erroneous conclusions about the evolution of novel domain architectures of multidomain proteins.2.In view of our observation that sequence errors and confusion of epaktologs with other types of homologs significantly distorts the evolutionary history of the DA of multidomain proteins, it is important to re-examine the conclusions of earlier studies that neglected the influence of these errors. As emphasized in the accompanying papers, these errors not only increase the apparent rate of DA change but they also introduce a strong positional bias in favor of terminal over internal DA changes.In the case of bacterial genomes (where it is justified to neglect misprediction) it was convincingly shown that terminal changes are significantly (more than 10-fold) more frequent than internal ones . Interesvs. internal changes and this was taken as evidence for gene fusion. This point may be illustrated by the work of Weiner et al. [We suggest that the absence of this shift is due to the fact that in most studies the high proportion of incomplete, abnormal or mispredicted sequences of higher eukaryotes increased the rate of terminal r et al. . These ar et al.  have alset al. [In their genome-scale studies on DA evolution, Ekman et al.  have useDespite the fact that the use of erroneous sequences and other types of methodological errors casts doubt on the results of such analyses the view that the major genetic operations leading to novel arrangements are fusion of existing genes and terminal loss of domains is gaining popularity since it is propagated in several recent review papers ,28.et al. [et al. also noted that the DA change occurs more frequently at the N-terminal than the C-terminal end. In a Research Highlight commentary of the work of Buljan et al. Marsh and Teichmann [Realizing the danger of confusing gene- and protein-annotation errors with true changes of DA, in a recent paper Buljan et al.  have choeichmann  have conet al. [et al. that explain this contradiction.Since these conclusions contradict our data and the widely accepted view that exon-shuffling played a major role in DA evolution of proteins unique to Metazoa we have carefully examined the evidence presented in the paper of Buljan et al. . Our anaThe first and most important problem is that the set of ‘high-confidence domain gain events’ does not properly represent the spectrum of DA changes . The authors acknowledge that the limited set (330 cases) of ‘high-confidence domain gain events’ may not properly represent the whole spectrum of DA changes: “even though we do not expect that the final set of high-confidence domain gains is biased towards any of the mechanisms, the total number of gain events in the set is relatively small and this could introduce apparent dominance of one mechanism over another”. To check this possibility they use a larger number of ‘medium-confidence’ domain gains (849 cases) to “test whether a larger set of domain gains would support the observed distribution of characteristics of gained domains”. Although they note that the “major difference between the two sets was in the number of middle domains coded by one exon” they dismiss this warning sign (pointing to the role of exon-shuffling) by saying that “we believe that this is largely due to false domain gain”.et al. are probably best illustrated by the fact that many of the best-known, well-documented cases of domain-gains are missing from the list (Table S1 in [The problems with the data set of Buljan le S1 in ). If we et al. is that the conclusions are sometimes erroneous as a consequence of the fact that TreeFam sometimes confuses paralogs and epaktologs. As discussed in an accompanying paper , this type of error is encountered most frequently in the case of epaktologous proteins that contain tandem repeats of the same domain-type. For example, in tree TF326157 (Family Name: Complement factor H-related protein precursor) complement factor H and factor XIIIb that arose in vertebrates (consisting of tandem sushi domains) are claimed to belong to the same family as some invertebrate proteins  simply because they also contain multiple tandem sushi domains.Another important problem with the analysis of Buljan etc. that all contain multiple SRCR domains. In contrast with this interpretation, neurotrypsins are paralogs of the plasminogen-activator branch of trypsin-like proteins (based on the evolutionary affiliations of its kringle and protease domains) and the present DA of neurotrypsin evolved through the gain of internal mobile SRCR domains by an ancestral protease and not through the gain of a terminal trypsin domain by an ancestral CD5-like protein. Similarly, in the list of ‘High confidence domain gain events’ in TreeFam family tree TF329295 ENSMUST00000084509  is listed as an example of domain gain, claiming that this protein arose from an ancestor with multiple SRCR domains by gaining a terminal PF00100 (Zona_pellucida) domain, rather than from an ancestor with a Zona_pellucida domain through acquisition of mobile SRCR domains.As a consequence of the contamination of some TreeFam trees with epaktologs the evolutionary history of the DA of proteins and the conclusions drawn from this history may be erroneous. The case of TF329295 is especially instructive. The common feature of proteins listed in this tree is that they contain tandem SRCR domains, including CD5-, CD6-, SRCRL-, C163A-, DMBT1- and NETR-related sequences, thus the tree implicitly assumes that they are all descendants of a common ancestor with multiple SRCR domains. Consequently, the tree implies that additional domains that are present only on some branches of the tree (zona_pellucida domain in DMBT1-related sequences and trypsin in NETR-related sequences)  were gaA third major source of errors is that the authors rely on EnsEMBL sequences, therefore the analysis inherits the problems (presence of erroneous sequences) of this database. For example, when we analyzed the dataset by blasting the EnsEMBL entries against UniProtKB we identified several cases where the corresponding UniProtKB entry was annotated as ‘no protein product’ or ‘retired entry’.Macaca mulatta and XP_002926069.1 of Ailuropoda melanoleuca) also arose by (in silico) fusion of the orthologous tandem genes CELR3 and S26A6 cannot be used to support gene fusion; it just illustrates the danger of error-propagation.Sometimes the authors draw conclusions based on analysis of abnormal transcripts. In Additional file 8 of  the authAnalysis of the case CELR3/S26A6 ‘fusion’ thus has several important messages. First, since co-transcription of tandem genes is quite general , there iAs an example of erroneous conclusion based on errors in gene prediction we may mention the case of TreeFam family TF351422 that the authors highlight as an example of a domain gain after segmental duplication and exon joining. This TreeFam family contains only primate sequences and it is claimed that after a gene duplication event one branch of the family has gained the PTEN_C2 (PF10409) domain . A closAs another example, we may mention the case of TreeFam family TF330855 . In the list of ‘High confidence domain gain events’ (Table S1 of ) the autet al. failed to achieve this goal. First, the limited set of ‘high-confidence domain gain events’ does not properly represent the spectrum of DA changes. Second, the dataset relies on EnsEMBL therefore the analysis inherits the problems associated with errors in gene prediction (mispredicted or non-existent genes). Third, the authors rely on TreeFam where many trees contain  epaktologs.In summary, although one could argue that it could have been worth paying the price of small-scale analysis in order to avoid errors, a closer examination of the cases of ‘high-confidence’ DA changes indicates that the analysis of Buljan 3.We have shown that earlier conclusions that DA changes of Metazoan multidomain proteins occur preferentially at termini (and that the major mechanism for gains of new domains in metazoan proteins is gene fusion) are unwarranted since they reflect the fact that the databases used in these studies  were significantly contaminated with incomplete, abnormal and mispredicted sequences and that the authors failed to separate paralogs and epaktologs. Our studies that eliminated these problems  confirmed that shuffling of mobile domains had a major role in the evolution of multidomain proteins of Metazoa and especially those formed in early vertebrates.


Here we report the crystal structure of the middle domain of H1N1 PB2 with or without m7GTP at 1.9Å and 2.0Å resolution, respectively, which has two mutations  to increase electrostatic potential and solubility. Here we report the m7GTP has unique conformation differ from the reported structure. 7-methyl-guanine is fixed in the pocket, but particularly significant change is seen in ribose and triphosphate region: the buried 7-methyl-guanine indeed binds in the pocket forming by H357, F404, E361 and K376 but the triphosphate continues directly to the outer domain. The presented conformation of m7GTP may be a clue for the anti-influenza drug-design.Influenza pandemics with human-to-human transmission of the virus are of great public concern. It is now recognized that a number of factors are necessary for human transmission and virulence, including several key mutations within the PB2 subunit of RNA-dependent RNA polymerase. The structure of the middle domain in PB2 has been revealed with or without m Orthomyxoviridae family, characterized by eight segments of viral RNA. In humans, seasonal influenza virus causes respiratory inflammation, high fever, head and muscle aches, and fatigue. However, during the 1918 pandemic, the influenza virus (known as the Spanish influenza) also caused severe pneumonia, and an estimated 50 million died worldwide [Influenza virus is an RNA virus belonging to the orldwide . The polorldwide .7GTP showed that the cap-binding sites is the middle domain of PB2 [7GTP, human H3N2 middle domain with m7GTP and human H5N1 middle domain with m7GTP [The structure of the RNP complex were investigated using cryogenic electron microscopy ,5, howevn of PB2 . Recentlth m7GTP .7GTP (7GTP is different from the reported one. We discuss about the m7GTP conformation which would be important for the drug design.Here we report PB2 middle domain structure (318-483) belonging to subtype H1N1 human influenza with m7GTP , which h7GTP soaked crystal in 0.5 mM m7GTP for 2.5 hours, at 1.93 Å (1/2 [Rwork (Rfree) of apo and m7GTP-bound form were 18.6% (22.8%) and 17.8% (21.2%), respectively.We initially purified His-tagged PB2 middle domain using a nickel chelate column . Howevert 1.93 Å . The upp3 Å (1/2 . The fin315SHMRISS321 (315SHM317) are additional residues derived from pET15b vector. The side chain of H316 interacts with S336 and it seems to stabilize this α-helix. On the other hand, by m7GTP soaking, the α-helix deformed to a flexible structure as describe later. The RMSD value is 1.00 Å between native and m7GTP using all identical atoms. A homology structural search using DALI [There was one molecule in an asymmetric unit . This crMRISS321 . The firing DALI  showed n7GTP solution for 2.5 hours. The 2Fo-Fc electron density of m7GTP was explicitly visible  the side chain of R355 and K339 changed the direction to hold the triphosphate. The feature is not seen in other PB2 middle domain structures with m7GTP (2). The side chain of H357 moves from apo to holo state to interact with 7-methyl-guanine via π-π interaction (3). The conformation of m7GTP in 2VQZ shows the interaction with N429, M431 and H432, but the presented structure shows no interaction with these residues.The crystal was soaked in 0.5 mM m visible . Compare visible , it show visible . In our  in 2VQZ . Conformst human isolate in 1940 and 1918, respectively)) and from avian to H2N2/H3N2 ) [In tively)) . These stively)) . In thistively)) , though tively)) .7GTP we presented will be important clue for drug design targeting the cap-binding site. It should be noticed again that two mutations (P453H and I471T) we introduced are totally opposite surface against cap-binding site, thus they does not influence the conformational polymorphism of m7GTP  and novel site (this report)). The novel conformation of m7GTP by crystal soaking, the conformation of soaked compound may be restricted by crystal packing. Thus we checked next points about the phosphate binding site: (1) Original site was preserved in our crystal statically: 7-methyl guanine occupies the same site, but phosphate binds in different manner. There is no direct disturbance to bind phosphate in original site in our crystal. In the original site, important residues for the phosphate binding (H432 and N429) did not change the position (2). Original site was preserved in our crystal dynamically: In comparison with B-factor plot of four different PB2 structure with m7GTP, there are two regions (338 region and 424 loop region) with large difference  are suppressed upon m7GTP binding , the solvent content are ~54%. Average B-factor of our crystal might be the relatively high B-factor compared with 4ES5 (H5N1), but is almost similar compared with 4EQK (H3N2) . This is binding . In the first PB2 middle domain structural paper, it suggested the possibility of the different conformation of triphosphate in the context of a capped oligonucleotide. In electrostatic surface of the cap-binding site , we coulpb2 gene (RIKEN) was inferred from the amino acid sequence of influenza virus H1N1 (A/Puerto Rico/8/1934). We used DNA encoding residues 318-483 subcloned into pET15b vector between the NdeI and BamHI sites. The PB2 middle domain (318-483) was then expressed with an N-terminal histidine-tag and a thrombin cleavage site in E. coli BL21-CoDonPlus-RIL cells (Agilent Technologies) growing in LB medium. The protein was purified by affinity capture on a Ni-IDA 2000 column (MACHEREY - NAGEL). After elution with imidazole, the protein was cleaved with thrombin and loaded onto an Uno S column (Bio-Rad) and eluted with a salt gradient (0.5 M - 1 M NaCl). The eluted PB2 middle domain was not dissolved well under 500 mM NaCl concentration. To obtain more soluble domain, we introduced two mutations on the surface of this domain: P453H and I471T. The final protein was then concentrated to 10 mg/ml in 50 mM Tris-HCl (pH 8.0), 200 mM NaCl and 2 mM DTT and stored at -80°C. At N-terminal and C-terminal, there are additional four residues each 314,GSHM317 and 481GSGC484, respectively.The Crystals were grown using the hanging-drop vapor diffusion method. The protein solution was mixed with an equal volume of well solution (1.2 M NaCl and 2.5% (v/v) ethanol) and incubated at 4°C.RAXISVII. The diffraction images were indexed, integrated and scaled using HKL2000 program [R32. Initial phase was obtained by molecular replacement (phaser) using H3N2 cap-binding domain structure 2VQZ [Rmerge, Rmeas, Rpim and CC1/2 were obtained by Xtriage of phenix [Crystals were picked up using a nylon loop. They were then dipped in mother liquor containing 20% (v/v) ethyleneglycol as a cryoprotectant and plunged into a nitrogen-gas stream at 100 K. The data were collected using an X-ray wavelength of 1.54 Å using Micro7HFM  program . The cryure 2VQZ . The strure 2VQZ . Model bure 2VQZ . Compariure 2VQZ . Data stf phenix .481GSGC484  in native structure and 314GSHMRS319  and 481GSGC484  in m7GTP structure, respectively.The missing residues of electron density are G314  and 7GTP and with m7GTP are 3WI0 and 3WI1, respectively. Additionally, wild type of PB2 middle domain without m7GTP has been deposited with the accession number 4J2R .Coordinates and structure factors of PB2 middle domain with two amino acids mutation (P453H and I471T) have been deposited in the Protein Data Bank. The accession numbers of the structure without m


Enterobacteriaceae (SPATEs) constitute a large family of proteases secreted by Escherichia coli and Shigella. SPATEs exhibit two distinct proteolytic activities. First, a C-terminal catalytic site triggers an intra-molecular cleavage that releases the N-terminal portion of these proteins in the extracellular medium. Second, the secreted N-terminal domains of SPATEs are themselves proteases; each contains a canonical serine-protease catalytic site. Some of these secreted proteases are toxins, eliciting various effects on mammalian cells. Here, we discuss the biogenesis of SPATEs and their function as toxins.Serine Protease Autotransporters of  Enterobactericeae) is a family of extracellular proteases produced by the Enterobacteriaceae. As their name indicates, they are “autotransporters” (ATs), meaning they are secreted by the Type Va secretion system from gram-negative bacteria. ATs are very diverse in their function  [sec-dependent, signal-peptide required for targeting to- and export through the inner membrane, followed by a “passenger domain” , and a C-terminal β-domain (or ‘translocator” domain), which folds as a β-barrel in the outer membrane (OM). The C-terminal β-domain is necessary for translocation of the passenger domain through the OM [SPATE  , but it h the OM ,3. The rh the OM ,3.Pseudomonas aeruginosa harbors a globular, α-helical passenger domain, with no β-helix domain -encoded), initially designated PssA (protease secreted by STEC), and EpeA (EHEC plasmid-encoded autotransporter) from enterohaemorrhagic E. coli (EHEC) [plasmid-encoded toxin) from enteroaggregative E. coli (EAEC) [protease involved in intestinal colonization) from EAEC, uropathogenic E. coli (UPEC) and Shigella [EPEC secreted protein C) and Hbp (hemoglobin protease or hemoglobin binding protein) from enteropathogenic E. coli (EPEC) [secreted autotransporter toxin) from UPEC [temperature-sensitive hemagglutinin) and Vat (vacuolating autotransporter toxin) from avian pathogenic E. coli (APEC) [ETEC autotransporter A) from enterotoxinogenic E. coli (ETEC) [E.coli secreted protease, island-encoded) from Shiga toxin producing E. coli (STEC) [E. coli strain [Shigella flexneri: SepA (Shigella extracellular protein A) [Salmonella bongori: Boa (bongori autotransporter) [et al. also reported the identification of additional SPATEs in Citrobacter and E. coli strains E22, B7A, and F11 [The SPATE subfamily of autotransporters is currently -composed of: EspP (i (EHEC) ,15,16, Pi (EAEC) , Pic  ,21, Sat rom UPEC , Tsh (tei (APEC) ,24, EatAi (ETEC) , EspI (Ei (STEC) , EaaA ani strain ; as wellotein A) , SigA [2otein A) , and onesporter) . Yen et  and F11 .Proteins belonging to the SPATE family display the typical characteristics of autotransporters: they are composed of a signal sequence, a passenger domain secreted in the extracellular medium, and a C-terminal β-domain necessary for translocation of the passenger domain through the outer membrane. The crystal structure of one SPATE passenger domain has been solved (Hbp), as well as that of one SPATE β-domain (EspP) ,11. LikeSPATEs have been grouped in a family based on several criteria:(1) They display a very conserved β-domain. In pair-wise comparison, sequence identity among SPATE proteins ranges from 25 to 55%. However, the conserved residues are not equally spread over the length of the proteins. The passenger domains are variable in length (between 954 and 1050 residues) and show between 23 and 50% amino-acid identity. In contrast, the β-domains are all exactly 277-residues long and 60 to 99% identical. A few SPATEs are more conserved: EaaA and EaaC, as well as Tsh and Hbp are almost identical . In addition, Vat and Tsh/Hbp are 77.5% identical and SepA and EatA are 72.8% identical. In these pairs, both the β-domains and the passenger domains are conserved, with most of the divergence occurring at the N-terminus of the protein. SGS where S is the catalytic serine) at the N-terminus of their passenger domain (between residues 250–270). (2) As their name specifies, SPATEs are proteases. They all ccontain a conserved serine-protease motif (GD3) Whereas some autotransporters remain intact in the outer membrane after secretion, others are cleaved between the passenger domain and the β-domain, thus releasing the passenger domain into the extracellular milieu. All SPATEs are cleaved between the passenger domain and the β-domain after translocation of their passenger domain through the outer membrane. The cleavage occurs at a conserved FxxEVNNLNK site located in the linker domain, with the processing always occurring between the two asparagines. Various mechanisms have been involved in autotransporter cleavage [ Whereas (4) All SPATE proteins have unusually long (>49 amino-acids) signal sequences. This type of signal sequence is found in ~10% of autotransporters, as well as in proteins secreted by the “two-partner secretion system” (or type Vb secretion system). (5) SPATEs are usually among the most abundant proteins secreted by the parental strain, at least in laboratory conditions.Salmonella [eaaC) located on a prophage, could be experimentally transferred between two E. coli strains by transduction [et al. proposed that most ATs instead evolved through speciation and duplication events rather than horizontal gene transfer [eaaA and eaaC in the ECOR-9 strain of E. coli indeed suggests that duplication of SPATE genes occurred [SPATE-encoding genes are located on mobile genetic elements, such as plasmids . Recombromosome ,40. UltiespC transcription is also regulated by quorum sensing, through the Per/Ler regulatory pathway in EPEC. Although Ler is also present in EHEC, it does not influence EspP expression [The exact mechanism by which SPATE expression is regulated has not been studied in much detail. Their expression seems to be regulated by environmental stimuli  as well as by quorum sensing. In contrast with Pet, for which expression does not seem to be affected by temperature , SigA, Ppression . Autotransporters, including SPATE proteins, are transported through the inner membrane via the Sec-translocon. Once in the periplasm, the C-terminal domain folds as a β-barrel in the outer membrane, forming a pore. The passenger domain is then translocated through the outer membrane, cleaved from the β-barrel and released in the extracellular medium, where it will fulfill its function, usually related to the virulence of the producing strain. After synthesis, SPATE proteins are targeted to the Sec-translocon which catalyses their transport through the inner-membrane. To be targeted to the Sec-translocon, presecretory proteins need to harbor a N-terminal, cleavable, “signal sequence”. These sequences are usually ~20–30 residues long, highly variable in sequence and organized in three domains: a N-terminal, relatively basic “N-domain”, a “H-domain” rich in hydrophobic residues, and a “C-domain” where the cleavage by signal peptidase occurs. In the case of SPATE proteins, the signal sequence is at least 49 residues long and is comprised of a ~25 residues N-terminal conserved extension and a C-terminal non-conserved region, which resembles a conventional signal peptide . The orisignal recognition particle) pathway, or through a SecB-dependent pathway. The SRP ribonucleoprotein complex promotes cotranslational targeting of protein by interacting with highly hydrophobic signal sequences when they emerge from the ribosome. In the SecB-dependent pathway, presecretory proteins are targeted to the inner membrane post-translationally by a mechanism that requires the SecB chaperone to keep the protein in a secretion-competent state within the cytoplasm. Both EspP and Pet are secreted post-translationally, in a SecB-dependent manner. However, the deletion of the N-terminal extension increases the Pet rate of inner membrane secretion and reroutes EspP to the SRP pathway [The C-terminal “classical” signal peptide is necessary and sufficient for inner membrane translocation. In contrast, the N-terminal extension is not a targeting signal by itself, and is not necessary for secretion through the inner membrane ,48,49,50 pathway . The difet al. showed that deleting the N-terminal extension severely impairs EspP transport through the outer membrane [in vitro, without forming aggregates [in vitro [In vitro folding is nevertheless very different from in vivo events, where secreted proteins might encounter chaperones in the periplasm. Indeed, two SPATE “stalled” mutants have recently been used to identify potential partners of SPATE proteins during secretion. Using this strategy, Hbp was found to interact with the periplasmic chaperone SurA, whereas EspP passenger domain cross-linked with both SurA and Skp [E. coli mutant strains further suggest that these periplasmic chaperones are important for SPATE secretion. Indeed, EspP secretion was reduced in surA, skp, or degP mutants of E. coli, and Hbp secretion was affected in a surA strain [surA deletion had a much more pronounced effect on Hbp biogenesis than with EspP. In a surA strain, EspP is translocated through the OM and cleaved, but the level of cleaved EspP passenger domain found in the extracellular medium was slightly reduced compared with the level found in a wild-type E. coli strain [surA E. coli strain [et al.) that Hbp extension does not tether the protein to the inner membrane like it apparently does for EspP. In the absence of such tethering, Hbp might be more dependent on chaperone like SurA to avoid misfolding in the periplasm, which would explain the more pronounced effect of surA mutation on Hbp biogenesis compared with that of EspP. Thus, SPATEs apparently need to be kept in a secretion-competent conformation in order to be secreted properly through the outer membrane. This is apparently possible either through a prolonged interaction with the Sec-translocon or via interaction with periplasmic chaperones, or both. The question of what exactly is a “secretion-competent” state for autotransporters is still a matter of debate (see below).In addition to a role in protein targeting, it was also proposed that the conserved N-terminal extension could affect later steps in biogenesis. Indeed, despite the fact that the signal sequence is cleaved when the protein crosses the inner membrane, Szabadi membrane . To explmembrane . Whethergregates . Howeverin vitro . In vitr and Skp ,55. Alth and Skp . In addiA strain ,56. Intei strain . Hbp secOnce in the periplasm, the autotransporter β domain is inserted into the outer membrane (OM), where it forms a β-barrel. The passenger domain is then transported to the extracellular medium. Deletion of an AT β domain abolishes the translocation of the passenger domain through the OM; this domain is thus essential to the translocation. However, its exact role is still unknown. Because β-barrels can form pores in the OM, it was initially proposed that AT passenger domains are translocated through the OM via the pore formed by their associated β-barrel (hence the name “autotransporters”) . Accordi(1) In the “hairpin” model, the passenger domain is transported through the pore of the β-barrel, with the C-terminus of the passenger first. This model implies that the passenger is unfolded when it crosses the OM.Neisseria, showed that this particular AT is able to form multimers in the OM, which led to a second model in which the passenger domain translocation would occur through a central channel formed by the walls of 6 or more β-barrels [(2) A study with IgA protease, an AT from -barrels . Because(3) Finally, a third model proposes that AT are inserted in the OM via the general OM-protein assembly machinery Omp85/YaeT ,58,61, aet al. found that although the Hbp passenger domain folds to a certain extend in the periplasm, tightly folded domains attached to this passenger can block translocation [SPATEs β-domains for which information on oligomerization states is available (EspP and Tsh) were found to be monomeric ,61,62. Tlocation . Finallylocation ,55,56. AShigella flexneri), is cleaved by an outer membrane protease homologous to OmpT (IcsP), and some autotransporters are cleaved by other ATs  [in vitro and that mutations in EspP β-barrel abolish cleavage. Specifically, we identified two positions that are crucial for cleavage: Asp1120, which is located in the β-domain and is facing the pore of the barrel, and Asn1023, which is the residue just upstream from the cleavage site [Whereas some autotransporters remain intact in the outer membrane after secretion  . All SPAs IgA-P) ,18,42. Vs IgA-P) ,28,42,47s IgA-P) ,17,28,42age site . The impage site . As a coage site . In such a mechanism, the cyclization of the catalytic Asn results in the formation of a succinimide intermediate, which is then hydrolyzed into either asparagine or iso-asparagine . Accordiet al., found that, like in EspP, mutation of the Asn at position P1 of the cleavage site abolishes cleavage in Tsh [Bordetella pertussis [Bordetella ATs, which have very low homology with SPATE β-domains. In theory, intramolecular cleavage would be faster than intermolecular cleavage. Thus, this intramolecular cleavage mechanism may have been favored in ATs for kinetic reasons. However, no comparison of cleavage rate among ATs has been performed yet. Also, whether an intramolecular cleavage catalyzed by the β-domain is faster than one catalyzed by the passenger domain will depend mainly on the folding state of the passenger domain during secretion. Indeed, cleavage in SPATEs occurs immediately after passenger translocation is completed and is probably not dependent upon passenger domain folding. In contrast, cleavage by the passenger domain requires the passenger domain (or at least domain 1) to be folded in the extracellular space. If this domain is not folded during translocation, then cleavage would have to “wait” for folding of the proteolytic domain. Considering the slow folding rate of the autotransporter in vitro, this might considerably delay cleavage [The residues important for cleavage are conserved in all SPATEs, suggesting that all members of this family are processed by the same autoproteolytic mechanism. Although this has not been confirmed, Kostakioti e in Tsh . In addie in Tsh ,66. Howee in Tsh ,66. Inteertussis . The cleertussis . The pasertussis . Considecleavage . Thus, tE. coli and Shigella). Although SPATE genes have now also been detected in non-pathogenic strains of E. coli [in vitro has provided valuable information about their putative role in pathogenesis.SPATEs were initially identified as proteins secreted by bacterial species that are pathogenic to humans or animals  passenger domain [Haemophilus and Neisseria, which also displays a serine protease domain in the passenger domain. IgAP is involved in the degradation of type 1 immunoglobin A (IgA1), which is found in humans and great apes. Specifically, IgAP cleaves after proline residues located in the hinge region between the Fc and the Fab domains of IgA1. The structural similarity between Hbp and IgAP has led some authors to include IgAP in the SPATE family [Enterobacteriaceae. Furthermore, whereas IgAP specifically cleaves the hinge region of IgA1, none of the SPATEs for which this substrate has been tested do. Thus, despite structural homology, these enzymes show a clear difference in substrate specificity. The variation in substrate specificity is probably due to the presence of a loop in IgAP, which is absent from SPATEs [As previously mentioned, all SPATEs possess a serine protease motif in the first third of their passenger domains. The actual proteolytic activity of this domain has been demonstrated for most of the family members . SPATEs are indeed able to cleave various substrate proteins in vitro . In addid His127 . These td His127 . This dor domain . IgAP isE family . Howeverm SPATEs ,69. Thism SPATEs . Also, tm SPATEs ,69. Although Hbp is the only SPATE for which a passenger domain structure is available, homology modeling of EspP and Pet suggests that these SPATE passenger domains adopt a very similar fold compared with Hbp ,68. In pin vitro both on synthetic oligopeptides labeled with paranitroanilide (p-AN) or on entire proteins. As can be seen in The serine protease activity of SPATE passenger domains is not involved in the processing of the passenger domain from the β-domain and is not related functionally to the proteolytic activity of the β-domain. In contrast to the β-domain proteolytic activity, which is apparently only required for the release of the passenger domain in the extracellular space, the proteolytic activity located in the passenger domain might be important for the virulence of the producing strain. The proteolytic activity of SPATE passenger domains has been tested in vitro is relevant to their role in vivo is mostly unknown. It has been proposed that the cleavage of mucin might help pathogenic bacteria degrade mucus and thus adhere to the mucosa [in vivo target is still unknown. Whether the ability of SPATEs to cleave the proteins tested e mucosa . The clee mucosa . Finallye mucosa ,71. HoweSPATEs have been divided into two sub-groups based upon phylogenic criteria. One group includes SPATEs that display cytotoxic activity  and, which probably have intracellular targets. The other group includes SPATEs that do not have demonstrated cytopathic activity on cells, but which might be important in pathogenesis because they affect an extracellular target . Here, wEaaA/EaaC: eaaA and eaaC are two genes that have been identified in the non-pathogenic E. coli strain ECOR-9 . The two genes are located on prophages and are linked to genes encoding non-SPATE, trimeric autotransporters involved in non-immune IgG and IgA binding (eib “E. coli Ig binding” genes). eaaA and eaaC are 99.4% identical at the nucleotide level and encode proteins homologous to the SPATE proteins. The actual expression of eaaA and eaaC in ECOR-9 or the role of these genes has not been tested [E. coli suggests that SPATE proteins might not all be virulence factors per se. Certain SPATEs might be important for colonization of the gastrointestinal tract by commensal strains, but this needs to be tested.n tested . AlthougEatA: EatA is produced by enterotoxigenic strains of E.coli (ETEC), which are a major cause of diarrhea in developing countries. EatA is 73% identical to SepA and displays similar enterotoxic effects in the rabbit ileal loop model of infection. In a manner that is similar to a Shigella sepA mutant [eatA mutant caused less fluid accumulation and mucosal destruction in this model than the wild-type strain would. However, after 16 h of infection, no difference was observed between the mutant and the parental strain (sepA effects 16 h after infection were not reported [p-nitroanilide (AAPM-pNA: 100% activity) in vitro, as well as AAPL-pNA (80%), AAPF-pNA (10%), VPF-pNA (30%) and FLF-pNA (10%) [in vitro are also substrates for cathepsin G  and SepA. However, despite a strong homology with EatA, SepA levels of activity on these substrates are different: 50% for AAPM-pNA and AAPF-pNA: 50%, 100% for VPF-pNA, and 80% for FLF-pNA [A mutant , at 7 h reported ). These NA (10%) . The sub FLF-pNA . EpeA: epeA was identified by sequencing the plasmid pO113 transfer region from non-LEE EHEC [et al. that shows that EpeA belong to the non-cytotoxic group of SPATEs [LEE EHEC . EpeA exLEE EHEC . This isf SPATEs .EspC: Upon infection, EPEC form characteristic “attaching and effacing” (A/E) lesions on the intestine mucosa. These lesions are characterized by the effacement of microvilli and the formation of an actin pedestal underneath the adherent bacterium. Although the role of these lesions in pathogenesis is not clear, factors involved in their formation are important for virulence. EspC is secreted by EPEC and was initially shown to associate with HeLa cells in vitro, suggesting that it might have a role in virulence. However, an espC deletion mutant was not different from its isogenic EPEC parent in terms of adherence or invasion of HeLa, HEp-2, or polarized Caco-2 cells. In addition, EspC was shown not to be involved in the formation of A/E lesions: first, the ΔespC mutant was not affected in EPEC-mediated epithelial cell signaling, cytoskeletal rearrangements, or Tir-phosphorylation, all of which are necessary for A/E lesion formation [Hafnia alvei or Citrobacter freundii, which also form A/E lesions, indicating that EspC is not required for this phenomenon to occur [in vitro [in vitro and the cleavage sites recognized by EspC in fodrin are different from those recognized by Pet. Also, whereas fodrin cleavage by Pet triggers foldrin redistribution in HeLa cells, such effects were not observed upon incubation of cells withEspC [in vivo or whether the cytotoxic effects induced by EspC are due to fodrin cleavage has not been determined. It is currently not known if the cleavage of other EspC substrates identified in vitro  is physiologically relevant and has any role in pathogenesis. Finally, EspC cytotoxic effects are strictly dependent upon the protein internalization in the cytoplasm of epithelial cells. Purified EspC can be internalized by pinocytosis in a receptor-independent fashion when added to epithelial cells at high concentration for a long (8 h) incubation time [Vibrio cholerae T6SS VgrG protein: both of these proteins form long β-helices that are able to perforate membranes without the need of a pore or a dedicated translocase [ormation . Second,to occur . EspC alto occur . Howeverto occur . Also, pto occur  as well in vitro . The effin vitro . Whetherin vitro . In contin vitro . A potenwithEspC . Howeverion time . Howeverion time . It is gion time . Since Eion time , the intion time , which inslocase . Also, Vnslocase , is ablenslocase . Thus, βEspI: espI was identified as one of the ORFs present in a novel pathogenicity island found in the STEC subgroup that lacks the locus of enterocytes effacement (LEE). Like EspP, EpeA, EspC, Pet, and Pic, purified EspI is able to digest pepsin A1. However, like EpeA, it is unable to elicit toxic activity when applied to Vero cells [et al. [in vivo is not known [ro cells , suggest [et al. . Interesot known .EspP/PssA: EspP/PssA is produced by EHEC [in vivo, since the cytopathic effects observed with EspP/PssA are similar to the ones observed with EspC and Pet, both of which target fodrin. In addition, a role for EspP/PssA in intestinal colonization of cattle was suggested by isolation of the pssA mutant in a signature-tagged mutagenesis screen [E. coli O157:H7 colonization of intestinal mucosa and adherence based upon experiments done with an E. coli O157:H7 espP::kanR mutant, which was shown to be slightly affected in adherence to cultured cattle rectal cells in vitro [ΔespP derivative of the E. coli O157:H7 strain EDL933, when compared with the parental strain, was also deficient in adherence to T84 colonic adenocarcinoma cells in vitro [espP was also one of the genes isolated in a screen for genes involved in biofilm formation [ by EHEC ,15. EspPs screen . EspP/Psin vitro . In a moin vitro . Howeverormation . The rolHbp: Hbp “hemoglobin protease” or “hemoglobin binding protein” was initially identified in an E. coli strain (EB1) isolated from a patient presenting a wound infection [in vitro, but has better affinity for hemoglobin than heme. Hbp can also degrade hemoglobin, by a mechanism dependent upon its serine protease activity. Although it has been proposed that the Hbp heme-binding domain could be located between residues 608 and 644, this has not been tested experimentally [E. coli ChuA. However, because Hbp binds heme with high affinity, the release of free heme in the media would probably need to occur after all Hbp molecules have been converted to holo-Hbp. Assuming that heme-binding does not affect Hbp proteolytic activity (which to our knowledge has not been tested), cleavage of hemoglobin by holo-Hbp would release free heme in the environment (because no empty heme binding pocket is available on Hbp). Because hemoglobin triggers release of Hbp from hemin-agarose [Bacteroides fragilis, which does not express Hbp [B. fragilis to capture heme from E.coli Hbp is probably one of the reasons for the synergy of abscess formation in intra-abdominal infections caused by E. coli and B. fragilis. Indeed, Hbp is essential for abscess formation in the context of coinfection by E. coli and B. fragilis. Immunization of mice with Hbp prevented such abscess formation [nfection . Hbp canmentally . The hemmentally . Thus, nmentally . Because-agarose , it is aress Hbp . The abiormation .B. fragilis suggest a role for Hbp as a hemophore. Growth of B. fragilis can be made heme-dependent in certain medium [B. fragilis has a specific receptor for holo-Hbp and can transfer heme from holo-Hbp. Whether E. coli EB1  also has such a receptor remains to be determined.Whether Hbp acts as a hemophore or only as a hemoglobin-protease has not been clearly established yet. However experiments performed with n medium . In thesn medium . These rE.coli EB1. Finally, the last possibility would be that holo-Hbp transfers heme to another hemophore with higher affinity for heme and that this second hemophore then shuttles the heme back to the bacteria. However, such a mechanism has never been described and we are not aware of any other heme-binding protein secreted by The ability to cleave hemoglobin has been tested only for a few of the other SPATEs; whereas Sat and EspI do not process hemoglobin ,40, EspCB. fragilis, suggesting that it also has the ability to cleave hemoglobin and/or bind heme. However no report of such activity is available [Finally, Hbp and Tsh only differ by two residues (Q209K and A842T). Tsh, like Hbp was able to induce abscess formation in a mouse model of coinfection with vailable . Howevervailable , suggestPet: Pet was the first autotransporter for which an enterotoxic activity was reported [reported . Specifireported ,86. Pet reported . In a mareported . After ereported . Once inPic: Pic is produced by Shigella flexneri, EAEC, and UPEC [pic mutant in colonization of the urethra, bladder, or kidney [pic mutant or a strain expressing Pic with no proteolytic activity (042PicS258A) [ex vivo, thus suggesting another role for Pic. Interestingly, it was shown that 042PicS258A is affected in its ability to grow in the presence of mucin in vitro. It was therefore proposed that Pic might have a nutritional role in colonization. Specifically, Pic may allow bacteria to use mucus or mucin as a nutrients source when other sources are not available [and UPEC ,19. Pic and UPEC . Pic alsand UPEC . No effeand UPEC . Howeverr kidney . In conticS258A) . Howevervailable .Sat: Sat is produced by UPEC, a leading cause of urinary tract infection. Sat is cytotoxic on VERO kidney cells, HK-2 human bladder, and HEp-2 cell lines. Like Pet or EspC, Sat contact with culture cells leads to cell elongation and detachment from their support [in vitro suggests that, like EspC and Pet, Sat might target fodrin in vivo. No difference between wild-type and sat mutant were observed in a short-term assessment of colonization in a mouse model of ascending urinary tract infection [sat mutant than in those infected with the wild-type strain, thus suggesting that Sat acts mainly as a toxin [sat gene is also found in Shigella and DAEC. In DAEC, Sat was shown to promote lesions in tight junctions between intestinal epithelial cells. This activity was dependent upon the proteolytic activity of Sat, but the exact target in unknown [ support . Sat als support . This ac support . The abinfection . However a toxin . In cont a toxin . The sat unknown .SepA: SepA is one of the SPATEs produced by Shigella flexneri, the agent of Shigellosis, a disease in which bacteria invade the colonic mucosa and trigger a strong inflammatory response. SepA is 72% identical to EatA, an enterotoxin produced by ETEC (see above). SepA is not involved in bacterial entry in HeLa cells, plaque formation on Caco-2 cells, or dissemination between cells [sepA mutant is attenuated in the rabbit model of ligated ileal loop. Infection with this mutant indeed causes reduced fluid accumulation, reduced mucosal atrophy and decreased tissue inflammation compared with the wild-type strain 8h after inoculation [in vitro on synthetic peptides, and showed similarity to cathepsin G. However, in contrast with cathepsin G, SepA was not able to activate platelets, cleave thrombin receptors, fibronectin, collagen or angiotensin I. This indicates that despite in vitro similarity, the SepA in vivo target is probably not one of the cathepsin G substrates [in vivo target either [en cells . Howeverculation . Howeverculation . Thus, iculation . SepA substrates . As disct either ,70.SigA: SigA is produced by Shigella flexneri. A culture supernatant containing SigA caused damage to HEp-2 cell culture (cell rounding and detachment). However, the effects were less pronounced than the similar effects seen for Pet. Addition of a protease inhibitor abolishes toxicity, indicating that the damage is caused by the ability of SigA to cleave a host target [in vitro and in situ [in situ by SigA causes its redistribution within the cell [t target . Indeed, in situ . The clethe cell . In contTsh: The Tsh-encoding gene was initially identified as a gene from avian pathogenic E. coli (APEC) strain χ7122. The gene confers hemagglutination properties to E. coli K12 [E. coli that cause extraintestinal infection in poultry. APEC infections affect the respiratory tract, but can also become generalized and cause fatal septicemia in animals. Tsh hemagglutination activity was not abolished in a strain expressing a mutant where the Tsh catalytic serine was changed to threonine or alanine, suggesting that the hemagglutinin activity is independent from the proteolytic activity [B. fragilis in a mouse model of intra-abdominal abscess formation [B.fragilis. Indeed, Tsh was shown to bind hemoglobin [tsh knockout mutant of E.coli χ7122 causes fewer and less pronounced lesions in these organs in comparison with the parental strain [coli K12 . APECs aactivity  Interestormation . This sumoglobin . Tsh alsmoglobin . Tsh, inmoglobin , but wasl strain . Tsh is l strain .Vat: APEC causes extraintestinal infection in poultry, such as respiratory diseases, cellulites, or septicemia. Vat, like Sat and EspC, has vacuolating activity: it induces the formation of intracellular vacuoles in cell culture. No vacuolating activity was reported for Tsh despite being 75% identical to Vat. A vat mutant was attenuated in a cellulite model of infection in chicken [ chicken , suggestin vitro that suggest they are somehow involved in the pathogenesis of their parental strain. Currently, the determination of the role of SPATEs in pathogenesis is limited by the lack of small-animal models of infection. Hopefully, the development of new animal models will permit precise understandings of the roles these proteins play in establishment of disease. Meanwhile, a lot of questions regarding the evolution and biogenesis of SPATEs remain to be answer, including: what is the common ancestor of SPATEs? What exactly is the role of the N-terminal extension, and why is it so conserved in SPATEs? What is the role of the autocleavage? Why do SPATEs not use their serine protease activity to cleave their passenger domains, like other serine protease autotransporters? What is the molecular basis for substrate recognition and specificity? How is SPATE expression regulated? What differentiates SPATEs that are internalized in host cells from SPATEs that are not?Autotransporters are an expanding family of secreted proteins from gram-negative bacteria. Over the past two decades, numerous studies have focused on elucidating the secretion mechanism of these proteins. However, so far, the mechanism by which these proteins cross the outer membrane remains controversial. The discrepancy of data obtained between different studies actually suggests that different autotransporters might use different secretion mechanisms. It is already known that these proteins show variations in their biogenesis  and function . Thus, differences in secretion would not be that surprising. The AT variety is particularly striking in the SPATE family of autotransporters. Although SPATEs are much more conserved than other autotransporters, discrepancies are also observed between studies addressing their secretion mechanism. Whether this is due to variation in experimental conditions or not will need to be addressed in the future. In addition, SPATEs show huge diversity in function. All these proteins are serine proteases, and probably adopt a similar fold, but still, they do not share the same targets. In addition, their effect on cells varies: some SPATEs are cytotoxins, while others are not. The mechanism SPATE cytotoxins use to enter host cells also varies. Their trafficking routes inside these cells also vary. Still, all these proteins have effects Considering that most SPATEs are proven or putative virulence factors, understanding the details of their biogenesis and function would be particularly useful in designing new antimicrobial therapies.We are grateful to Travis Barnard and Todd Holyoak for their help in constructing


Pseudomonas aeruginosa is responsible for a plethora of biofilm mediated chronic infections among which cystic fibrosis pneumonia is the most frightening. The long-term survival strategy of P. aeruginosa in the patients lungs is based on a fine balance of virulence vs dormant states and on genetic adaptation, in order to select persistent phenotypes as the small colony variants (SCVs), which strongly correlate with antibiotic resistance and poor lung function. Recent studies have coupled SCV with increased levels of the signaling molecule cyclic di-GMP, and demonstrated the central role of the diguanylate cyclase YfiN, part of the tripartite signaling module YifBNR, in c-di-GMP dependent SCV regulation. YfiN, also called TpbB, is a multi-domain membrane enzyme connecting periplasmic stimuli to cytosolic c-di-GMP production by an allosteric inside-out signaling mechanism that, due to the lack of structural data, is still largely hypothetical. We have solved the crystal structure of the catalytic domain (GGDEF), and measured the enzymatic activity of the cytosolic portion in real-time by means of a newly developed method. Based on these results we demonstrate that, unlike other diguanylate cyclase, YfiN does not undergo product feedback inhibition, and that the presence of the HAMP domain is required for dimerization and catalysis. Coupling our structural and kinetic data with an in silico study we are now able to propose a model for the allosteric regulation of YfiN. Pseudomonas aeruginosa is responsible for a plethora of biofilm-mediated chronic infections among which cystic fibrosis (CF) pneumonia is the most frightening [P. aeruginosa undergoes specific genotypic adaptation to the host environment and, after a yearlong persistence, it develops small-colony variants (SCVs) [The majority of chronic infections involve a biofilm stage. In most bacteria, the synthesis of the ubiquitous second messenger cyclic di-GMP (c-di-GMP) represents a common principle in the formation of otherwise highly diverse and species-specific biofilms –4. Thereghtening . During s (SCVs) –8. SCVs,s (SCVs) –11, are s (SCVs) –14; theis (SCVs) ,12,15. As (SCVs) .P. aeruginosa is highly complex: 42 genes containing putative diguanylate cyclases (DGCs) and/or phosphodiesterase are present [in vitro as well as obtained from clinical isolates contain mutations that upregulate the activity of a specific DGC, i.e. YfiN , are called YfiB and YfiR, respectively. Formation of SCVs depends on enhanced c-di-GMP output by YfiN, which elevates transcription of the pel operon [P. aeruginosa cells in CF lungs. Jenal and coworkers [P. aeruginosa, that the activity of YfiN (and the occurrence of the SCV phenotype) is under continuous cycles of positive and negative selection; the same group proposed that this mechanism may contribute to the in vivo fitness of P. aeruginosa during chronic lung infections.The c-di-GMP metabolism in  present . It has led TpbB , encodedled TpbB ,19,20, il operon ,21. The P. aeruginosa chronic infections. A third component of the system is the YfiB protein, spanning the outer membrane and the peptidoglycan and involved in binding YfiR, thus relieving the repression of YfiN activity  of YfiN, completed its multi-domain structure by homology modeling, and performed a biochemical characterization of two constructs of the cytoplasmic portion of YfiN. We also measured the enzymatic activity by using a new approach for c-di-GMP detection in real-time . Here weorescens . CouplinHAMP-GGDEF; residues 183-435). This truncated construct resulted monomeric in solution. After extensive crystallization trials we could finally collect a complete data set at 2.8 Å resolution from one single hexagonal crystal. The crystal belonged to the P6522 space group. Surprisingly, analysis of the unit cell solvent content (Matthews coefficient) clearly indicated that only one of the two domains of the protein could be physically present in the crystal lattice since fitting both domains in the cell volume would result in a solvent content of 11%, which is too low for a protein crystal. The solved structure confirmed that YfiNHAMP-GGDEF had actually undergone proteolysis and that only the GGDEF domain had crystallized (YfiNGGDEF). The quality of the diffraction data is good and electron density is clearly visible for all main chain atoms spanning from residue 254 to 414 of the GGDEF domain , which is present in all the homologues structures  flanked by five α-helices (αA to F) . YfiNGGDescentus ,28; WspRruginosa ,30; XCC4mpestris  and A1U3quaeolei ) with thP. aeruginosa, containing a degenerated GGDEF domain [P. putida, that displays a PilZ domain [stair-motif interaction and is recurrent in protein/DNA complexes [glue two domains through a double stair-motif interaction. In the case of the I-site of DGCs the first arginine is provided by the primary I-site (Ip) of the GGDEF domain (the conserved RxxD motif), while the second may be recruited from the secondary I-site (Is) of another GGDEF domain [s or another domain, is also needed.It is a general feature of DGCs to undergo a negative feedback inhibition caused by the product binding to the so-called I-site. In particular, c-di-GMP binds as a mutually intercalated dimer with sub micro-molar affinity to the DGCs that display a conserved I-site ,28,30 anF domain , or PP43Z domain . In all omplexes ,36. EachF domain  or from F domain  or the rF domain  and PP43F domain ). Conseqp , while the Is appears degenerated. In particular, the second arginine necessary to form an inactive GGDEF/GGDEF dimer, is substituted with Asp-273 2 by an additional stair-motif interaction [s (α-A) is shifted with respect to the corresponding helix of PleD, WspR and A1U3W3, which all display product feedback inhibition. The shift is due to the hindrance of Tyr-379 side chain  and no binding was observed . To verify whether c-di-GMP could in any way hamper or negatively affect substrate binding to YfiNHAMP-GGDEF, the GTP binding experiment was also repeated in the presence of an excess of product: no influence of c-di-GMP on the binding affinity of the substrate was observed , the formation of a transient catalytic dimer must be assumed. Indeed, the real time kinetics, as monitored with this new method, displays an interesting sigmoidal behavior (currently under investigation), which may well be related with such a mechanism. To verify the role of the HAMP domain in transient dimer formation, we produced a shorter construct containing only the GGDEF domain . This construct, which as expected is monomeric . Likewise, Wood and co-workers have shown that in vitro feedback inhibition for full-length YfiN is observed only at c-di-GMP concentration higher than 200 μM [The enzymatic activity of YfiNur group . We obseonomeric , althougonomeric , indicatn 200 μM .vice versa [Thus, the YfiBNR signaling system appears to be an ON/OFF switch, with the output of the module (i.e. c-di-GMP production) responding only to external stress signals and not to endogenous c-di-GMP levels. It as been shown that the domain architecture of YfiN represents a widespread module to connect periplasmic stimuli to a cytosolic response or ce versa ,37–39. IP. fluorescens adopts a novel fold, consisting of a V-shaped, domain-swapped dimer  that shde: 3pjv . Consistde: 3pjv . As in Lde: 3pjv ), while de: 3pjv ), for alde: 3pjv ). stalk helix) to the GGDEF domain. The final effect is the unlocking of the C-terminal domains, which are now able to adopt a catalytically competent dimeric conformation  to tAs an additional indirect support to this hypothetical mechanism, we mapped the sequence conservation of YfiN and the position of known activating/inactivating mutations  on the fxNlxLiaRsxxYTxEaavvFxD; Strikingly, the accessible central gorge of the LapD-like periplasmic domain, presumably involved into the interaction of the periplasmic domain with YfiR, is characterized by a well-conserved helix spanning residues 44-72  . The impFinally, the C-terminal GGDEF domain is also characterized by a large evolutionarily conserved surface region, which comprise the active site GGDEF motif (residues 319-338: RexDxVaRlGGDEFavllxp), and the adjacent helix-turn-helix region  . These aHAMP-GGDEF is not able to bind c-di-GMP. On the other hand, YfiNHAMP-GGDEF binds GTP with sub-micromolar affinity, and is able to condensate it into c-di-GMP. These data point to the conclusion that YfiN does not undergo product feedback inhibition as other DGCs and, therefore, functions as ON/OFF cyclase responding solely to periplasmic signals.We have shown that YfiN displays a degenerated secondary I-site and that the conserved primary I-site (RxxD) has no counterpart supplied by the HAMP domain, since YfiNPseudomonas adaptation. Of course, availability of structural data represents the bottleneck for an efficient drug design approach: understanding the structural details of the allosteric control of DGC activity is highly desirable yet challenging. By assuming a LapD-like fold for YfiN periplasmic portion, we could speculate that its allosteric regulation is similar to the P. fluorescence receptor [HAMP-GGDEF, we suggest that the periplasmic domain of the full-length protein, by assuming a LapD-like fold that is based on domain-swapping, could function as the driving force for dimerization. A key role in the conformational transition appears to be played by the region connecting the HAMP to the GGDEF domain. We propose that this linker loop may act as a hinge whose locking/unlocking equilibrium, driven by the conformation of the HAMP domain helices, controls the catalysis by keeping the two GGDEF domains separated or allowing their facing  together with of the catalytic domain could become a winning strategy to fight biofilm-mediated infections. This is especially true in the case of the YfiBNR system, which functions as an entry point for different environmental signals during receptor . Normal r facing . CatalysYfiNHAMP-GGDEF and YfiNGGDEF fragments were amplified from a pET24b plasmid harboring a synthetic YfiNfl gene (Geneart). The purified PCR products, verified by sequencing, were ligated  in frame with a C-terminal His-tag into a pET24b vector (Novagen) and transformed into BL21-(DE3) E. coli strain for expression.Both the Both construct were expressed as described in . BrieflyGGDEF; Mw = 23.5 kDa) indicated an apparent molecular mass of 28 kDa consistent with a monomeric state, while for the YfiNHAMP-GGDEF resulted in an ambiguous apparent molecular mass of 41 kDa, in between a monomeric (28 kDa) and a dimeric (56 kDa) form in solution. Therefore, further investigation of the aggregation state of was conducted on YfiNHAMP-GGDEF by analytical ultracentrifugation (AUC)  equilibrated with 10 mM Tris–HCl, pH 8.0, 250 mM NaCl, 10% glycerol; the proteins were eluted with 100 mM imidazole, in the same buffer. Finally, the purified proteins were loaded on an FPLC column , and eluted with 10 mM Tris–HCl pH 8.0, 100 mM NaCl, 2% glycerol. Size exclusion chromatography (SEC) analysis for the shorter construct (YfiNon (AUC) .HAMP-GGDEF in solution was assessed in sedimentation velocity experiments carried out on a Beckman XLI analytical ultracentrifuge using absorbance optics. The experiments were conducted at 35,000 rpm and 20 °C at a protein concentration of 2 mg/mL in 250 mM NaCl, 10 mM Tris-HCl pH 8.0, 10% glycerol. Radial absorbance scans were obtained at 280 nm at a spacing of 0.003 cm with three averages in a continuous scan mode. Sedimentation coefficients were calculated using the software Sedfit [s20,w) using standard procedures. Sednterp software (http://sednterp.unh.edu/) was used to calculate the buffer density and viscosity. The sedimentation coefficient (S) of YfiNHAMP-GGDEF was 2.3 for 98% of the protein, consistent with a molecular mass of 21 kDa, pointing to a monomeric state of YfiNHAMP-GGDEF in solution.Size distribution of YfiNe Sedfit  and wereHAMP-GGDEF were screened using a crystallization robot , by mixing 300 nL of 3.7 mg/mL protein solution in 0.1 M NaCl, 10 mM Tris pH 8 and 2 % glycerol with equal volumes of screen solution. No positive hit was observed during the first three month. After seven month one single hexagonal crystal was observed in the droplet corresponding to solution n.17 of Crystal-Screen2 (Hampton) containing 0.1 M Sodium Citrate dehydrate pH 5.6 and 35% v/v tert-butanol. The crystal was flash frozen in liquid nitrogen, without any cryoprotectant, and diffracted to 2.77 Å resolution . Data were processed with XDS [522 space group with the following unit cell constants: a=b=70.87 Å; c=107.62 Å.Crystallization condition for YfiNwith XDS . The cryHAMP-GGDEF was 1.38 Å3Da-1 with a solvent fraction of 0.11, pointing to the assumption that only the GGDEF domain (YfiNGGDEF) was present in the crystal lattice (Matthews coefficient for YfiNGGDEF was 1.93 Å3Da-1 with a solvent fraction of 0.36). Phases were obtained by molecular replacement using the GGDEF domain of PleD (PDB ID: 2wb4) as template with Molrep [The Matthews coefficient for YfiNh Molrep . Cycles h Molrep  and Refmh Molrep , model gh Molrep  and MolPh Molrep . Final sHAMP-GGDEF protein sample with either GTP or c-di-GMP, and YfiNGGDEF with GTP. Nucleotide stock solutions were prepared in water and diluted into ITC buffer . Protein solution was diluted into the same buffer lacking glycerol. Titration with c-di-GMP were carried out by injecting 1.5 μL aliquots of 90 µM c-di-GMP to a 3 μM protein solution at 25° C; titration with GTP was carried out by injecting 1.5 μL aliquots of 170 µM GTP to 14 μM protein solution at 25° C. The same experiment has been repeated by incubating both GTP and protein samples with 40 µM c-di-GMP. Injection of nucleotides into buffer was also performed as control, under the same experimental conditions. If indicated, data were fitted as described in [ITC experiments were carried out using an iTC200 microcalorimeter , by titrating YfiNribed in . All meaHAMP-GGDEF or YfiNGGDEF in 150 mM NaCl, 20 mM Tris/HCl pH 7.5, 10 mM MgCl2, 2.5 mM MnCl2 and 1% glycerol. C-di-GMP formation was monitored following the CD signal at 282 nm, using a 1 cm quartz cuvette (Hellma) on a JASCO J-710 spectropolarimeter at 20° C.YfiN activity was measured by circular dichroism (CD) spectroscopy as described in . In briePseudomonas aeruginosa was retrieved from the Uniprot database . UniRef50 was used to find sequences closely related to YfiN from the Uniprot database. 123 orthologous sequences displaying a minimum percentage of sequence identity of 50% were obtained. Each sequence was then submitted to PSI-Blast .The YfiN protein sequence from , 0.0001 ), to ret, 0.0001  at EBI (http://npsa-pbil.ibcp.fr/), and Psi-Pred . A consA fold prediction-based approach was utilized to gain some structural insights into the domain organization of YfiN and related proteins. Although three-dimensional modeling performed using such techniques is seldom accurate at the atomic level, the recognition of a correct fold, which takes advantage of the knowledge available in structural databases, is often successful. The programs Phyre2  and HHPRP. fluorescens  was constructed using the MODELLER-8 package , using ade: 3pjv ); the HAde: 3pjv ); Sensorde: 3pjv ); diguande: 3pjv ).Pseudomonas aeruginosa (PDB Code: 3i5c) to predict the structure and orientation of the linker region ; 2) the helical stalk motif of 3i5c (residues 157-170) was then superposed to the C-terminal helix of the HAMP domain of the aerotaxis transducer Aer2 (residues 138-156), to predict the structure and orientation of the HAMP domain of Yfin (residues 182-146); 3) the orientation of the TM helices of Sensor protein qseC (PDB Code: 2KSE) with respect to the hydrocarbon core of the lipid bilayer was derived from the OPM server [A full YfiN dimeric model was built starting from the crystal structure of the cyclase domain (GGDEF – present work) and performing a backward multi-step homology modeling approach, in which each new predicted domain has been linked to the previously obtained model by following the orientation of its structural template. The structural templates were oriented as follows: 1) GGDEF domain of YfiN (residues 254-414) was initially superposed to the GGDEF domain of WspR from M server ; the N-tM server : the modNormal mode analysis and hinge regions predictions were carried out by using the “HingeProt” server, using as cutoff distances for GNM and ANM the default values 10 Å and 18 Å, respectively .Evolutionary sequence conservation was mapped onto the accessible surface of the best model by means of CAMPO , using tFigure S1GGDEF.Residues visible in the crystal structure of YfiN The predicted HAMP and GGDEF domains are underlined in purple and orange respectively. The residues that are visible in the electron density are highlighted in green (254-414). The linker region between the HAMP and the GGDEF domains, where proteolysis conceivably occurred, is coloured in blue.(TIFF)Click here for additional data file.Figure S2HAMP-GGDEF in the presence of c-di-GMP.Binding of GTP to YfiNRepresentative microcalorimetric titration of 14 μM enzyme with GTP (170 μM in the syringe) in the presence of 40 µM c-di-GMP in both solutions. Upperpanel: Raw ITC data. Lowerpanel: Integrated peak areas (black square). Fit with the one-binding-site model of ORIGIN provided by MicroCal (continuous lines) is also depicted.(TIFF)Click here for additional data file.Figure S3Best templates for homology modelling of full length YfiN.Sequence alignments based on secondary structure prediction of the different domains of YfiN with the most significant structural templates according to two different fold prediction servers (Phyre2 and HHPRED).(TIF)Click here for additional data file.Figure S4Sequence conservation.Multiple sequence alignment of 53 non-redundant orthologous of YfiN sequences, from other Pseudomonas strains and from more distantly related sequences from other bacteria.(PDF)Click here for additional data file.Figure S5HAMP-GGDEF and YfiNHAMP-GGDEF in solution.Determination of the aggregation state of YfiNA) Size exclusion chromatography (SEC) of YfiNHAMP-GGDEF (green) and YfiNGGDEF (blue) after the affinity chromatography purification step. The proteins elutes with an apparent molecular mass of 41 kDa and 28 kDa respectively. B) Calibration curve obtained using the following standards: BSA 66 kDa; Carbonic Anhydrase 29 kDa; Myoglobin 18 kDa; Ribonuclease A 13.7 kDa and Aprotinin 6.5 kDa. C) Sedimentation velocity experiment to determine the size distribution of YfiNHAMP-GGDEF in solution. The sedimentation coefficient (S) was 2.3 for 98% of the protein, consistent with a molecular mass of 21 kDa, and indicating a monomeric state of YfiNHAMP-GGDEF in solution. D) The YfiNHAMP-GGDEF , the results of the SEC analysis indicates that the two domains of the protein are mobile, thus displaying a large hydrodynamic volume. On the contrary, YfiNGGDEF displays an apparent molecular mass consistent with a monomer, as illustrated in the scheme.(TIF)Click here for additional data file.Movie S1Normal Modes Analysis on YfiN model.The animation illustrates the rigid parts of YfiN the hinges connecting them, together with the direction of the fluctuation of each residue in the slowest two modes as predicted by the server HingeProt [ingeProt . Two ort(MOV)Click here for additional data file.