Diictodon feliceps. Distorted Diictodon crania can be categorized into seven typical styles of deformation: lateral compression, dorsoventral compression, anteroposterior compression, “saddle-shape” deformation , anterodorsal shear, anteroventral shear, and right/left shear. In simulated morphometric datasets incorporating known “biological” signals and subjected to uniform shear, deformation was typically the main source of variance but accurate “biological” information could be recovered in most cases. However, in empirical datasets, not only was deformation the dominant source of variance, but little structure associated with allometry and sexual dimorphism was apparent, suggesting that the more varied deformation styles suffered by actual fossils overprint biological variation. In a principal component analysis of all anomodont therapsids, deformed Diictodon specimens exhibit significant dispersion around the “true” position of this taxon in morphospace based on undistorted specimens. The overall variance associated with deformation for Anomodontia as a whole is minor, and the major axes of variation in the study sample show a strong phylogenetic signal instead. Although extremely problematic for studying variation in fossil taxa at lower taxonomic levels, the cumulative effects of deformation in this study are shown to be random, and inclusion of deformed specimens in higher-level analyses of morphological disparity are warranted. Mean morphologies of distorted specimens are found to approximate the morphology of undistorted specimens, so we recommend use of species-level means in higher-level analyses when possible.Taphonomic deformation, the distortion of fossils as a result of geological processes, poses problems for the use of geometric morphometrics in addressing paleobiological questions. Signal from biological variation, such as ontogenetic trends and sexual dimorphism, may be lost if variation from deformation is too high. Here, we investigate the effects of taphonomic deformation on geometric morphometric analyses of the abundant, well known Permian therapsid  Geometric morphometrics is a well-established tool for addressing biological questions related to shape . The disTaphonomic deformation includes all sources of postmortem shape change in a biological structure, but is mostly used to refer to distortion as a result of geological processes in the surrounding rock . AlthougFor geometric morphometric analyses in particular, it is currently uncertain to what degree taphonomic deformation can “overwrite” the underlying signal of biological shape variation. Does distortion obscure variation intraspecifically, interspecifically, or even at higher clade levels? To address these questions, study systems comprising taxa represented by large sample sizes known from a variety of localities and stratigraphic intervals are needed. Although a frequent target for morphometric and other disparity-based analyses, few groups of terrestrial vertebrates satisfy these criteria. A noteworthy exception, however, can be found in the non-mammalian Synapsida.Non-mammalian synapsids are one of the major groups of Paleozoic terrestrial vertebrates, and dominated ecosystems in both abundance and richness from the Pennsylvanian through the Middle Triassic . Non-mamAlthough a number of synapsid species are represented by large sample sizes ranging into the hundreds of specimens , the groGiven these potential difficulties, it is important to address certain questions regarding taphonomic deformation in morphometric studies, focusing here on the synapsid fossil record as a case study. For example, is deformation of synapsid specimens random, and if not, does the directionality of deformation change with geographic and/or stratigraphic position? Do deformed specimens alter the structure of morphospace within groups and/or between groups? Does the inclusion of numerous deformed specimens significantly alter measures of disparity in a study group? Is biological variability overwhelmed by taphonomic variability, and if so, at what taxonomic levels?Diictodon feliceps. We have four main objectives:Diictodon. The range of variation attributable to deformation in Diictodon crania will be described and the possible effects of this deformation on morphometric analysis discussed.Description of the major forms of deformation in the cranium of Diictodon will be constructed using the results of a principal components analysis of cranial landmark data, and variation in this morphospace as related to biological and deformational variables will be examined. The null hypotheses are that deformation is random and that random deformation adds noise to the data but does not overwhelm known biological signals.Test the effects of deformation on intraspecific morphospace analyses. An empirical morphospace for Diictodon fossils; Use simulations featuring known types and amounts of deformation to investigate the circumstances when deformation is likely overwhelming our ability to extract biological signal from a dataset. We will focus on correctly recovering signals reflecting ontogenetic variation and sexual dimorphism  does not overwhelm interspecific sources of morphological variance.Test the effects of deformation on morphospace analyses at higher taxonomic levels. Samples of deformed and undistorted Here, we address these questions using a combination of simulation and empirical analyses centered on the abundant dicynodont therapsid Diictodon feliceps  offers the opportunity to address the potential problem of changing styles of deformation as a result of changes in the sedimentary geology of the Karoo Basin over the course of the Permian . Additionally, a nearly complete ontogenetic series is known for this taxon, ranging from probable hatchlings  with total skull lengths measuring less than two cm to large adults  with skulls nearly 15 cm in total length. Finally, Diictodon has been the subject of intense taxonomic scrutiny over the past two decades. Several studies based on qualitative comparisons as well as traditional and geometric morphometric analyses have concluded that Diictodon represents a single morphospecies, D. feliceps  were examined for this study. Of these specimens, 485 were complete enough to have landmarks digitized on at least one side of the skull in dorsal view, with 387 of those complete enough for a bilaterally-symmetric configuration of dorsal landmarks, and 464 specimens were complete enough to have landmarks digitized in lateral view . Specimen images were digitized using ImageJ and TPSDig  antWe analyzed two permutations of the dorsal view dataset. In the first dataset , we reflected bilaterally symmetric landmarks across the midline and averaged the positions of the resulting pairs of landmarks to create a series of “half specimens.” In cases where a symmetric landmark was not preserved on one side of the skull, the coordinates of the single preserved landmark were used. This approach follows common practice for dealing with incomplete specimens in paleontological datasets, but the reflecting and averaging process can affect both the biological and taphonomic signals in a dataset including distorted specimens  . TherefoFor the lateral view, we digitized 11 landmarks : (1) AntDiictodon , the tusks are just erupting—all known smaller skulls are tuskless. Because the sex of these presumed juveniles cannot be determined, they were excluded from analyses using this variable. The specimens excluded from analyses of sex make up the “small” size class, consisting of all specimens with a total skull length less than 5 cm. The “medium” size class consisted of specimens with total skull length ranging between 5 and 9 cm, and the “large” size class consisted of specimens with total skull length in excess of 9 cm. Assemblage zone data were available for 419 specimens used in the dorsal analysis, 337 specimens used in the dorsal complete analysis and 391 used in the lateral analysis. Assemblage zone data were derived from specimen labels, Tropidostoma AZ, with 216 dorsal, 178 dorsal complete, and 208 lateral landmarked specimens. The second largest sample is from the Cistecephalus AZ, with 112 dorsal, 89 dorsal complete, and 102 lateral. From the Tapinocephalus AZ there are 47 dorsal, 37 dorsal complete, and 40 lateral. From the Daptocephalus AZ there are 24 dorsal, 16 dorsal complete, and 22 lateral. Lastly, from the Pristerognathus AZ there are 20 dorsal, 17 dorsal complete, and 19 lateral specimens. Deformation style was determined qualitatively a priori (see “Patterns of Deformation” below). Many Diictodon skulls are subject to multiple forms of distortion, in which case the dominant style of deformation was given precedence for binning. Undeformed specimens were recognized based on consistent cranial symmetry, uniformity of outline in easily distorted structures like the orbits and foramen magnum, and sutural integrity .Specimens were grouped by four variables: sex, size class, assemblage zone (AZ), and deformation style . Landmark protocol for this analysis was identical to that for the within-group Diictodon study, with “dorsal complete” landmarking used for skulls in dorsal view. Anomodont specimens were binned into the higher-level taxa described by Procrustes superimposition and principal components analysis (PCA) of landmark data were performed using the program MorphoJ . MeaningDiictodon datasets. In contrast, the analyses focusing on biologically relevant variables  were carried out using only the specimens in the “undeformed” class from each dataset. When a variable was determined to be significant, pairwise post-hoc tests were carried out to determine whether significant differences in mean shape existed among the classes. Significance was judged at a Bonferroni-corrected alpha level that was appropriate for the number of comparisons.Procrustes ANOVAs were carried out in geomorph  to assesA series of simulations was conducted using the program DefCat  and because landmarks could be digitized on it in each view. For the dorsal complete dataset, the “large” specimen was SAM-PK-K6041 (skull length approximately 10.1 cm). The “large” specimen in both the dorsal and lateral datasets was USNM 25157 . DefCat was then used to generate as many simulated specimens as we had empirical specimens. This meant 485 dorsal and 464 lateral simulated specimens generated within each simulation. This evenly divides the size and shape differences between the endpoint specimens, creating a simulated ontogenetic series of undeformed specimens. A small amount of identical independent Gaussian noise was added to the data to simulate individual variation among the specimens.To make the simulated dataset with an ontogenetic signal, two undistorted Diictodon. Following the hypothesis of Diictodon are likely male and tuskless individuals are likely female, two undeformed specimens of either sex were used to create a series of simulated specimens that represent only one sex or the other. For the dorsal complete datasets, BP/1/293 and SAM-PK-K1650 were chosen to represent males, and BSPG 1934-VIII-48 and UCMP 42837 were chosen to represent females. For the dorsal view datasets, NHMUK PV OR 47052 (the holotype of Diictodon feliceps) and SAM-PK-K1650 were chosen to represent males, and BSPG 1934-VIII-48 and TM 299 were chosen to represent females. For the lateral view datasets, NHMUK PV OR 47052 and TM 373 were chosen to represent males, and BSPG 1934-VIII-48 and SAM-PK-K11484 were chosen to represent females. In each case, the number of simulated specimens reflect those in each category for the empirical dataset, resulting in 251 simulated females and 220 males for the dorsal analysis, 194 females and 179 males for the dorsal complete analysis, and 238 females and 214 males for the lateral analysis. A small amount of identical independent Gaussian noise was added to the data to simulate individual variation among the specimens.The second series of simulated datasets was created to test the effects of deformation on sexual dimorphism. Here, two datasets were created using sets of either likely male or likely female specimens, so that each dataset would be representative of either male or female Diictodon (see below), it provides an intuitive starting point for investigating the potential effects of deformation on morphometric data for the species. The deformation model has two main parameters, θ and a. The θ parameter alters the ratio of the long axis to the short axis of the strain ellipse, whereas the a parameter varies the strain ellipse ratio and the orientation of the strain ellipse . Groups 4 and 5 had the same parameters as Group 1 except that the orientation of the specimens relative to the strain ellipse was constrained . Groups 6 and 7 had the same parameters as Group 1, but deformation amplitude (a) was allowed to vary . All parameters were allowed to vary in Groups 8 and 9  of a distorted and undistorted specimen in the same orientation. These differences are caused by deformation-induced changes in angulation of various structures in the distorted specimen, resulting in landmarks that appear more distant or closer together in the digitized image than they are in the actual specimen. Each major style of deformation incurs both kinds of artifacts, the details of which are described below.Although the amount of distortion in ormation . Most DiDiictodon skull are illustrated in Diictodon skull and generally display little change in compressed skulls, but extreme dorsoventral compression can cause these structures to bend anteriorly.The typical styles of deformation in the More unusual styles of deformation are shown in USNM 22948  represenIn addition to compression, various forms of deformation attributable to shear are observed in the study sample. Most distorted dicynodont specimens exhibit some degree of asymmetry as a result of shear . For lateral compression, the primary positional artifacts in dorsal view are medial movement of the set of landmarks along the lateral skull surface , bringing them physically closer to the midline landmarks 8, 9, and 10. Greater proximity of landmarks along the ventral margins of the orbit and temporal fenestra  to those on the dorsal margins  is also observed, but this can be attributed largely to perspective artifacts related to verticalization of the postorbital bar and orbit. For example, in lateral view, landmarks 4/13 and 6/15 of a laterally compressed skull are separated by either the same distance as in undistorted skulls or a slightly greater distance , but in dorsal view the straightening of the postorbital bar puts these landmarks in nearly the same plane , sharply decreasing the distance between them in the projected image.For dorsoventral compression, most of the changes in landmark position are the opposite of those for lateral compression , but with some peculiarities. In lateral view, greater proximity between the set of landmarks along the base of the skull  and those higher up  in projected images represents a combination of artifacts of perspective  and position . Anterodorsal displacement of landmarks 2 and 4 is also sometimes observed, in the extreme cases where the tusk or caniniform process has bent anteriorly.As noted above, “saddle-shape” deformation results in decreased intertemporal length and increased snout length in dorsal view (artifacts of perspective), bringing the midline landmarks either closer together (9 relative to 10) or further apart (8 relative to 1) than in an undistorted skull. In lateral view, the most affected landmarks are those around the dorsal margin of the orbit , which become displaced ventrally as a result of the collapse of the skull roof and postorbital bar. For anteroposterior compression, the primary change in both dorsal and lateral views is reduction in distance between all landmarks along the long axis of the skull. In lateral view, the skull also appears taller than in undistorted specimens , resulting in greater separation between ventral  and dorsal  landmarks.Anterodorsal shear results in a shorter observed snout in dorsal view , but no change in observed length of the intertemporal bar . In lateral view, dorsal landmarks  are generally displaced anteriorly relative to the ventral  ones. The opposite is the case for anteroventral shear. Left/right shear results in variable landmark movement in lateral view, depending on which side of the skull is being digitized. Dorsally, it results in displacement of the pairs of symmetric landmarks on opposing sides of the skull relative to one another, a result only captured in the dorsal complete analysis.Although they account for just 49.8% of the cumulative variance in the data set, only the first two lateral PC axes contain meaningful structure under the broken-stick criterion. PC1 accounts for 26.6% of variance in the lateral dataset. This PC describes relative skull height, snout depth, and angulation of the temporal region . SpecimeDeformation style is the only variable that shows clear structure in the PC1 vs. PC2 morphospace . UndistoOnly the first two PC axes derived from the dorsal data set contain significant structure under the broken-stick criterion, and together they account for 58.3% of cumulative variance in the data. PC1 accounts for 38.4% of variance in the dorsal data. This PC describes snout length and position of the postorbital bar . SpecimeAs with the lateral data, the only variable showing clear structure in PC1 vs. PC2 morphospace is deformation style . DirectiOnly the first two PC axes derived from the dorsal complete data set contain significant structure under the broken-stick criterion, and together account for 47.0% of the variance in the data. PC1 accounts for 29.1% of the variance in the dorsal complete data. PC1 and PC2 both describe information about length and breadth of the skull, though in different ways. PC1 describes differences in anteroposterior length, showing changes associated with shortening of the back of the skull for specimens with high positive scores . It alsoDaptocephalus AZ do tend to mostly have higher values on PC1 and lower values on PC2, but they are broadly overlapped by specimens from the other assemblage zones on these axes.Like the lateral and dorsal data, the predominant variable showing clear structure in PC1 vs. PC2 morphospace for dorsal complete is deformation style . Lateralhttps://doi.org/10.5061/dryad.5tb2rbp1x). Only size class was found to be significant in the dorsal (p = 0.021) and dorsal complete (p = 0.048) datasets (undeformed specimens only), although sex and AZ approached significance in some cases . The pairwise comparisons revealed that the different deformation styles usually have significantly different mean shapes , and also differ significantly from the undeformed specimens in the majority of cases , even under the rather stringent Bonferroni-corrected alpha level of 0.002. The cases where the mean shapes did not significantly differ are generally logical when one considers the way in which the deformations styles tend to alter specific aspects of shape. For example, laterally compressed and right/left sheared specimens do not have mean shapes that differ significantly from undeformed specimens in the lateral view datasets because these deformation types tend to have relatively mild effects on shape when viewed from the side. Among the pairwise comparisons for the size classes, only medium and small specimens in the dorsal complete dataset had a significantly different shape at the Bonferroni-corrected alpha of 0.02.Deformation style was found to explain a significant amount of variation in all three empirical datasets, but significant results were rarer for the biologically relevant factors , however.The results of the all-anomodont lateral PCA including deformed ed above . Deformee on PC1 . Procruse on PC1 ) does noD. feliceps specimens, the first four PC axes account for 71.1% of cumulative variance in the dataset. The first three PC axes show strong phylogenetic signal based on qualitative observation of the morphospace. PC 1 accounts for 39.0% of variance in the data and describes relative skull length . Procrustes variance for Anomodontia as a whole does not change significantly whether only undistorted or all Diictodon specimens are included, however (0.2741 vs. 0.2702 respectively).Including deformed We calculated the morphological disparity present in each of our simulated deformed datasets and mapped it to observed disparity in the empirical datasets. For the first four sets of parameters in the dorsal and lateral datasets, and the first five in the dorsal complete dataset, disparity in the simulated datasets was lower than in the empirical datasets at low values of θ and higher at high θ values. However, simulated disparity was close to empirical at values of θ in the 7.0–15.0 range, with the greatest similarities usually in the range of 8.0–10.0. These simulated datasets did not show significantly different disparity from the observed empirical datasets. For parameter sets 6–9 simulated disparity was always higher than in the empirical datasets. We consider the simulated datasets with disparities close to empirical levels to be the most relevant for further tests of whether we can accurately recover biological signals from datasets including deformed specimens, with simulated datasets with higher or lower disparity levels representing “worst case” and “best case” scenarios, respectively.In the trajectory analysis, each simulated deformed dataset was compared to its simulated non-deformed counterpart. Specifically, we considered three parameters of the ontogenetic trajectories: (1) their lengths (trajectory size); (2) their orientations in relation to each other ; and (3) the paths their trajectories take through shape space (trajectory shape) .For the majority of the datasets, there was no significant difference between the ontogenetic trajectories of the deformed and non-deformed datasets. The aspect that showed the largest difference between trajectories was trajectory size. In the lateral datasets, 6.7% of the datasets showed significant differences in disparity between empirical and simulated datasets. In the dorsal datasets, 9% showed significant differences, and in the dorsal complete datasets, 5.8% showed significant differences. Combined, 21.5% of the datasets showed significant differences in trajectory size between the empirical and simulated datasets, and 78.5% showed no significant difference. In the lateral datasets, differences were most often found between θ 1.0 and 2.5 and above θ 6.0, but was less common between θ 2.5 and 6.0. Differences were found for each parameter group except for parameter group 2, and were most often seen in groups 6–9. In the dorsal and dorsal complete datasets, differences were found for each parameter group except for group 5. Just as with lateral, differences were most often found in parameter groups 6–9.The second most common difference was trajectory shape. In the lateral datasets, 4.6% of the data showed significant differences between the non-deformed and deformed trajectory shapes. In dorsal, 3.5% showed significant differences, and in dorsal complete, 4.4% showed significant differences. In total, 12.5% of the data showed significant differences in trajectory shape, with 87.5% showing insignificant differences. In the lateral datasets, differences were found for parameter groups 3 and 5–9, but not 1, 2, or 4. For dorsal, differences were found in parameter groups 4 and 6–9, but not 1–3 or 5. In dorsal complete, differences were found in all parameter groups except group 2. As with trajectory size, trajectory shape showed most prevalent difference in parameter groups 6–9.Significant differences in trajectory orientation were very uncommon. There were only three instances of significant differences in trajectory orientation in the lateral view datasets , two instances in the dorsal view datasets (θ 3.5 in parameter set 8 and θ 10.0 in parameter set 9), and none in the dorsal complete datasets.Diictodon dataset.The analytical procedure for the homogeneity of slopes test was the same as for the ontogenetic trajectory analysis, with the slope of the deformed dataset being compared to that of its non-deformed counterpart. The results of the analysis were similar to those of the trajectory analysis, with significant differences in slope rarely occurring between simulated deformed and non-deformed datasets. In the lateral datasets, significant differences occurred only 3 times. In dorsal, significant differences occurred 10 times, and in dorsal complete, significant differences occurred 11 times. In total, significant differences were found in 5.5% of all of the datasets, and there was no significant difference in slope for 94.5% of the datasets. There was no real trend for the thetas at which these differences would occur, but it was more common in parameter groups 5–9 in the dorsal and dorsal complete datasets. Taken together, these results imply that it may be possible to recover an accurate ontogenetic signal from therapsid datasets that include deformed specimens, even in cases where the amount of disparity contributed by deformation exceeds what is observed in the empirical Our analysis of the effects of deformation on a sexual dimorphism signal focused on whether we could recover a significant difference in shape between sets of simulated deformed male and female specimens. The results of our Procrustes ANOVAs comparing deformed male and female datasets were very straightforward: a significant difference between males and females was preserved in all of the deformed datasets, regardless of the starting deformation parameters. This finding indicates that it may be possible to identify instances of sexual dimorphism in therapsid morphometric datasets even when deformed specimens are included.Diictodon crania strongly indicate that deformation is the dominant source of morphological variance in the lateral, dorsal, and dorsal complete empirical datasets. The Procrustes ANOVA results corroborate these observations, with deformation always having a significant effect, and deformation styles having significantly different mean shapes in the majority of cases. Deformation style exhibits considerable structure in PC1 vs. PC2 morphospace, with almost all major styles of deformation occupying a characteristic region of morphospace. Left/right shear is the sole exception to this pattern, with specimens more evenly distributed in morphospace. This likely stems in part from averaging of symmetric landmarks during the construction of the lateral and dorsal datasets, which is expected to remove some effects of shear deformation  of the bones in question. Although architectural properties of elements clearly have some influence on proclivity to distortion and the effects thereof  exhibit seemingly random occupation of morphospace. Diictodon skulls in lateral view using a smaller sample of more or less undeformed specimens and a slightly different configuration of landmarks in lateral view. The absence of an obvious size-shape relationship in the empirical data here suggests that it was overprinted by deformation, mirroring the conclusion of Psittacosaurus dataset did not preserve an original allometric signal. It also appears that size class and sex have only a weak effect on overall skull shape in Diictodon under even the best circumstances. These factors typically explained only about 10–15% of the shape variance among the undeformed specimens in our datasets.In contrast to the strong signal from deformation style, Daptocephalus AZ specimens displayed lower scores on PC2 for the dorsal and dorsal complete datasets, and mostly higher scores on PC1 for the dorsal complete dataset. These positions can be accounted for by the absence of any anterodorsally sheared specimens in the Daptocephalus AZ sample. We interpret the lack of anterodorsal deformation among Daptocephalus AZ specimens to be an artifact of small sample size, because anterodorsal shear is one of the rarest styles of deformation , and the Daptocephalus AZ is the second smallest sample. Moreover, several specimens of the Daptocephalus AZ index taxon Daptocephalus leoniceps clearly exhibit anterodorsal shear , allowing us to reject a change in basin-wide deformation style as the source of the pattern. The morphometric heterogeneity of specimens from the different assemblage zones provides some corroboration of the hypothesis that only a single species of Diictodon (D. feliceps) is present in the Karoo Basin  has been greatly expanded by the deformed specimens, with especially troubling implications for between-group measures of disparity at lower taxonomic levels.Anomodont morphospace is dominated by phylogenetic signal in the sense that specimens belonging to the higher-level clades used in binning tend to cluster together. However, the relationships between these clades , the likelihood of anomodont singletons preserving the undistorted cranial morphology of their species is low. Nevertheless, our morphospace results indicate that even if a deformed specimen does not occur precisely where it would if it was not deformed, the majority of deformed specimens still fall in the region occupied by their larger clade. This result is encouraging for analyses that seek to maximize taxonomic inclusivity. It is notable, however, that even at this broad scale, some variation due to taphonomic overprinting can be discerned, with PC4 from the all-anomodont dorsal complete analysis being related to specimen shear.Given the strong apparent phylogenetic signal in anomodont morphospace, the inclusion of deformed taxonomic singletons is warranted in clade-level analyses of morphological disparity. Because undistorted specimens are so rare . This pattern was observed in a number of undistorted skulls in association with other features, suggesting that it is not the result of a taphonomic overprint. In a sample of only dorsoventrally or laterally compressed skulls, however, this pattern would not be discernible with confidence, given the strong influence of these deformation styles on skull breadth.Like the empirical datasets, deformation increased the disparity of the simulated datasets. However, in most or all cases we were able to recover an accurate (simulated) ontogenetic or sexual dimorphism signal from the simulated datasets, whereas there was no obvious structure associated with these factors in the empirical datasets that included deformed specimens. A possible explanation for this difference in the empirical and simulation results is the fact that the simulations utilized only a single kind of deformation (shear), in contrast to the multiple types of deformation experienced by the empirical specimens. The latter case might overprint the shapes of specimens to a greater degree than a more stereotyped deformation style, even if the various deformation styles tend to segregate into specific regions of morphospace. Therefore, although the simulation results raise the possibility that it might be possible to recover accurate biological signals from datasets including deformed specimens, this likely will be difficult in practice. One potential approach for trying to extract biological information from deformed specimens would be to conduct the analyses on groups of specimens sharing a similar deformation style, which might result in datasets more similar to our simulated data. Somewhat counterintuitively, it seems this is a case where a more restricted sample is more likely to recover biological signal. However, even a restricted sample could be misleading if the direction of deformation overlaps with the biological signal. As an example, Diictodon , so we do not think we have seriously mischaracterized the biological signals included in the simulated datasets. Instead, we consider it more likely that better preserved biological signals in the simulated deformed datasets is a reflection of the simpler style of deformation applied in the simulations.Alternatively, given that deformation in both our simulated and empirical datasets seems to add variance in a roughly even fashion in all directions in morphospace, it may be that our simulated datasets have clearer biological signals than would be the case in the empirical data even if deformation was absent. We used empirical specimens as the starting points for our simulated datasets, but the specimens chosen were relatively extreme shapes , which could result in an exaggerated “biological” signal. However, the simulated ontogenetic signal in our lateral view dataset is quite similar to that documented by Diictodon is the single most common dicynodont in the Permian rocks of the Beaufort Group in the Karoo Basin . Therefore, we recommend caution in intraspecific analyses of variation in non-mammalian synapsids, as differences interpreted as biological variation may simply represent deformational artifact. This concern may be broadly applicable for fossil taxa, as demonstrated by other empirical and theoretical analyses (Distortion of analyses . Less ca


Porphyromonas gingivalis, the major human pathogen associated to periodontal diseases, utilizes the Bacteroidetes-specific type IX secretion system (T9SS) to export virulence factors. PorE is a periplasmic multi-domain lipoprotein associated to the outer membrane that was recently identified as essential for T9SS function. Little is known on T9SS at the structural level, and in particular its interaction with peptidoglycan. This prompted us to carry out structural studies on PorE full length as well as on its four isolated domains. Here we report the crystal structure of the C-terminal OmpA_C-like putative peptidoglycan-binding domain at 1.55 Å resolution. An electron density volume was identified in the protein cleft, making it possible to build a naturally-occurring peptidoglycan fragment. This result suggests that PorE interacts with peptidoglycan and that PorE could anchor T9SS to the cell wall. Porphyromonas gingivalis is a pathogen highly associated with periodontal diseases. P. gingivalis can locally invade periodontal tissues, disrupt the tooth-supporting structure and evade the host defense mechanisms. Importantly, several studies have revealed that there are cause-and-effect relationships between periodontal diseases and rheumatoid arthritis, heart disease, diabetes, Alzheimer and other systemic diseases7. Tissue damages caused by P. gingivalis infection are mainly induced by a cocktail of specialized toxin proteins secreted by the bacterium, the gingipains8. Several studies have demonstrated that P. gingivalis employs the Type IX secretion system (T9SS) to translocate gingipains across the outer membrane (OM)10. Therefore, it is important to gather structure/function data on T9SS to fight P. gingivalis virulence.Periodontal disease, or periodontitis, is a chronic inflammatory disease that destroys periodontal tissue and alveolar bone, representing a major public health concern. The Gram-negative anaerobic bacterium Fibrobacteres-Chlorobi-Bacteroidetes superphylum (CFB group). It has been mainly studied in the oral pathogen P. gingivalis and in the gliding bacterium F. johnsoniae. The P. gingivalis T9SS recruits and delivers virulence factors, such as gingipains, to attack to the host periodontal cells, while the F. johnsoniae T9SS secretes adhesins that promote gliding motility. Effectors secreted by the T9SS use a two-step mechanism. Substrate proteins have cleavable N-terminal signal peptides that target them first to the Sec translocon for export across the inner membrane (IM). They also harbour a conserved carboxy-terminal domain (CTD) comprising approximately 80 residues that is necessary for recruitment to and transport across the OM by the T9SS11. T9SS is composed of at least 14 different proteins. Worth noticing, the location of the P. gingivalis T9SS subunits have been determined within the cell envelope: two are IM embedded proteins (PorL and PorM), six are OM embedded proteins , five are OM-associated proteins  and one is a periplasmic protein (PorN)19. However, few is known at the structural level. It has been shown by cryo-electron microscopy (cryo-EM) that PorK and PorN polymerize to form a large ring-shaped complex20. Recently, the crystal structures of the periplasmic domain of PorM from P. gingivalis and its homologue GldM from F. johnsoniae have been solved21, proposing a schematic model of their function. Finally, the cryo-EM structure of the SprA protein (Sov homolog in F. johnsoniae) was solved in complex with its partner proteins, PorV, PPI (lipoprotein peptidyl-prolyl cis-trans isomerase) and Plug protein22. The structure showed that SprA folds as a large 36-strand single polypeptide transmembrane β−barrel. This β−barrel is gated by a plug and displays a lateral opening that might be important for effector translocation.T9SS is present in the PorE, initially named PG1058 (Uniprot code F5HF80)23. PorE locates in the periplasm and is anchored to the inner leaflet of the OM through its lipid modification17. In the same study, it was shown that PorE is essential for T9SS function. Indeed, the porE mutant in P. gingivalis displays phenotypes characteristic of non-functional T9SS: defect in colonial pigmentation, defect in surface-associated proteolytic activity and absence of a visible electron dense surface layer (EDSL) containing gingipains and other substrate proteins by cryo-EM17. Sequence analysis and computer modelling indicated that PorE could be divided into four domains: i) a tetratricopeptide repeat domain ; ii) a β–propeller domain ; iii) a carboxypeptidase regulatory domain-like fold  and iv) an OmpA_C-like putative peptidoglycan-binding domain (residues 534–668). The TPR and WD40 domains present at the N-terminus of PorE are structural scaffolds often involved in protein-protein interactions25, suggesting that PorE may have a role as an essential scaffold linking the periplasmic and OM components of the T9SS. The OmpA-like C-terminus domain is similar to peptidoglycan binding proteins in other bacteria, suggesting that PorE N- to C-terminus could establish a bridge between the OM and the peptidoglycan.In 2016, a new T9SS subunit was identified, L-Ala-γ-D-Glu-meso-DAP-D-Ala-D-Ala is the common pentapeptide, in which DAP represents diaminopimelate, a unique bacterial amino acid27. It was shown only recently that P. gingivalis peptidoglycan contains meso-DAP and not the stereochemical variant LL-DAP as believed for a long time29. PG is located between the IM and OM of gram-negative bacteria. The OmpA family proteins have been proposed to interact noncovalently with PG30. There are three different groups in the OmpA family proteins: OM proteins , PG-associated lipoproteins , and proteins associated with large machineries 34. While OM proteins and PG-associated lipoproteins connect the OM with the PG network and hence stabilize the OM, proteins associated with multiprotein machineries usually help to proper position and immobilize the apparatus35.Peptidoglycan (PG) is the major component of the bacterial cell wall and shapes the cell by giving structural strength. PG is a polymer consisting of repeating disaccharide units of N -acetylglucosoamine (NAG) and N-acetylmuramic acid (NAM). A PG strand is cross-linked to the other neighbouring PG strands through a pentapeptide stem, which is appended to the NAM moiety. In gram-negative bacteria, P. gingivalisPorE T9SS-associated lipoprotein. The full-length mature PorE protein and its four truncated domains were cloned, produced and one domain was crystallized. The crystal structure of the OmpA_C-like putative peptidoglycan-binding domain was solved in complex with a part of the naturally-occurring peptidoglycan peptide stem, bound to each monomer of the tetrameric arrangement in the asymmetric unit.In this study we focused on the PorE as well as the four isolated domains defined by Heath et al.17. The full-length protein and the β–propeller (WD40) domain were insoluble. The production yield of the tetratricopeptide repeat (TPR) domain was very low. By contrast, the carboxypeptidase regulatory domain-like fold (CRD) and the OmpA_C-like putative peptidoglycan-binding constructs were produced in high yield and were soluble.We attempted to produce the full length PorE OmpA_C-like domain was solved by single-wavelength anomalous diffraction (SAD) at 1.55 Å resolution . However, this oligomerization state results from a crystallization artefact as Size-Exclusion Chromatography Multi-Angle Light Scattering (SEC-MALS) analysis indicated that the protein in solution is monomeric with a molecular weight of 17.7 kDa  consists of a three-stranded β-sheet (β1-3) and five α-helices (α1–5) with connectivity α1β1α2β2α3α4α5β3 .The structure of the OmpA_C-like domain of β-sheet β-3 and fiE. coli OmpA36 or Neisseria meningitides RmpM37.To note, the structure of the OmpA_C-like domain features a relatively large bulge containing the two small α-helices, α3 and α4, which are not connected by a disulfide bond as it is the case in some other well-known peptidoglycan-associated proteins, for examples, L-Ala-γ-D-Glu-meso-DAP-D-Ala  are conserved among OmpA family proteins, such as the OmpA and Pal proteins from Bacteroidetes phylum. To fit the specific needs of each bacterium, T9SSs have evolved to secrete various substrates such as toxins involved in pathogenesis, S-layer to confer protection, or adhesins for gliding motility. A number of researches have shown that more than 14 proteins are involved in this still ambiguous process, with some playing roles in the post-translational modification of T9SS substrate proteins9.Secretion systems in Gram-negative bacteria are large multiprotein complexes comprising proteins from the IM and OM. In this study we focus on the last discovered type IX secretion system (T9SS), a complex translocon only found in species of the et al.17 have shown that porE inactivation in P. gingivalis caused a loss of cell-associated proteinase activity, while immunoblotting and proteomic analyses revealed that virulence factors precursor proteins accumulated in the periplasm and were not targeted to the membrane. This provided strong evidence that PorE is a component of the T9SS in P. gingivalis essential for the proper secretion of T9SS substrates. Furthermore, sequence analysis revealed that PorE is a lipoprotein anchored in the OM through N-terminal acylation, and is composed of four domains, from N to C-terminal: a tetratricopeptide repeats domain, a five bladed β-propeller domain, a predicted carboxypeptidase regulatory domain-like fold, and an OmpA_C-like domain. The C-terminal domain was predicted to be a peptidoglycan binding protein, suggesting that PorE could connect the OM and the peptidoglycan mesh.Heath PorE reported here shows strong three-dimensional evolutionary relationship with known OmpA_C-like domains found in other bacteria. Moreover, a fragment of PG could be built into the electron density. This fragment, containing four residues of the stem peptide (L-Ala-γ-D-Glu-meso-DAP-D-Ala), penetrates deeply in the PG binding groove. Indeed, PG binding in PorE is very similar to that has already been described for other PG binding domain within the OmpA_C-like family. Thus our work brings experimental evidence that PorE interacts with the stem peptide that is present in the PG of P. gingivalis, which suggests that PorE could serve as a bridge between the OM and the PG network  to confirm that PorE could anchor T9SS to the cell wall.The structure of the C-terminal domain of ork Fig. . InteracPorE are present in 174 species of the Bacteroidetes phylum17. In many species, including P. gingivalis, PorE is unique. For instance, a search with the BLAST program in the genome of Duncaniella muris, a species highly abundant in the murine intestinal tract38 and that potentially possesses a functional T9SS as PorK-L-M-N homologs are present in its genome, results in one PorE homolog. Indeed, this protein shares the same architecture as PorE (46% sequence identity with 98% sequence coverage), it shares the three main residues involved in PG binding, suggesting that it actually interacts with PG, and it is predicted to be a lipoprotein. On the other hand, multiple PorE homologs are present in some species. For instance, fourteen PorE homologs are present in the genome of Tannerella forsythia, another periodontal pathogen39 that potentially possesses a functional T9SS . Noteworthy, more than twenty PorE homologs are present in the genome of F. johnsoniae . However, while these proteins have a signal peptide, except one, suggesting that they are translocated in the periplasm as PorE, none is predicted to be a lipoprotein. Further investigations would be necessary to reveal whether one of these F. johnsoniae proteins could have a function in T9SS homologous to PorE. Nevertheless, this result highlights that anchoring of T9SS into the cell wall might be different in the two bacterial models used for T9SS study, P. gingivalis and F. johnsoniae.As previously pointed out, sequences homologous to PorE :188594840) and its four protein domains were cloned using the Restriction Free method40. The full length sequence of PorE except the signal peptide and the first cysteine proposed to be acylated (residues 25–672) was amplified from P. gingivalis ATCC3327 chromosomal DNA using primers 5′-CCGAGAACCTGTACTTCCAATCAAAGAGCGTGAAGTTGAAAGATGCGG and 5′- CGGAGCTCGAATTCGGATCCTTATTATTAACGCAACTCTTCTTCGATCAC. The sequences of PorE individual domains starting from the N-terminus to C-terminus: i) tetratricopeptide repeat domain , ii) β–propeller domain , iii) carboxypeptidase regulatory domain-like fold , and iv) OmpA_C-like putative peptidoglycan-binding domain  were also amplified from P. gingivalis ATCC3327 chromosomal DNA. All constructs were cloned into the pLIC03 expression vector that is a pET-28a+ derivative (Novagen) carrying a cassette coding for a His6 tag and a Tobacco Etch Virus (TEV) protease-cleavage site followed by the suicide gene sacB flanked by BsaI restriction sites downstream the start codon.The full length 41. E. coli T7 transformants were grown in TB medium at 37 °C until reaching an Optical Density at λ = 600 nm (OD600nm) of 0.6–0.8. The expression was induced by adding 1 mM IPTG and the bacterial growth was pursued for 18 h at 17 °C. The cells were harvested by centrifugation at 2,500 × g for 15 min at 6 °C and the dried pellet was stored at −80 °C. After thawing, the cell pellet was resuspended in lysis buffer  and the cells were lysed by sonication on ice. The pellet and soluble fractions were separated by centrifugation (16 000 g for 30 min). The His6-tagged protein was purified from the soluble fraction by immobilized metal ion-affinity chromatography using a 5 ml HisTrap Crude  Ni2 + -chelating column equilibrated in buffer A . The protein was eluted with buffer A supplemented with 250 mM imidazole and was further purified by size-exclusion chromatography  equilibrated in 10 mM HEPES pH 7.4, 200 mM NaCl. For crystallization trials, the purified protein was concentrated by centrifugation to 35 mg.mL−1 using an Amicon 10 kDa cut-off concentrator.Protein expression was carried out as previously described−1, using an Ultimate 3000 HPLC system (Fischer Scientific). Detection was performed using an eight-angle light-scattering detector  and a differential refractometer .For the Size-Exclusion Chromatography Multi-Angle Light Scattering (SEC-MALS) analysis, the purified, concentrated protein was loaded on a Superdex 200 Increase 10/300 GL column  equilibrated in 10 mM HEPES pH 7.4, 200 mM NaCl at a flow rate of 0.6 mL.minE. coli T7 cells cultured in SeMet minimal medium42 at 37 °C until reaching an OD600nm of 0.6–0.8. The expression was induced by adding 1 mM IPTG and the bacterial growth was pursued for 18 h at 17 °C. Cell lysis and protein purification were performed with the same protocol as for native OmpA_C-like domain.Selenomethionine-labeled (SeMet) OmpA_C-like domain was produced in Crystallization screens using the purified samples of native and SeMet OmpA_C-like proteins were performed by the sitting-drop vapour diffusion method at 293 K in 96-well Swissci-3 plates with Stura Footprint (Molecular Dimensions), Wizard I and II (Rigaku) and Structure I and II (Molecular Dimensions) screens. The reservoirs of the Swissci-3 plates were filled by a TECAN pipetting robot, and the nanodrops were dispensed by a Mosquito robot (TTP Labtech). Crystals of OmpA_C-like domain appeared in several conditions with polyethylene glycols and sodium ions. The diffraction quality crystals for both native and SeMet OmpA_C-like domains achieved the maximum size after 7 days by mixing the protein solution with 0.1 M sodium acetate, 0.1 M MES pH6.5, and 30% (w/v) PEG 2000. Crystals were mounted in cryo-loops  and were briefly soaked in crystallization solution supplemented with 20%(v/v) ethylene glycol. The crystals were flash-cooled in a nitrogen-gas stream at 100 K using a home cryocooling device (Oxford Cryosystems).43. The structure of the OmpA_C-like domain was determined by single wavelength anomalous diffraction (SAD) using data collected at the selenium X-ray absorption peak (0.97911 Å) from a selenomethione-derived crystal. The substructure determination for four methionine sites and the phase calculation were performed in Phaser44 as embedded in Phenix45. Model building was performed by the multivariate algorithm in Crank2 that simultaneously combines the structure determination steps, including the scaling and extracting the anomalous signal, finding the heavy atom sites, and solvent flattening46. The initial structure model was improved through iterative refinement with AutoBuster47 and manual refitting with COOT48. The validity of the refined structure was assessed by MolProbity49. Data collection and refinement statistics of the PorE OmpA_C-like domain are reported in Table X-ray diffraction data were collected at Soleil synchrotron Proxima1 beamline and were processed using XDS packageSupplementary information.


Paenibacillus sp. strain URB8-2, isolated from the rhizosphere of wild grass in Kobe, Japan, revealing that this bacterium is related to Paenibacillus rhizophilus 7197, a novel species collected recently in Inner Mongolia, China, and that it possesses two gene clusters for distinct types of nitrogenases.We report here the complete genome sequence of nitrogen-fixing  Paenibacillus sp. strain URB8-2, isolated from the rhizosphere of wild grass in Kobe, Japan, revealing that this bacterium is related to Paenibacillus rhizophilus 7197, a novel species collected recently in Inner Mongolia, China, and that it possesses two gene clusters for distinct types of nitrogenases.We report here the complete genome sequence of nitrogen-fixing  The plates were incubated at 30°C for 5 days to form transparent slimy colonies. Two colonies were randomly selected and streaked onto the nitrogen-free plates in order to isolate independent colonies. The colony exhibiting the most prominent growth was designated URB8-2.We suspected that microorganisms with higher nitrogen-fixing capacity might be present in the weed settlements growing on barren sandy soil. Strain URB8-2 was isolated from the rhizosphere of wild grass from the Kobe University campus in Japan, as described previously , with sohttps://github.com/rrwick/Porechop). Reads with a Q score of <9 and size of <500 bp were discarded using NanoFilt v2.7.0 , SAMtools v1.9 (URB8-2 was grown in tryptic soy broth (TSB)  at 30°C for 48 h with shaking at 200 rpm. The genomic DNA (gDNA) was isolated using conventional phenol-chloroform extraction (ols v1.9 , and minPaenibacillus. The closest strain to URB8-2, sharing 98.89% identity, was P. rhizophilus 7197, which is a novel species isolated in Inner Mongolia, China (Paenibacillus (The findings suggested that URB8-2 belongs to the genus a, China . URB8-2 bacillus .Paenibacillus sp. strain URB8-2 has been deposited in the DDBJ/ENA/GenBank database under the accession number AP023239. The raw sequence data are available under SRA accession numbers DRX225195 and DRX225196.The complete genome sequence of


Porphyromonas gingivalis is a secondary colonizer of the oral biofilm and is involved in the onset and progression of periodontitis. Its fimbriae, of type-V, are important for attachment to other microorganisms in the biofilm and for adhesion to host cells. The fimbriae are assembled from five proteins encoded by the mfa1 operon, of which Mfa5 is one of the ancillary tip proteins. Here we report the X-ray structure of the N-terminal half of Mfa5, which reveals a von Willebrand factor domain and two IgG-like domains. One of the IgG-like domains is stabilized by an intramolecular isopeptide bond, which is the first such bond observed in a Gram-negative bacterium. These features make Mfa5 structurally more related to streptococcal adhesins than to the other P. gingivalis Mfa proteins. The structure reported here indicates that horizontal gene transfer has occurred among the bacteria within the oral biofilm.The Gram-negative bacterium  Porphyromonas gingivalis that is a secondary colonizer of the oral biofilm. They find a von Willebrand factor domain and two IgG-like domains in this structure. This study suggests that horizontal gene transfer may have occurred among the bacteria within the oral biofilm.Heidler et al. report a structure of the N-terminal half of a bacterial fimbrial protein, Mfa5, from  They usually contain several protein subunits encoded by the same gene cluster, resulting in the assembly of a long shaft of repetitive proteins decorated by ancillary tip proteins4. For some gene clusters, flanking transposons have been identified indicating that horizontal gene transfer can occur5. In Gram-negative bacteria, these filamentous structures are assembled in a non-covalent manner assisted by multi-protein complexes that span either the outer membrane or both the outer and inner membranes. In contrast, Gram-positive fimbrial proteins are covalently linked to each other by intermolecular isopeptide bonds, which are amide bonds between a lysine side chain of one subunit and the carboxyl group of a C-terminal threonine of the next subunit. The formation of these bonds is mediated by specific sortases that are encoded by the same gene cluster7. To date six different types of fimbriae have been classified: (1) type-I, also called the chaperone-usher type, (2) type-IV fimbriae, (3) type-IV secretion fimbriae, (4) type-V fimbriae, (5) curli fibers, and (6) sortase-mediated fimbriae, all of which are essential for bacterial virulence4.Fimbriae  are long filamentous protein polymers that project from the bacterial surface and are crucial for attachment to other microorganisms, host cells, and surfacesPorphyromonas gingivalis is a Gram-negative, rod shaped, anaerobic bacteria that belongs to the Bacteroidetes phylum8. P. gingivalis colonizes the oral biofilm as a secondary colonizer using primary colonizers, mainly commensal streptococci, as attachment partners10. The proliferation of P. gingivalis can lead to a shift from a healthy biofilm composed mainly of commensals to a more pathogenic biofilm8. P. gingivalis causes chronic inflammation (periodontitis)13 and is believed to be involved in the onset of systemic diseases such as various cancers, rheumatoid arthritis, heart diseases, diabetes, pregnancy complications, and Alzheimer’s disease20.P. gingivalis expresses two antigenically distinct but structurally homologous type-V fimbriae that are important both for attachment to other bacteria and for invasion of different host tissues25. FimA fimbriae are up to 2 µm long and Mfa1 fimbriae are ~100-nm long27, and both have been shown to be involved in disease progression28. The mfa1 and fimA gene clusters encode five proteins each, including one major stalk subunit (Mfa1 or FimA), a cell-wall anchor (Mfa2 or FimB), and three ancillary tip proteins (Mfa3-5 or FimC-E)29. The individual proteins harbor an N-terminal signal peptide that leads them to be transported into the periplasm via the Sec pathway30. Within the periplasmic space, the proteins are acylated, processed by signal peptidase II, and transported to the outer membrane by the localization of lipoprotein export pathway31.P. gingivalis, the gingipains. Those are proteases crucial for processing other virulence factors in addition to causing severe host tissue destruction. Blocking the type-IX secretion system hampers gingipain activity, and is therefore considered an important target for the development of novel antibacterial substances32. Proteins translocated by this system are selected by a conserved C-terminal signal domain and among the fimbrial proteins such a C-terminal signal domain is only found in Mfa5. Blocking the type-IX secretion system in P. gingivalis inhibits export of Mfa5 to the outer membrane, whereas the other fimbrial proteins are not affected33, indicating that they are transported over the membrane using another, unknown pathway. Mfa5 is also unique among the fimbrial proteins in that it is exceptionally large (1228 amino acids), and whereas the Mfa1–4 proteins consist of two β-sandwich domains, Mfa5 is predicted to contain several domains, one of which is a von Willebrand factor (vWF) domain.Bacteria of the Bacteroidetes phylum have a specific secretion system, type-IX, which transports proteins and many virulence factors across the outer membrane. Among these are the most important virulence factors in In this study, we present the crystal structure of the N-terminal half of Mfa5, residues 99–664, at 1.8 Å resolution. Our high-resolution structure reveals unforeseen similarity to adhesins hitherto only observed in Gram-positive bacteria. As predicted, the structure contains a vWF domain with a complete metal ion-dependent adhesion site (MIDAS) as well as two IgG-like domains. Intriguingly, one of the IgG-like domains is stabilized by an intramolecular isopeptide bond, which to our knowledge is the first such bond observed in a Gram-negative bacterial surface protein.21–1044, was designed and expressed in E. coli , forms a Rossman fold classified as a vWF domain .From the crystallized 70 kDa fragment a model that spans residues 99–664 could be built. The structure consists of three domains (D1 to D3) that build up an elongated shape with dimensions of 46 Å × 48 Å × 164 Å Fig. . This moin Figs. 34. The v35. Interestingly, an intramolecular isopeptide bond connects Lys111 of the first β-sheet to Asn518 of the second β-sheet , has an IgG-like fold composed of a β-sandwich with three and five β-strands, respectively. Intriguingly, the vWF domain is inserted between strands B (first sheet) and C (second sheet) of the D2 domain. The overall topology of D2 is similar to CnaA, which is one of the common building blocks of Gram-positive adhesinset Figs. .35; however, the generally high B-factor and comparisons to other IgG-like domains led to the assumption that D3 might be incomplete.D3, encompassing amino acids 547–664, comprises six β-strands in a twisted tube-like β-sheet that can be classified as a ubiquitin-like roll. A topology plot shows that there is a similarity to the IgG-like CnaB fold of Gram-positive bacterial adhesins Figs.  and 3c35138–435, that will be referred to as Mfa5D1, is folded into a vWF domain consisting of a typical Rossman fold comprising a six-stranded β-sheet surrounded by three helices on each side. This globular domain, which has a wrench-like form, has the dimensions 44 Å × 52 Å × 79 Å and includes the MIDAS motif, which is a central feature for interaction with various targets. In the Mfa5 MIDAS site, the five residues coordinating the metal ion are situated on the wrench head on top of the β-sheet. Mfa5 has a classical MIDAS motif36 in which the side chain oxygens of Ser152, Ser154, and Thr224 coordinate the metal directly and the side chains of Asp150 and Asp252 coordinate the metal via a water molecule. The crystallization conditions and the coordination distances (average 2.1 Å) suggest that this is a Mg2+ ion  and GBS104 (PDBID: 3txa39) from Streptococcus pneumoniae and Streptococcus agalactiae as well as to human integrins such as the collagen-binding α2-I domain (PDBID: 1dzi40). A superposition of their respective vWF domains to the vWF domain of Mfa5 gives a good match to the streptococcal tip fimbriae (rmsd 1.9 Å (151 of 296 Cα-positions)) and the α2-I vWF domain (rmsd 2.5 Å (88 of 296 Cα-positions)). This was unexpected based on their low sequence similarity and identity to Mfa5, 19/10% for the streptococcal adhesins and 21/9% for the α2-I protein. A structure-based sequence alignment of the vWF domain confirmed that the MIDAS site is conserved  between the fourth β-strand and the fourth α-helix of the domain (amino acids 257–355). In Mfa5, ARM2 folds into a small domain comprised of long loops centered around a β-sheet of three very short strands. Two helices, separated by a proline, form an L-shaped structure that covers one side of the sheet. One of the loops in the ARM2 domain coordinates the second metal in the structure. The metal is coordinated by the main chain carbonyls of Asn298, Thr301, and Leu303 and by the side chain oxygens of Asn298 and Thr301. In addition, the side chain of Asp336 from another loop also coordinates the metal. The conformation of the loop is additionally constrained by the three prolines Pro299, Pro302, and Pro305. Based on the coordination distances (average 2.4 Å), this metal has been modeled as a calcium ion. Interestingly, the whole ARM2 domain is rich in prolines, 13 of 98 residues, which contributes to the rigidity of the loops . In contrast, the ARM2 domain seems to mimic parts of the αV integrin domain, which together with β3 integrin forms a cleft that binds to fibronectin , thus verifying the formation of an isopeptide bond . This has led to the development of novel antibacterial treatments that have gone as far as clinical trials4. P. gingivalis, on the other hand, belongs to the Bacteroidetes phylum and accordingly its two fimbriae, Mfa1 and FimA, are of type-V. Recent publications describing the X-ray structures of recombinant proteins building up type-V fimbriae describe a common core structure of two domains, both comprising a β-sandwich46 where the first strand of the protein is removed by gingipain proteases when the fimbriae are polymerized. These structures led to the suggestion that type-V fimbriae also depend on a donor-strand exchange mechanism for bioassembly; however, the exact nature of this mechanism is still not fully understood47. The mfa1 gene cluster encodes five proteins where the first four all have the classical type-V fold. The fifth protein, Mfa5, is different from the other Mfa proteins in many ways. It is considerably larger (1228 residues) than Mfa1–4 (324–663 residues), and contrary to Mfa1, 3, and 4 it does not appear to be dependent on gingipains for maturation33. Instead, Mfa5 is the only fimbrial protein that has been found to be dependent on the type-IX secretion system for translocation across the outer membrane. Indeed, the crystal structures of the three N-terminal domains of Mfa5, that we present here, do not resemble the other Mfa proteins. The three domains include one vWF domain (D1) and two IgG-like domains (D2 and D3). Surprisingly, despite being unrelated in sequence, Mfa5 shows a striking similarity to the Gram-positive adhesins RrgA and GBS104, which both have a vWF domain and a number of IgG-like domains that appear to function as the stalk to project the VWF domain toward ligands, presumably those presented on host cells. The first Mfa5 IgG-like domain, D2, is formed from two segments of the protein, residues 105–138 that form the first two β-strands and residues 422–546 that constitute the rest of the domain. An intramolecular isopeptide bond between Lys111 and Asn518 links the first segment with the second. Keeping in mind that the Mfa5 D1, D2, and D3 domains presented here only comprise half of the full-length protein, it is possible to imagine that the full-length stalk might be constructed from several additional IgG-like domains. The D3 domain folds like an incomplete IgG-like domain, and comparison to the IgG-like domains in RrgA and GBS104 lead us to speculate that strands are missing from this domain. Hypothetically, extra strands might have been donated by a putative D4 domain if the protein had not been crystallized in a degraded form. In this model, there are also ~75 residues missing from the N-terminus, which may form a segment long enough to contribute with multiple β-strands both to the D2 and D3 domains. Such a segment would hypothetically have the potential to provide more stability to the protein. Furthermore, Mfa5 constitutes the tip of the fimbria together with Mfa3 and Mfa4, and the association of these proteins in the fimbrial spatial arrangement is likely to have a stabilizing effect on the large Mfa5 protein compared to when it is expressed recombinantly on its own. The vWF domain is folded from 276 residues located between the two segments of the D2 domain. vWF domains are most commonly expressed by eukaryotic organisms but have also been found in some bacteria. In the human integrin α2-I, the vWF domain has its collagen-binding MIDAS motif exposed at the top of the protein, whereas bacterial vWF domains have extra segments (ARM1 and ARM2) folding as subdomains on the sides of the MIDAS motif. The streptococcal adhesins RrgA and GBS104 have two ARMs, whereas Mfa5 only has one. Superposition of the vWF domain of Mfa5 onto the human α2-I domain indicates that ARM2, in the present conformation, would interfere with collagen binding. Superposition of the vWF domain of Mfa5 onto integrin αVβ3 indicates a closer mimic to its fibronectin-binding cleft. Thus the vWF domain is a clear indication that the D1 domain of Mfa5 functions as an adhesin, but the ligand has yet not been determined.Fimbriae are protein polymers projecting from the bacterial surface, and these make the first contact with the targeted host. Therefore, it is of great interest to understand the structure, assembly mechanism, and ligand specificity of fimbriae because their adherence mechanisms and biogenesis are potential targets for the development of novel targeted antibacterialsmfa5 gene has been obtained from a streptoccocal ancestor. It has been shown that horizontal gene transfer between microorganisms happens frequently in the oral biofilm where hundreds of species live in close physical contact48. Over the course of evolution, Mfa5 has retained crucial structural features from its Gram-positive ancestor, such as the stabilizing intramolecular isopeptide bond and the conserved MIDAS motif, and has adapted to its Gram-negative host by adding a C-terminal domain that targets the protein to the type-IX secretion system, which is unique to the Bacteroidetes phylum. While the assembly mechanism of type-V fimbriae is not fully understood, it is known that the fimbrial proteins, that build up the final fimbria, are expressed as lipidated precursors that are transported to the outer membrane via the lipoprotein export system31, followed by gingipain-dependent removal of the first β-strand. This cleaved fimbrial protein can then polymerize through a donor-strand replacement mechanism in which a segment from the next fimbrial protein is predicted to participate46. Integration of Mfa5 into the fimbriae is not expected to follow this mechanism because Mfa5 is not dependent on the presence of gingipains; however, it has been indicated by in vivo studies that the incorporation of Mfa5 into the mature fimbriae requires a functional vWF domain33. This needs to be studied further because the truncation of the vWF domain might obstruct the correct formation of the other domains and thus its ability to bind to other fimbrial proteins.The Mfa5 structure suggests that the ancestor of the P. gingivalis, as analyzed by SDS gels, revealed protein bands that were identified as Mfa5 by Hasegawa et al.42. In this study, we could further confirm that antibodies derived from recombinant Mfa5 specifically stain these bands. In negative staining images, the antibody against Mfa5 connects multiple fimbriae together, verifying the assumption that Mfa5 is indeed located at the tip of the fimbriae. The different conformations visible in the images also indicate high flexibility, which might be related to the linker regions between the different proteins and their domains. A structure of the full-length native fimbria would answer the remaining questions about the localization of the different subunits and their biogenesis.Native fimbrial purifications from P. gingivalis is a key pathogen and is strongly associated with the periodontitis that affects a large part of the population worldwide. The presence of P. gingivalis is believed to contribute to the onset of other systemic diseases such as Alzheimer’s disease, rheumatoid arthritis, and oral and pancreatic cancer, and its capacity to bind early colonizers of the oral biofilm and to extracellular matrix proteins on host cells is deemed to be an important factor. Therefore, further development of anti-adhesive substances to block the vWF-binding cleft or the development of blocking antibodies will enable future tools to hinder the establishment and proliferation of this pathogen. Hindering the fimbriae from attaching to the primary colonizers of the oral biofilm is crucial because if P. gingivalis cannot colonize the mouth it will never cause the chronic inflammation that burdens the immune system and it will not have the opportunity to spread to other, non-oral parts of the body.56. Based on the combined results from all analyses, the full-length Mfa5 was amplified by polymerase chain reaction from the P. gingivalis ATCC 33277 genome. Additional constructs, containing amino acids 21–1044, 138–435, 99–664, and 99–664 (K111A) were subsequently cloned by polymerase incomplete primer extension57 into a pET-ZZ-1a58 vector with flanking NcoI/XhoI sites (primers are presented in Supplementary Table The primary Mfa5 sequence (GenBank AUR48966.1) was analyzed for conserved features using the web-based SIGNALP, PROSITE, INTERPRO, and Conserved Domains Database, GOR4, SCRATCH, PSIPRED, and PHYRE2 toolsE. coli C41 (DE3) (Mfa521–1044) or BL21 (DE3 pLysS) (other constructs). Cells were cultivated to an OD600 of 0.8 at 37 °C in 2–4 L Luria Bertani media supplemented with 50 μg·mL−1 kanamycin. The temperature was shifted to 20 °C and protein expression was induced by the addition of 0.25 mM IPTG. Cells were harvested 16 h later by centrifugation for 20 min at 4000 × g, flash frozen in liquid nitrogen, and stored at −80 °C until further processing.The recombinant proteins were expressed in g for 30 min, and the supernatant was applied to Ni-IDA resin (TaKaRa Bio). After two wash steps, the protein was eluted with PBS and 250 mM imidazole. Tobacco Etch Virus protease or α-chymotrypsin at a 1:100 (w/w) ratio was added to the eluted sample, and the mixture was dialyzed against PBS overnight at 4 °C. Next, the sample was applied to a fresh Ni-resin column for re-chromatography. The cleaved protein was concentrated with an Amicon Ultra centrifugal filter  and run on a Superdex200 16/60 column  in 20 mM Tris pH 7.4 and 100 mM NaCl. Peak fractions were pooled, concentrated, and stored at −80 °C until further use.Cell pellets were resuspended in 50 mL cold phosphate-buffered saline (PBS) containing additional 260 mM NaCl, 10 mM imidazole, 2 mM β-mercaptoethanol, 1% Triton X-100, and protease inhibitors (Pierce) and lysed by sonication. Cell debris was removed by centrifugation at 64,000 × P. gingivalis strains JI-1 fimA-deletion mutant (ΔfimA) and mfa1-deletion mutant (Δmfa1), both derived from P. gingivalis strain ATCC 33277, growing on blood agar medium were used to inoculate tryptic soy broth (TSB) supplemented with 2.5 g·L−1 yeast extract, 5 mg·L−1 menadione, 2.5 mg·L−1 hemin, and 0.01 mg·L−1 DTT overnight as previously described9. The cultures were preincubated at 37 °C in an anaerobic chamber with an atmosphere of 85% N2, 5% CO2, and 10% H2. Aliquots of 250 mL of anaerobic TSB were subsequently inoculated with 5 mL (2%) overnight cultures of the two P. gingivalis mutants for 16–18 h and further processed.Colonies of the P. gingivalis culture was harvested by centrifugation at 4000 × g for 20 min. The cells were resuspended in 30 mL 20 mM Tris pH 7.4, 10 mM MgCl2, 1.5 M NaCl, 10% sucrose, 0.1 mM DTT, and DNase and lysed by four passes through a French press at 900 psi. Cell debris was separated by centrifugation at 9000 × g for 10 min, and the supernatant was cleared at 143,000 × g for 90 min. The supernatant was saturated with 50% NH4SO4 at 4 °C, and a follow-up centrifugation at 15,000 × g for 30 min was used to collect the precipitated protein. The pellet was resuspended and dialyzed overnight in 20 mM Tris pH 7.4 and 20 mM NaCl. A 1:2 diluted sample was applied on a HiTrap Q1 column  and eluted in a step gradient with 20 mM Tris pH 7.4 and 1 M NaCl. Protein-containing fractions were pooled and applied on a HiPrep Sephacryl S-400 HR 16/60 column . Fractions corresponding to the peak were collected and applied to a Superose 6 Increase 10/300 column . Peak fractions were collected, and aliquots were flash frozen in liquid nitrogen for storage at −80 °C.A total of 1 L of Mfa proteins were detected in fimbrial purifications by western blotting. An SDS gel was loaded with 50 ng detectable protein per lane. After electrophoresis, the gel was incubated together with blotting paper and membrane  in Towbin buffer  methanol) for 5–10 min. Next, the blot was assembled in a semi-dry blotter and run at 15 V for 15 min. The membrane was blocked with 5% milk in TBST  for 40 min at 37 °C and for 20 min at room temperature. The primary antibodies, anti-Mfa5-vWF or anti-Mfa3 (Agrisera AB), were diluted 1:20,000 in 3% milk in TBST and incubated with the membrane on a shaker for 60 min. After washing for 10 min three times with TBST, the secondary antibody  was applied at a 1:50,000 dilution in 3% milk in TBST for 60 min. After washing three times with TBST, the bound secondary antibody was detected by chemiluminescence using BCIP/NBT (Promega).21–1044 (longest construct) at 12 mg·mL−1 and Mfa5138–435 (predicted vWF domain) at 18 mg·mL−1. Crystallization was performed by sitting-drop vapor-diffusion at 18 °C using commercially available screens from Molecular Dimensions and Hampton Research with a Mosquito robot (TTP Labtech). Mfa521–1044 crystals grew in 12–17% PEG 3350, 80 mM Na2PO4 pH 6, 3 mM CaCl2, and 3 mM MgCl2 and were cryoprotected in mother liquor supplemented with 25% glycerol and flash-cooled in liquid nitrogen. A native dataset was collected at beamline MX14.1 at the Berlin Electron Storage Ring Society for Synchrotron Radiation (Germany). One single crystal of Mfa5138–435, grown in 20% PEG 500, 10% PEG 20,000, 0.1 M Sodium HEPES/MOPS pH 7.5, and 0.1 M Glu/Ala/Gly/Lys/Ser-mix was obtained. The crystal was directly flash-cooled without additional cryoprotectants. A high redundancy sulfur single-wavelength anomalous dispersion (SAD) dataset was collected at beamline ID29 at the European Synchrotron Radiation Facility 59. All datasets were processed with XDS60, and the sulfur SAD data were combined with BLEND61. Phase determination, refinement, and automated model building were performed in the PHENIX suite62. The initial solution of Mfa5138–435 was used as a molecular replacement model for Mfa521–1044, which was built by BUCCANEER63 and with cycles of manual building in COOT64 followed by refinement with PHENIX-refine. The final structures were validated using MOLPROBITY65 and PDBSUM66, and analyzed with CATH34, SALIGN67, and DALI37. Figures were prepared with CCP4MG68.Initial screening was performed using Mfa599–664 and its isopeptide mutant Mfa599–664 (K111A), were analyzed for thermal stability. The protein samples at 1 mg·mL−1 were mixed with Tris buffer pH 8.5 and SYPRO Orange (Molecular Probes), to a final concentration of 20 mM and 5×, respectively. The assay was conducted using a stepwise temperature increase from 10 to 95 °C in a CFX Connect instrument (BioRad) at a rate of 2 °C/min. Data points were collected with the FAM channel (excitation 450–490 nm/detection 510–530 nm) after each 0.5 °C with 3 s equilibration time. Plotting the first derivative of the measured fluorescence at each data point versus the temperature in Microsoft Excel allowed the calculation of the inflection point, and the minima were referred to as the melting temperatures (Tms). Each point was measured in triplicate, and their average value was used.A stable construct representing the long form of the Mfa5 structure, Mfa599–664 and its K111A mutant was determined by electrospray ionization time of flight (ESI-TOF) mass spectrometry. Purified protein, 4 μL at 10 mg·mL−1, was separated on a C8 column using a gradient of 0.1% aqueous formic acid to 0.1% formic acid in acetonitrile over 3 min on an Agilent 1200 HPLC system. The sample was subsequently ionized in an acquisition mode operated at 4 GHz on an Agilent 6230 LC-TOF/MS. The acquired total ion chromatogram was deconvoluted in the MassHunter Qualitative Analysis software .The accurate molecular mass for Mfa5P. gingivalis fimbriae  was incubated 1 h at 4 °C with anti-Mfa5-vWF antibody serum (12 µL) in 20 mM Tris pH 7.4, 100 mM NaCl and applied to Superdex200 10/30 . Peak fractions were directly transferred (3.5 µL) to a carbon-coated and glow-discharged copper grid  and incubated for 3 min. The excess sample was removed by blotting, and the grid was washed with two drops of water and stained with 1.5% w/v uranyl acetate (UA) for 30 s. For gold-labeled samples, the sample was transferred to the grid and after 3 min incubation washed in two drops of water. Secondary anti-rabbit 5-nm-gold-conjugated IgG antibody (5 µL) (Abcam) was added directly to the grid and incubated for 5 min. Grid was blotted dry and washed in two drops of water and was further stained with 1.5% UA for 30 s. A Ceta CMOS camera  connected to a Talos L120C transmission electron microscope  operating at 120 kV was used to examine the negative-stained samples. Images were recorded using the TIA software (FEI).A reaction mixture of purified 69 were redrawn in ESPRIPT70 for better visibility.Sequence alignments calculated in MULTALIGNThe source data underlying Fig. Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Supplementary FilesSupplementary Data 1Reporting Summary


Dimerization of the BCAP Toll/IL1 domain is required for function.BCAP TIR modulates the oligomerization state of TLR signaling adaptor MAL.TIG domains are a promiscuous dimerization module in gene expression and signaling. The B cell adaptor protein (BCAP) is a multimodular regulator of inflammatory signaling in diverse immune system cells. BCAP couples TLR signaling to phosphoinositide metabolism and inhibits MyD88-directed signal transduction. BCAP is recruited to the TLR signalosome forming multitypic interactions with the MAL and MyD88 signaling adaptors. In this study, we show that indirect dimerization of BCAP TIR is required for negative regulation of TLR signaling. This regulation is mediated by a transcription factor Ig (TIG/IPT) domain, a fold found in the NF-κB family of transcription factors. We have solved the crystal structure of the BCAP TIG and find that it is most similar to that of early B cell factor 1 (EBF1). In both cases, the dimer is stabilized by a helix-loop-helix motif at the C terminus and interactions between the β-sheets of the Ig domains. BCAP is exclusively localized in the cytosol and is unable to bind DNA. Thus, the TIG domain is a promiscuous dimerization module that has been appropriated for a range of regulatory functions in gene expression and signal transduction. Toll-like receptors are pattern recognition receptors that respond to conserved microbial stimuli, such as LPS from Gram-negative bacteria. These stimuli induce dimerization of the receptor Toll/IL-1R (TIR) domains that act as a scaffold for the recruitment of downstream signal transducers, leading to the activation of NF-κB. Although receptor and adaptor TIR domains are known to engage in homotypic and heterotypic interactions, the stoichiometry and assembly of the TIR signalosome remains unsolved. However, residues and interfaces in the TIR domains of the TLRs, MyD88, and MAL adaptor proteins that are required for signal transduction have been mapped . This haMore-recent studies found that MyD88 and MAL have the ability to form filaments in vitro, similar to other pattern recognition receptors such as NOD-like receptors (NLR), inflammasomes, and antiviral RIG-I–like receptor (RLR) complex pathways . This fiAn important regulator of TLR signaling is the B cell adaptor protein (BCAP). BCAP is categorized as a negative regulator of TLR signaling because BCAP-deficient macrophages produce higher amounts of TLR-induced inflammatory cytokines IL-12, IL-6, and TNF-⍺ . On a moDrosophila protein Dof, the BCAP B cell scaffold protein with ankyrin repeats (BANK1), and BCAP. The DBB domain, along with the ankyrin repeat domain, has been suggested to drive dimerization of BCAP  domain of BCAP is required for TIR domain interactions with MAL and MyD88 as well as the negative regulation of TLR signaling . The DBB of BCAP , 15.In this study we present a structural and functional analysis of the BCAP DBB domain and its role in the TLR signalosome. We show that the TIR domain of BCAP is sufficient for interaction with MAL and that the DBB domain is essential for the negative regulation of TLR signaling both in vivo and in vitro. Using a combination of biophysical and structural techniques, we show that dimerization of BCAP TIR by the DBB domain drives negative regulation of TLR signaling. The structure of the BCAP DBB domain reveals that it shares the same fold and dimerization interface as the transcription factor Ig (TIG) domains found in the NF-κB family of transcription factors (TF). However, the BCAP TIG domain does not bind to DNA.l-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin; all from Invitrogen). THP-1 cells were differentiated to macrophages using 10 ng/ml PMA (Sigma-Aldrich) for 12 h, followed by rest for 24 h in complete RPMI 1640 medium. HEK293T cells (ATCC) were maintained in DMEM . Expi293F cells (Thermo Fisher Scientific) were cultured in Expi293 Medium (Life Technologies) at 140 rpm, 37°C, and 8% CO2.THP-1 cells and Ramos B cells  were maintained in RPMI 1640 medium , TIR-DBB (1–311), and TIR-DBB-ANK (1–426) domains into a pcDNA3.1–3X-FLAG vector using KpnI and BamHI sites. Full-length BCAP (2–805) was cloned into pcDNA3.1(+) with an N-terminal His-Avi-TEVFor bacterial expression, the TIG2α (179–288), TIR-TIG2α (7–288), DBB-ANK (179–404), MBP-ANK (333–467), and MAL TIR domains (79–221) containing a C-terminal Strep-tag were cloned into pMCSG7 or pMCSG9 (DNASU) using ligation-independent cloning as described .g for 10 min at 4°C. The supernatant was collected for immunoblotting or immunoprecipitation with EZview Red FLAG M2 beads (Sigma-Aldrich) according to the manufacturer’s recommendation. For immunoblotting, Myc and FLAG-tagged proteins were visualized using anti-Myc  and anti-FLAG M2  primary Abs, respectively. Anti-mouse-HRP IgG  was used as secondary Ab.Various Myc and FLAG-tagged protein constructs were transiently transfected into HEK293T cells (ATCC) using JetPEI (Polyplus Transfection SA) according to the manufacturer’s recommendations. Twenty-four hours posttransfection, cells were washed with 1× PBS and lysed using 300 μl of Tris lysis buffer  supplemented with 1× protease inhibitor mixture . After 30 min of lysis at 4°C with agitation, the mixture was centrifuged at 16,000 × 4 cells per well. At 70–80% confluence, JetPEI (Polyplus Transfection SA) was used according to the manufacturer’s instructions to transiently transfect cells with the NF-κB reporter vectors pBIIX-luc and pCMV-Renilla-luc, and cells were cotransfected as described with Myc-MAL, Myc-MyD88, and FLAG-TIR, FLAG-TIR-DBB, FLAG-TIR-TIG2α, FLAG-TIR-DBB-ANK, or FLAG-BCAP, totaling 100 ng of DNA per well in 100 μl of DMEM. At 24 h posttransfection, cells were washed in 1× PBS and subsequently lysed in Passive Lysis buffer (Promega). The lysates were assayed for luciferase activity using the Dual-Glo luciferase kit (Promega). Luciferase activity shown is firefly luciferase relative to Renilla luciferase and normalized to cells transfected with empty vectors.HEK293T cells were seeded in a 96-well plate at 1.5 × 10TIG2α and the DBB-ANK domain of human BCAP were expressed in BL21(DE3) cells (Novagen) grown in Luria broth medium. After Ni-NTA purification using immobilized metal ion affinity chromatography (IMAC) buffer  and elution in IMAC buffer containing 500 mM imidazole, samples were treated with tobacco etch virus (TEV) protease to remove the hexa-histidine tag. Further purification was performed using Superdex 200  size exclusion chromatography in 20 mM TRIS, 100 mM NaCl, 1 mM TCEP (pH 7.5). The MAL TIR domain, MBP-ANK, and GST-CRKL were expressed and purified as described above, but TEV protease digestion was omitted.For selenomethionine incorporation, the DBB domain was expressed in BL21(DE3) cells grown in 1 l of M9 minimal medium supplemented with 0.05 g of selenomethionine, 0.1 g of lysine, 0.1 g of threonine, 0.1 g of phenylalanine, 0.05 g of leucine, 0.05 g of isoleucine, and 0.05 g of valine. Purification of selenomethionine-containing DBB domain was performed as described for the unmodified protein.clBCAP. At a density of 4 × 106 cells/ml, 6 μg/ml linear PEI Max (Polysciences) was used to transfect 1.5 μg/ml plasmid DNA. Fresh medium was added to double the volume of the culture 24 h after transfection. Cells were harvested 3 d posttransfection by centrifugation. BCAP(FL) Ni-NTA purification was performed in high-salt IMAC buffer  supplemented with 5 mM sodium orthovanadate, 50 mM sodium fluoride, 60 mM β−glycerophosphate and protease inhibitor mixture . TEV protease digestion and size exclusion chromatography were performed as described above.For the expression of BCAP(FL), Expi293F cells were transiently transfected with HisAviTEV2PO4, 0.7 M K2HPO4, and 0.1 M sodium acetate (pH 4.5). The selenomethionine-containing DBB domain crystallized at 2.2 mg/ml in 0.9 M NaH2PO4, 0.9 M KH2PO4, and 0.1 M sodium acetate (pH 4.5).Crystals were obtained using the sitting-drop vapor diffusion methods at 19°C. The DBB-ANK domain crystallized at a concentration of 9 mg/ml in 1.1 M malonate, 0.5% jeffamine ED-2003, 16% glycerol, and 0.1 M HEPES pH 7.5. Crystals of native DBB were obtained at a concentration of 5.5 mg/ml in 1.3 M NaHhttp://www.rcsb.org/).DBB-ANK diffraction tests were conducted at the i02 beam-line . Native DBB diffraction data were collected at ID30A-3 beam-line . The DBB single wavelength anomalous dispersion (SAD) dataset was collected at Proxima 2A . Diffraction data were indexed and scaled using XDS . Initial+ (Wyatt technology) instruments, respectively. Data analysis was performed using ASTRA (v6.1) software (Wyatt technology).For size exclusion chromatography combined with multiangle light scattering (SEC-MALS) analysis of DBB and DBB-ANK domains, 50-μl protein samples at a concentration of 2 mg/ml were injected onto a Superdex 200 Increase 10/300 GL column . Differential refractometry and multiangle light scattering data were collected using Optilab T-rEX (Wyatt technology) and DAWN8g for 10 min and subsequently analyzed by SDS-PAGE.Starting from a stock concentration of 10 μM, MAL and various BCAP constructs were mixed at a molar ration of 1:10. The protein mixture was then incubated at 30°C for 1 h to induce filament formation of the MAL TIR domain. Soluble and insoluble fractions were separated by centrifugation at 16,000 × Cells were fixed in 3.7% formaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 5 min. After blocking with 2% BSA in 1× PBS, cells were immunostained with goat anti-BCAP  primary Ab and Alexa Fluor–conjugated secondary Ab . Cells were mounted using Vectashield DAPI (Vector Shield) and visualized using EVOS M5000 .A total of 10 μg of purified BCAP(FL), DBB-ANK, GST-CRKL, or high-mobility group B1 protein (HMGB1)  was applied to 20 μg of DNA cellulose (Merck) pre-equilibrated in 500 μl of binding buffer . Samples were then incubated for 1 h at 4°C with shaking. Samples were applied to small gravity flow columns, and the flowthrough was collected. The DNA cellulose was repeatedly washed with 500 μl of binding buffer containing increasing amounts of NaCl to a final concentration of 600 mM. The resulting flowthrough was subjected to a TCA precipitation, and samples were analyzed on SDS-PAGE.Complementary 81-bp DNA strands were annealed at 95°C for 5 min to generate dsDNA. The duplex was diluted to 1 μM in binding buffer  and mixed with 10 μg purified BCAP(FL), DBB-ANK, GST-CRKL, or HMGB1 . After 15 min at room temperature, the samples were diluted in Hi-Density TBE sample buffer (Thermo Fisher Scientific) and ran on a 6% acrylamide DNA retardation gel (Thermo Fisher Scientific). The acrylamide gel was prerun in 0.5× Novex TBE running buffer (Thermo Fisher Scientific) at 100 V and 4°C for 1 h, and the samples were run at the same settings. The gels were fixed by washing in 35% ethanol and 5% acetone for 5 min followed by SYBR safe (Thermo Fisher Scientific) staining to visualize the DNA.In previous studies, constructs containing both the TIR and DBB domains were used to characterize BCAP as a negative regulator of TLR signaling . To deteTo further investigate the role of the DBB domain, we measured the ability of BCAP truncations to inhibit signaling by MAL and MyD88 in an NF-κB reporter assay. As expected, constructs containing the TIR-DBB domains, TIR-DBB-ANK, or full-length BCAP were able to inhibit NF-κB signaling induced by MAL and MyD88 , 1D. TheTo understand the functional importance of the DBB domain, we examined its oligomerization properties. A previous study has shown that full-length BCAP is dimeric in solution . FurtherA recent study has shown that the MAL TIR domain assembles into complex filaments in vitro and that some of the homotypic interactions seen in the structure of these filaments are critical for signal transduction . We therTo obtain structural information for the DBB dimerization interface, a DBB-ANK construct was crystallized. However, diffraction was limited to 7 Å and therefore insufficient to obtain an atomic model. Subsequently, the shorter TIG2α  construcThe DBB TIG fold exhibits structural similarity to TIG domains from several TFs despite low sequence similarity . It sharAlthough specific binding is mediated by N-terminal Rel homology domains, NF-κB, NFAT, and Ebf1 TIG domains make sequence-unspecific contacts with the DNA phosphate backbone through several arginine and lysine residues at the N-terminal loops of the TIG domain –23. BCAPThe paralogues BCAP and BANK1 exhibit analogous domain architectures, with an N-terminal TIR domain followed by a DBB domain and ankyrin repeat followed by a C-terminal unstructured region. Moreover, because of the high level of sequence similarity between the DBB domains , we hypoIn this study, we provide the first direct evidence, to our knowledge, that the BCAP TIR domain forms heterotypic interactions with the MAL adaptor protein. The BCAP TIR domain is monomeric in solution  and may + T cells. Interaction of BCAP with the TIRs of these receptors activates the PI3K–mTOR pathway and enhances Th17 and Th1 cell responses , systemic sclerosis, and rheumatoid arthritis in multipole genome-wide association studies –34. On aData Supplement