In lieu of comprehensive instrumental seismic monitoring, short historical records, and limited fault trench investigations for many seismically active areas, the sedimentary record provides important archives of seismicity in the form of preserved horizons of soft-sediment deformation features, termed seismites. Here we report on extensive seismites in the Late Quaternary-Recent  alluvial and lacustrine strata of the Rukwa Rift Basin, a segment of the Western Branch of the East African Rift System. We document examples of the most highly deformed sediments in shallow, subsurface strata close to the regional capital of Mbeya, Tanzania. This includes a remarkable, clastic ‘megablock complex’ that preserves remobilized sediment below vertically displaced blocks of intact strata (megablocks), some in excess of 20 m-wide. Documentation of these seismites expands the database of seismogenic sedimentary structures, and attests to large magnitude, Late Pleistocene-Recent earthquakes along the Western Branch of the East African Rift System. Understanding how seismicity deforms near-surface sediments is critical for predicting and preparing for modern seismic hazards, especially along the East African Rift and other tectonically active, developing regions. Despite recent events associated with devastating liquefaction and fluidization of near-surface sediments, such as the 2011 New Zealand and Japan earthquakes and the ongoing Lusi mud eruptions in Indonesia, the dangers to life and infrastructure from soft-sediment deformation are often overlooked. In 1910 7.5 million people lived in Tanzania when the most powerful earthquake in Africa of the twentieth century  method by Beta Analytic Inc. The samples were pretreated with an acid wash, and conventional radiocarbon ages were corrected for total fractionation effects and rounded to the nearest 10 years per conventions of the 1977 International Radiocarbon Conference. Calibrated ages were calculated using the IntCal13 database .Here we describe for the first time a 50 m-tall cliff face exposure of a spectacular, large-scale , soft-sediment deformation feature that we term the ‘Songwe Megablock Complex’ Figs . The SonA large-scale, soft-sediment deformation feature dominates the finer-grained Lake Beds Succession facies. Deformation is contained between the buttress unconformity and the top of the modern-day cliff face. A 10 m-tall x 20 m-wide block of intact siltstone from the unit immediately above the basal conglomerate appears to have been uplifted ~10 m. The block is supported by a massive, fine-grained foreign body of tuffaceous material, not correlative with the laminated strata on either side of the soft-sediment deformation. This same ‘parent material’ that supports the megablock extends upwards via irregular dykes that surround the displaced blocks of bedded, cohesive sediment. A 5 cm-wide clastic injection dyke emerges from the top of the basal Lake Beds Succession conglomerate, crosscutting the intrusive sediment and uplifted megablock, where it expands gradually into a “V”-shape, up to 2.5 m in diameter at the top of the outcrop. The lower dyke contains metamorphic cobbles (4–20 cm diameter) from the basal Lake Beds Succession conglomerate and abundant pumice clasts . MetamorThirty-five km to the northwest of the Songwe Megablock Site, at a locality called Ilasilo 6, dekameter-scale asymmetrical, recumbent folds occur in a 3 m-thick, horizontally bedded, tuffaceous siltstone unit , stratigThe Lake Beds Succession at the Songwe Megablock Site is characterized by older fluvial-dominated facies and younger lacustrine facies, separated by an erosional unconformity. Prior to the deposition of the lacustrine facies preserved on the north end of the outcrop, a portion of the fluvial units was deeply incised by erosional downcutting. Fluvial incision created a topographical depression, into which lacustrine sedimentation  records the presence of a network of small, isolated, shallow wetland ponds and lakes. This major decrease in grain size, coupled with a dramatic increase in volcaniclastic sediments  in the lThe megablock complex lies on top of the undisturbed, basal Lake Beds Succession conglomerate. This soft-sediment deformation complex displays a unique combination of seismogenically-remobilized sediment coupled with brittle deformation features. It is comprised of (1) an injected body of fluidized volcanic ash that hydraulically displaced 2) an equally sized, semi-consolidated block of intact strata (megablock); both of which were subsequently intruded by 3) a clastic injection dyke  and/or aftershocks  allowed lifting of the megablock by fluidized injected volcanic ash, defies traditional concepts of downward displaced blocks of coherent sediment, usually due to collapse from overloading, liquefaction, and density inversions. This unique seismite formation illustrates the powerful effects of overpressured systems and active seismicity in this region on near-surface sediments. Consequently, this deformation indicates significant surface stability hazards associated with seismicity in the region, and other parts of the East African Rift System.This megablock complex is significant because it is one of the few continental, outcrop examples of very large-scale soft-sediment deformation associated with injectite features , 27, 28,The large-scale asymmetric, recumbent folds at Ilasilo 6 record hydroplastic soft-sediment deformation in a quiet, shallow lake or pond. The orientation of the folds suggests a very low slope (0.5°-2°) at the time of deformation. The folded horizon is truncated, indicating that the deformed unit was at the sediment/water interface at the time of deformation.Seismites are horizons of secondary sedimentary structures generated close to the surface by earthquakes of great enough magnitude (M ≥ 5 ± 0.5) , 32–36 tHowever, the above conditions are not diagnostic, and often these criteria can also fulfill the requirements for triggers other than seismicity. In contrast, some workers advocate a context-based approach –43 to idThe following field-based evidence is used to meet the above criteria and to diagnose a seismic trigger for the soft-sediment deformation in the Lake Beds Succession in the southern Rukwa Rift Basin:Deposition of the volcaniclastic, Late Pleistocene-Recent Lake Beds Succession occurred simultaneously with active volcanism and active faulting in the southern Rukwa Rift . Refer tGrain size analysis suggests the deformed sediment is highly susceptible to liquefaction . The uppLarge-scale deformation features are correlated via radiocarbon dates over 35+ km Figs  and 8.Folds at Ilasilo 6 are overlain and underlain by undeformed, horizontally bedded siltstones. The megablock complex is positioned within undisturbed, horizontal bedding and preserves intact, correlative stratigraphy on either side Figs , 3 and 4Soft-sediment deformation horizons are not only a common and repeated occurrence in the Lake Beds strata, but have also been abundantly recognized in Permian–Paleogene strata of the Rukwa Rift Basin. For example, stratigraphic sections at Ilasilo 6 and elsewhere in the Rukwa Rift Basin record diverse forms of soft-sediment deformation, including: flame structures; cm- to m-scale folded beds; ball-and-pillow structures; syn-sedimentary faults; sand injection features; and m-dkm-scale clastic injection dykes . ClasticThe circumstantial evidence presented above strongly suggests a seismic triggering mechanism for the large-scale soft-sediment deformation in the southern Rukwa Rift Basin. However, because seismite criteria can be ambiguous, we also consider other likely triggering mechanisms. Sediment loading can be ruled out because of the homogeneity of tuffaceous, silt-sized lithology that dominates the outcrops at both localities, eliminating overloading and inverse density gradients as triggers. Additionally, truncation of the folds at the top of the deformed unit at Ilasilo 6 indicates formation near the sediment/water interface . LikewisLarge-scale (hundreds of meters to hundreds of kilometers) clastic injectite networks, extrudite deposits, load casts, and slumps have been previously identified, and their generation has been attributed to thermal destabilization , fluid oThe Songwe megablock complex and recumbent folds at Ilasilo 6 likely occur along the same stratigraphic surface, as indicated by two radiocarbon dates Figs  and 8, sWe suggest that earthquakes of M ≥ 6 were responsible for the soft-sediment deformation features we report here, based on minimum magnitude capable of liquefaction, and circumstantial evidence, as discussed below. However, relating seismites to earthquake magnitude is equivocal, due to complexities including distance from the causal fault, attenuation of ground motion, and sediment susceptibility to liquefaction, which could not be quantitatively measured here. Many studies , 32–36 hRodríguez-Pascua et al.  qualitatOutcrop exposure of exceptional, large-scale soft-sediment deformation, including the uplift of a bus-sized megablock by fluidized, injected material; fluidization of large cobbles at Songwe; formation of giant, recumbent folds at Ilasilo 6; and numerous smaller scale clastic dykes and seismites, occur over at least 35 km in the RRB. These unique features suggest potential for dangerous surface deformation related to the seismically active East African Rift System. Our documentation provides evidence for M ~6+ Late Pleistocene earthquakes, similar to the M7.4 earthquake at the same location in 1910, extending the record of large-magnitude earthquakes beyond the last century, and contributing valuable data to better constrain recurrence intervals of high magnitude earthquakes in the Western Branch of the East African Rift. Our discovery is consistent with high-resolution seismic reflection data from Lake Rukwa, which provided evidence for high-frequency changes in boundary fault activity that occurs with a periodicity of thousands to tens-of-thousands of years . Documen


Amorphous thin-film oxygen evolving catalysts, OECs, of first-row transition metals show promise to serve as self-assembling materials in solar-driven, photoelectrochemical ‘artificial leaf’ devices. This report demonstrates the ability to use high-energy X-ray scattering and atomic pair distribution function analyses, PDF, to resolve structure in amorphous metal oxide catalysts films, and is applied here to resolve domain structure differences induced by oxyanion substitution during the electrochemical assembly of amorphous cobalt oxide catalyst films. Amorphous thin film oxygen evolving catalysts, OECs, of first-row transition metals show promise to serve as self-assembling photoanode materials in solar-driven, photoelectrochemical ‘artificial leaf’ devices. This report demonstrates the ability to use high-energy X-ray scattering and atomic pair distribution function analysis, PDF, to resolve structure in amorphous metal oxide catalyst films. The analysis is applied here to resolve domain structure differences induced by oxyanion substitution during the electrochemical assembly of amorphous cobalt oxide catalyst films, Co-OEC. PDF patterns for Co-OEC films formed using phosphate, Pi, methylphosphate, MPi, and borate, Bi, electrolyte buffers show that the resulting domains vary in size following the sequence Pi < MPi < Bi. The increases in domain size for CoMPi and CoBi were found to be correlated with increases in the contributions from bilayer and trilayer stacked domains having structures intermediate between those of the LiCoOO and CoO(OH) mineral forms. The lattice structures and offset stacking of adjacent layers in the partially stacked CoMPi and CoBi domains were best matched to those in the LiCoOO layered structure. The results demonstrate the ability of PDF analysis to elucidate features of domain size, structure, defect content and mesoscale organization for amorphous metal oxide catalysts that are not readily accessed by other X-ray techniques. PDF structure analysis is shown to provide a way to characterize domain structures in different forms of amorphous oxide catalysts, and hence provide an opportunity to investigate correlations between domain structure and catalytic activity. PDF measurements provide a measure of atom pair distances ranging from bonded atom pairs to nanoparticle dimensions (Juhás E = 1.04 V (versus NHE), pH 7, found larger average CoPi domains corresponding to about 19 Co atoms 2·6H2O. The electrolyses were stopped after 2–5 h deposition. The black deposited films were rinsed with water, dried, scrapped off the electrode surface and loaded as powders into 1 mm diameter thin-walled (10 µm) glass or Kapton capillaries. High-energy X-ray  scattering patterns for the Co-oxide catalyst powders, capillaries and backgrounds were measured as a function of the scattered wavevector q, where q = 4π sin(θ)/λ and 2θ is the scattered angle, in the range 0.4 < q < 24 Å−1 at the Advanced Photon Source (APS) of Argonne National Laboratory at beamline 11-ID-B  were prepared by anodic electrochemical deposition from aqueous buffered solutions containing the oxyanions Pi, MPi or Bi, following the conditions described previously  using the program PDFgetX2 . The reduced scattering structure function F(q) was calculated from I(q) according tof(q) is the sum of the composite atomic form factors, G(r), was obtained by direct Fourier transform of the oscillatory F(q)F(q) extrapolated to F(0) below the experimental q range < 0.4 Å−1 and using qmax = 24 Å−1 and a Lorch dampening function to remove truncation effects  is related to the real space electron density distribution function according tor) is the spherical average of the real space electron density distribution function, ρ(r) and ρo is the average electron density of the sample  to be measured with a signal-to-noise of greater than 10 for the Co-OEC powders throughout the measured q range, 0.4 < q < 24 Å−1. The level of spatial resolution was found to be necessary to resolve contributions from edge or defect sites in the CoPi films.Experimental HEXS patterns were used to generate the pair distribution function 2, ICSD entry 172909  for the model domains were calculated from the atomic scattering parameters using the program solX  patterns were calculated from equation (1)et al., 2013et al., 2013et al., 2012Models for Co-oxide domains in the water-oxidation catalyst films were built by extracting coordinates of variable dimensions from reference crystal structures, including layered LiCoOSEM image and energy-dispersive X-ray analysis (EDX) data were collected with scanning electron microscopy  at the Electron Microscopy Center, Argonne National Laboratory. EDX data were acquired at 12 kV.3.G(r) functions for Co-OEC formed in the presence of phosphate (pH = 7.0), methylphosphate (pH = 8.0) or borate (pH = 9.2) as the proton accepting oxyanion, following procedures described previously  are shown in Fig. S1 of the supporting information. The morphologies and elemental compositions for the CoPi, CoMPi and CoBi OECs analysed in this study correlate to those reported earlier  occur at 1.91 (2) and 2.81 (2) Å, with uncertainty from PDF measurements on four different CoPi samples electrochemically deposited and processed using equivalent conditions. These values correspond closely to the first coordination shell Co—O, 1.89 (1) Å, and μ2-oxo-bridged Co—Co, 2.81 (1) Å, distances, measured by XAFS  pattern for the CoPi measured here reproduces the PDF features described previously  pattern for the CoPi in Fig. 2et al., 2012G(r) intensity pattern for the P—O and Co—O bonded pairs is consistent with Co:P elemental analyses that report Co:P ratios in the range 2:1 to 3:1  measurements for the CoMPi and CoBi in Fig. 2Figs. S5 shows representative PDF patterns measured for pairs of different CoPi, CoMPi and CoBi films formed from equivalent electrolysis conditions. Some variability is seen in relative peak intensities. This variability could arise from a variety of causes, including inconsistencies in experimental background and other data corrections, variation in domain structure induced by the rinsing and dying procedures used to make the ex situ powders for PDF measurements, and presumably also from the heterogeneous nature of the amorphous films which may yield slightly varying distributions of domains in individual experiments. In spite of these variances, the PDF patterns show a clear trend for increasing the dimension of the domains following the sequence: Pi < MPi < Bi. This indicates an important role for the anions, or possibly the accompanying pH and electrochemical conditions, in determining the average domain size during film deposition. The longest atom-pair distances in the domain model (2) used to fit the PDF pattern , used for the PDF analysis and measured for CoPi, CoMPi and CoBi OECs. The identification of markers for structure changes in unprocessed experimental I(q) patterns is a useful check on structure characterization as it provides a means to recognize elements of structure change without the data processing and parameterization needed for PDF analysis. The experimental I(q) measured for each Co-OEC shows characteristic features, including a scattering peak at about q = 1.36 Å−1 which appears with increasing amplitude in the CoMPi and CoBi OEC, but is absent for the scattering data for the CoPi.Fig. 3I(q) and PDF patterns between CoPi, CoMPi and CoBi can be interpreted by comparison to I(q) and PDF calculated for domain structures extracted from layered cobaltate mineral structures. For example, Fig. 4I(q) and G(r) calculated using the LiCoOO crystal structure  and G(r) were calculated for domain model 1 in monolayer, bilayer and trilayer stacked assemblies, keeping the domain size constant in each layer, and using layer positioning determined by the LiCoOO crystal structure. The domain model structures are illustrated in the inset in Fig. 4a), top. The interlayer lithium ions were excluded from the calculations. The calculated I(q) patterns, Fig. 4a), show that the introduction of domain stacking is correlated with the appearance of a new scattering peak at a small angle, q = 1.31 Å−1. The line shape and position of this peak is a function of the number of layers in the stack and the inter-layer distance. By extending the assembly beyond three layers, the interlayer interference is reinforced and the first scattering interference peak narrows and progressively evolves into a Bragg diffraction layer peak. The shape of the first scattering peak can be altered and additional scattering features can be introduced by adding slip-stack offsets along the crystallographic a and b directions or by varying the dimensions of individual layers in the bilayer and trilayer stacks. However, the symmetry of the experimental I(q) first interference peak for CoBi and CoMPi suggests that these effects are largely averaged out, and that the first interference peak in the I(q) provides an experimental marker for layering in amorphous cobalt oxides.The characteristic changes in the experimental Figs. S7 by a comparison of experimental scattering for the CoPi, CoMPi and CoBi OEC to calculated scattering curves using the reference model 1 monolayer, bilayer, trilayer domains, but applied to CoOOH and LiCoOO mineral structures. The stacking of CoOOH and LiCoOO differ by the inter-layer distance, 4.38 and 4.68 Å, respectively, and have different offset alignment between adjacent layers. For both models the introduction of domain stacking is associated with the appearance of an additional scattering peak in the low q region, with the peak position correlated to the inter-layer spacing. Model domains composed of four and more layers gave linewidths and intensities which are narrower and more intense than those seen in the CoMPi and CoBi data. Variations are seen in the amplitude of the layering peak in CoBi in different samples, indicating that the extent of domain layering in the CoBi OEC is variable in different sample preparations. The correlation between this layering variation and catalytic activity is significant, and will be described in a subsequent publication. Here we focus primarily on developing the protocol for mesoscopic scale structure analysis.The sensitivity of the scattering peaks as markers for domain stacking is shown in q = 1.36 Å−1 compared with the interference peak at q = 2.7 Å−1. The amplitude and shifted peak position of the layered interference peak for the CoMPi compared with CoBi OEC can be interpreted in this context as a lower extent of stacking and a shift toward the smaller bilayer form in CoMPi. The absence of this peak for the CoPi confirms the monolayer character of this domain. We note here that the first I(q) interference peak for CoBi, q = 1.36 Å−1, is intermediate, but somewhat better aligned to CoOOH, q = 1.38 Å−1, than LiCoOO, q = 1.31 Å−1, tri-layer stacks. I(q) calculated from comparable models using the NaCoOO and KCoOO minerals have much larger interlayer spacing of 5.50 and 6.91 Å, respectively, and could be ruled out as candidate models for CoMPi and CoBi. In addition, the I(q) comparison shown in Fig. S7 suggests that the CoOOH stacked model is somewhat better aligned to the CoBi experiment in the low q region 2.0 < q < 4.5 Å−1. However, at higher q the I(q) for LiCoOO appears to be qualitatively closer to CoBi. For example, the CoOOH models have a I(q) peak at 9.5 Å−1, which is absent in the LiCoOO model and in the CoBi data. Both higher angle scattering features and effects of layered lattice structures can be further analysed in the PDF.For the data shown in Fig. 3b) shows the progressive change in calculated PDF patterns for LiCoOO domain models with different extents of stacking. This sequence shows analogies to those seen in the PDF for the experimental sequence CoPi, CoMPi and CoBi OEC, Fig. 2e compared with d, Fig. 4b). This relative intensity change is also seen in the experimental PDF, Fig. 2g compared with f, is still detected in the PDF for the layered structures. This is further shown by the analogous comparison of monolayer, bilayer and trilayer LiCoOO stacking models, but using individual layers having the edge distortions shown as in model 2, Figs. S8. This confirms the presence of stacking within a LiCoOO model; layer stacking peaks do not interfere with the use of the PDF peak g as a marker for edge or terminal oxygen atom distortions. We note that the introduction of stacking in the LiCoOO model calculations is also correlated with several longer-range, inter-layer atom pair correlations. This pattern of additional pair correlations is not seen in the data. However, these longer-range pair correlations are dependent upon the details of the overlap, alignment and disorder parameters between adjacent layers. We expect that a quantitative modelling would need to include the effects of stacking alignment disorders.Fig. 4Fig. S9 shows the PDF patterns for the CoOOH domain model in monolayer, bilayer and trilayer stacks. Inter-layer pair correlation peaks are seen that overlap those from the lattice in the individual layers. The prominent distinction in PDF markers for stacking in CoOOH and LiCoOO domain models arises because of the differences in inter-layer a–b plane alignments in the mineral structures. These result in the shift in positions for the inter-layer atom pair distance peaks. Interestingly, these different PDF markers for domain stacking can be used to distinguish between CoOOH and LiCoOO domain stacking models. The comparison of experimental and calculated PDF patterns show that domains in the CoPi, CoMPi and CoBi OEC differ in domain size and extent of layering, and can be qualitatively described using a LiCoOO layered model. However, a quantitative model may need to consider structures with intermediate protonation states between CoOOH and LiCoOO.The qualitative agreement between the sequence of PDF patterns for CoPi, CoMPi and CoBi OEC and stacking in the LiCoOO model can be contrasted with the markers for stacking in the CoOOH domain model. a) shows the overlap of the lattice structures for CoOOH and LiCoOO using the domain model 1. CoOOH compared with LiCoOO has a slightly expanded domain size that occurs because of an increased di-μ-oxo linked Co—Co distance of 2.851 Å compared with 2.816 Å for LiCoOO. The PDF patterns for the two model domains are shown plotted in Fig. 5b), along with the PDF measured for the CoPi. Remarkably, the PDF measured for the CoPi distinguishes between the CoOOH and LiCoOO lattice models. In particular, the LiCoOO model is found to provide a better match of the experimental di-μ-oxo linked Co—Co distance, and longer range Co—Co distances, including that between Co1 and Co11 (or Co12) annotated in Fig. 5a), and positioned at 10.1 Å in the experimental PDF, compared with 10.15 Å for the LiCoOO PDF peak, and 10.26 Å for CoOOH. These distance differences are very small, but potentially of interest since they reflect structure changes arising from changes in electronic structure for the cobaltate layer. For this reason, the development of techniques that track the changes in amorphous oxide domain structures could provide insights into unravelling the sources for enhanced catalysis. Amorphous cobalt oxides are often described as cobalt oxyhydroxides (Farrow et al., 2013et al., 2011et al., 2013Finally, lattice layers for CoOOH and LiCoOO differ in the details of structure that are discernible as small differences in atom pair distances in the PDF. These can be compared with the PDF for the amorphous cobalt oxide OEC. Fig. 54.et al., 2013−1, and examined domain structures for cobalt OEC formed in the presence of the potassium salts of Pi, MPi and Bi. The films were found to be composed of analogous cobaltate domains that increase in size following the sequence of Pi < MPi < Bi, illustrated in Fig. 6The X-ray scattering and PDF analysis has characterized the changes in domain structure for amorphous cobalt oxide OEC upon replacement of Pi with MPi and Bi. Previous PDF analysis of CoPi and CoBi OEC found that the replacement of the oxyanion was accompanied by an increase in domain size for the CoBi compared with the CoPi, and was correlated with the appearance of a disordered layered domain structure in the CoBi (Farrow 10.1107/S2052520615022180/ps5049sup1.pdfSupporting information is provided with 9 figures as described in the text.. DOI:


Class A plexins (PlxnAs) act as semaphorin receptors and control diverse aspects of nervous system development and plasticity, ranging from axon guidance and neuron migration to synaptic organization. PlxnA signaling requires cytoplasmic domain dimerization, but extracellular regulation and activation mechanisms remain unclear. Here we present crystal structures of PlxnA  full ectodomains. Domains 1–9 form a ring-like conformation from which the C-terminal domain 10 points away. All our PlxnA ectodomain structures show autoinhibitory, intermolecular “head-to-stalk” (domain 1 to domain 4-5) interactions, which are confirmed by biophysical assays, live cell fluorescence microscopy, and cell-based and neuronal growth cone collapse assays. This work reveals a 2-fold role of the PlxnA ectodomains: imposing a pre-signaling autoinhibitory separation for the cytoplasmic domains via intermolecular head-to-stalk interactions and supporting dimerization-based PlxnA activation upon ligand binding. More generally, our data identify a novel molecular mechanism for preventing premature activation of axon guidance receptors. •Structural studies reveal a major ring-like conformation for PlxnA ectodomains•cis-interactions in vitro and on cell surfacePlxnA ectodomains make head-to-stalk •cis-interactions induces cell and growth cone collapseDisruption of PlxnA •PlxnA ectodomain structure and interaction enable autoinhibition and activation cis-interactions of the receptor ectodomains.PlxnA signaling, important in nervous system development and plasticity, requires multi-leveled regulation. Kong et al. reveal a novel mechanism for PlxnAs, in which autoinhibition pre- and activation post-ligand binding are achieved through distinct conformations and  The guidance of axons to their synaptic targets is an important event during neural circuit development. Axon guidance cues are also implicated in processes such as neuronal migration and polarization, neurite growth and pruning, and synaptogenesis . Cell suSemaphorins form the largest family of secreted and membrane-anchored axon guidance cues and generally require plexins as cell surface receptors in neurons . Plexins1-4, comprises the ligand-binding sema domain, followed by a PSI, IPT, and a second PSI domain  extracellular segments of PlxnA1, PlxnA2, and PlxnA4. Negative stain electron microscopy (EM) confirms this ring-like structure as the predominant form . Our crystal structures also reveal an intriguing intermolecular “head-to-stalk” interaction common to all PlxnAs. We confirm this interface mediates PlxnA–PlxnA interactions and occurs pre-semaphorin binding using structure-guided mutagenesis, biophysical assays, and fluorescence microscopy-based live cell imaging. Functional assays  confirm that intermolecular 1-10 that diffracted otherwise at best to 6 Å resolution (see 1-4 (3OKT), PlxnA21-2 (3AL9), and additional higher resolution crystal structures that we determined of portions of PlxnA1 and PlxnA2; that is, PlxnA24-5 to 1.36 Å resolution and PlxnA17-10 to 2.2 Å resolution , each interdomain interface having a small hydrophobic core. These substantial domain–domain interactions likely contribute to conservation of the overall curved ring-like structure among the different PlxnA members and crystal forms. Some flexibility in the stalk is evident on comparison of the 13 different structures and contributes to the ring diameter ranging from 84 Å to 99 Å . This relative compaction can, most likely, be attributed to the dehydration procedure applied to generate this crystal form. In all other structures, the plexin extracellular domains associate only via the consecutive domain-domain interactions. Thus, contrary to previous speculations on plexin ectodomain structure  together with domains 2–9  lie in a 230 Å long curve that forms a ring-like conformation A and S1A to 99 Å C and S1A to 99 Å B. The ritructure , in the 7-10. Possibly this distinctive orientation is stabilized by a loop  extending from IPT6 that provides hydrophobic interactions with three residues in domain IPT5, Trp1055, Ile1057, and Tyr1145  is positioned perpendicular to the ring formed by domains 1 to 9, and the IPT6 C terminus points away from the plane of the ring. This out-of-the-ring orientation of IPT6 is observed in all five PlxnA ectodomain structures for which the electron density is sufficiently well ordered to allow IPT6 to be resolved. The same relative domain orientation recurs in the structure of PlxnA1 Tyr1145 B and S1C1-10 with negative stain EM  conformations . From the sema domain onward, the stalk region extends in a range of curved conformations, resulting, as in the crystal structures, in some variation in ring circumference. A second species, which includes 30.5% of the particles, can be identified by its distinct chair-like conformation. The N-terminal half of the stalk region curves out from the sema domain as in the ring-like structures, but the C-terminal half appears twisted by a 180° flip so that it curves away rather than completing the ring. No clear class averages could be generated for the remaining 3.5% of the particles. The PlxnA1-10 crystal structures could be fitted to the class averages by assuming two flexion points, one between domains 1 and 2 (sema–PSI1) and another at the domain 5–domain 6 (IPT3–PSI3) junction  and a complementary electrostatic charge distribution  or the PSI2 domain (PlxnA11-10 F693N/E695S) can block the PlxnA1–PlxnA1 interaction using multi-angle light scattering (MALS) experiments coupled to size-exclusion chromatography  and the PSI2-IPT2 domains (PlxnA24-5); each segment encompasses one side of the putative “head-to-stalk” intermolecular interface. We found that PlxnA21-2 and PlxnA24-5 associated with an equilibrium dissociation constant (Kd) of 20.9 ± 0.38 μM (4-5 completely abrogated the interaction with PlxnA21-2 (1-10 (PlxnA11-10 F693N/E695S). Taken together, the results of our in solution assays point to a head-to-stalk mode of interaction between receptor ectodomains that is common to all PlxnAs.We then undertook surface plasmon resonance (SPR) binding equilibrium experiments to probe the sema–IPT2-PSI2 interface and determine the affinity of interaction. We engineered two separate segments consisting of the PlxnA2 sema-PSI1 domains  AIV. Thes of 0.16 AIII. Sim = 0.16) AV. Thesecis-interactions that we observed using various approaches could function to impose pre-signaling autoinhibition. To test this model at the functional level, we first used COS-7 cells as a heterologous system that mimics growth cone collapse (FL) displayed a normal, spread-out morphology  results in robust cell contraction  granule cells from the hippocampus were incubated with wild-type PlxnA14-5 (domains PSI2-IPT2) or its interaction-blocking mutant, PlxnA14-5 F693N/E695S  was predicted to bind full-length PlxnA molecules on the growth cone surface, thereby counteracting cis-interactions between PlxnA molecules  and leading to receptor activation; DG granule cells were used because they endogenously express various PlxnAs and are responsive to a wide range of PlxnA ligands, including Sema3s and Sema6s  class cell-surface receptors, and studies in the past decades have shown that their signaling is often triggered, upon ligand binding, by receptor dimerization followed by clustering. Despite considerable progress in our understanding of the structure and function of axon guidance receptors, the multifaceted regulatory mechanisms that must underlie axon guidance receptor signaling remain poorly understood. These include the mechanisms that prevent premature signaling or that translate ligand binding into the activation of downstream signaling. To address these questions, we have focused on semaphorins and their PlxnA receptors, which have been implicated in the control of various aspects of neural circuit development . Thus fa1-10 also shows this ring-like state to be the favored ectodomain conformation. The sequential arrangement of ten domains to form a well-defined, open ring is unexpected and striking. This distinctive ectodomain architecture immediately provides a satisfyingly simple solution to a longstanding puzzle. The precise arrangement of bivalent semaphorin interfacing with two diverging PlxnA1-4 ectodomain fragments is conserved for Sema6A- and Sema3A-containing complexes  of protein solution with 100 nl reservoir solution at 20.5°C or 4.0°C. Optimization experiments including lysine methylation of protein samples and crystal dehydration are detailed in the Mouse PlxnA1, PlxnA2, and PlxnA4 were produced using the pHLsec vector  and expr1-10 were first solved by molecular replacement in PHASER (1-4 (3OKT) (54% sequence identity with PlxnA11-4). To solve the structures of the remaining stalk region, homology models of PlxnA1 domains IPT2-IPT5 were placed by molecular replacement in PHASER, and the resulting structure was further optimized by manual rebuilding in COOT  was coupled to static light-scattering , differential refractive index , and Agilent 1200 UV (Agilent Techologies) detectors. Data analysis was done using the ASTRA software package (Wyatt Technology).AUC sedimentation velocity experiments were conducted using a Beckman Optima XL-I analytical ultracentrifuge and an An-60 Ti rotor (Beckman) in absorbance mode (280 nm incident light) at 40,000 rpm rotor speed. A total number of 100 scans were collected with one scan every 6 min. Scans 6–100 were analyzed with Sedfit  and a c. For the4-5 were enzymatically biotinylated with BirA via an engineered C-terminal tag and attached to streptavidin covalently coupled to the surface of the SPR chip. PlxnA2 was used for SPR experiments because the PlxnA21-2 and PlxnA24-5 segments were much better expressed than their PlxnA1 counterparts. Data analysis was done using Biacore™ T200 Software v2.0 with the nonlinear curve fitting of a 1:1 Langmuir binding model (d) and the maximum analyte binding value (Bmax) via equation bound” is measured in response units (RU) and CA is the concentration of analyte.SPR equilibrium experiments were performed on a Biacore T200 instrument  at 25°C in SPR running buffer  Tween 20) and regeneration buffer (2 M magnesium chloride). Wild-type and mutant PlxnA2ng model  to calcufD) were calculated as described in in the COS-7 cells expressing fluorescently tagged PlxnA2 were imaged using a SP8-SMD Leica microscope from Leica Microsystems  together with a 63×/1.4 oil immersion objective, a 440 nm pulsed laser at 40 MHz coupled with single photon counting electronics (PicoHarp 300) to excite mTFP1, and a Leica Hybrid external detector to record photons. To achieve high signal to noise and minimize artifacts, only cells with 700–1,000 photons per pixel and negligible amount of bleaching after a 2 × 2 image binning were included in the analysis . The mea4 cell/cm2 in a 12-well plate. At day in vitro (DIV) 1, cells were transfected with 0.5 μg of PlxnA4 constructs in pcDNA3.1 (+) plasmids together with eGFP using X-tremeGENE™ 9 (Roche) following the manufacturer’s instruction. One day after transfection, cells were fixed in 4% PFA and immunostained for GFP. Images were acquired with a 40× objective with AxioScopeA1 (Zeiss) microscope. Cell area was measured using ImageJ as described previously (COS7 cells were cultured in DMEM (high glucose) supplemented with 10% FCS, penicillin-streptomycin, and L-glutamine. Cells were plated on glass coverslips at a density of 0.5–1 × 10eviously .2. On DIV1, cells were treated with vehicle or wild-type or mutant PlxnA14-5 recombinant proteins (3 mg/ml) for 30 min at 37°C, fixed in 4% PFA and sucrose for 20 min at room temperature, and immunostained with anti-βIII-tubulin antibodies and phalloidin. Images were acquired using a 100× objective on an AxioScopeA1 (Zeiss) microscope and analyzed using a growth cone morphology matrix (DG granule cell cultures were prepared as described . In briey matrix  in an obY.K., B.J.C.J., T.M., R.J.P., and E.Y.J. designed the experiments. Y.K., B.J.C.J., and T.M. performed structural and biophysical measurements. Y.K. carried out negative stain EM and light microscopy experiments. R.K. helped to perform localization experiments, C.H.C. was involved in negative stain EM, T.N. was involved in AUC experiments, R.J.C.G. helped interpret EM and AUC data, and S.P.-P. guided and interpreted the FRET-FLIM experiments. V.R.V. conducted COS-7 cell and DG growth cone assays. T.M. and V.R.V. contributed equally to this work. Main authors responsible for writing and editing the paper are Y.K., B.J.C.J., T.M., R.G., S.P.-P., R.J.P., and E.Y.J.


C. diphtheriae have been determined at 1.87 and 2.5 Å resolution. The N-terminal domain is found to contain an isopeptide bond that forms slowly over time in the recombinant protein. Given its structural context, this provides insight into the relationship between internal isopeptide-bond formation and pilus assembly.Two crystal structures of the major pilin SpaD from  Corynebacterium diphtheriae, the cause of diphtheria in humans, expresses pili on its surface which it uses for adhesion and colonization of its host. These pili are covalent protein polymers composed of three types of pilin subunit that are assembled by specific sortase enzymes. A structural analysis of the major pilin SpaD, which forms the polymeric backbone of one of the three types of pilus expressed by C. diphtheriae, is reported. Mass-spectral and crystallographic analysis shows that SpaD contains three internal Lys–Asn isopeptide bonds. One of these, shown by mass spectrometry to be located in the N-terminal D1 domain of the protein, only forms slowly, implying an energy barrier to bond formation. Two crystal structures, of the full-length three-domain protein at 2.5 Å resolution and of a two-domain (D2-D3) construct at 1.87 Å resolution, show that each of the three Ig-like domains contains a single Lys–Asn isopeptide-bond cross-link, assumed to give mechanical stability as in other such pili. Additional stabilizing features include a disulfide bond in the D3 domain and a calcium-binding loop in D2. The N-terminal D1 domain is more flexible than the others and, by analogy with other major pilins of this type, the slow formation of its isopeptide bond can be attributed to its location adjacent to the lysine used in sortase-mediated polymerization during pilus assembly.The Gram-positive organism  One of the most prominent of these is the bacterial pilus, a long, adhesin-tipped protein filament that extends several micrometres from the cell and aids colonization in motile environments  near the C-terminus of the pilin subunit, cleaves this motif between Thr and Gly, and joins the new Thr carboxyl to the ∊-amino group of a conserved lysine in the N-terminal domain of the next subunit , they comprise an adhesin subunit located at the tip, referred to as a minor pilin, followed by many copies of a major pilin that forms the polymeric shaft and finally a second minor pilin that terminates pilus extension and mediates cell-wall anchoring at the base  side chains that form internal cross-links within each domain . For the SpaD D1 construct encoding residues 49–183, the following primers were used: forward, 5′-TTCCAAGGTCCGGAACGAAAGGGCTCGCTGA-3′; reverse, 5′-GAAAGCTGGGTGCTAGGTTTCGGTGTTCTTCGG-3′. Another round of PCR was performed using the generic primers containing attB sequences for Gateway cloning and the sequence encoding an N-terminal, human rhinoviral (HRV) 3C protease site: forward, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTCTCTGCAGGTACTCTTCCAAGGTCCG-3′; reverse, 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTG-3′. The final PCR product was first cloned into the entry vector pDONR221 (Invitrogen) via a BP reaction, and an LR reaction was then used to clone into the expression vector pDEST17 (Invitrogen) for expression of a His-tagged construct. The final constructs were transformed into Escherichia coli BL21 (DE3) cells. The full-length native SpaD was overexpressed in ZYP-5052 autoinduction media  and incubation at 18°C for a further 60 h. All of the culture medium were supplemented with ampicillin (100 µg ml−1) and chloramphenicol (34 µg ml−1).The DNA sequence of SpaD (gi:38232859) was amplified from 2.2.2+, and the His-tagged SpaD proteins were eluted with a gradient of 20–500 mM imidazole. To remove the His tag, the eluted proteins were mixed with human rhinovirus (HRV) 3C protease (1 mg per ∼50 mg SpaD), 10 mM DTT and 2 mM EDTA and dialyzed against 50 mM Tris–HCl pH 8.0, 300 mM NaCl overnight at 277 K. For SeMet-substituted SpaD, the buffers were supplemented with 0.4 mM DTT from this step to prevent selenomethionine oxidation. The dialyzed sample was passed through a charged HisTrap column to collect the flowthrough containing untagged SpaD. Further purification was carried out using size-exclusion chromatography (SEC) on a Superdex 75 10/300 column  with SEC buffer . The final product contained two additional N-terminal residues, Gly and Pro, after His-tag removal.The SpaD proteins were purified using similar procedures to those used for the purification of SpaA , 10% PEG 4000. Crystals started to appear after 1–2 weeks in 2 µl sitting drops consisting of a 1:1 mixture of protein solution and reservoir solution at 18°C. When cryocooled in liquid nitrogen using standard cryoprotection protocols (reservoir solutions containing either 25% ethylene glycol or glycerol for native SpaD and 40% MPD for the SeMet protein), these crystals showed severe anisotropy, diffracting to 3 Å in one direction but typically to 8 Å in the other. The Bragg spots were also too close together to be resolved owing to high mosaicity and a long unit-cell edge.SpaD was initially crystallized by vapour diffusion using sitting drops in 96-well Intelli-Plates (Hampton Research) at 291 K set up with a Cartesian Honeybee dispensing system (Genomic Solutions). After optimization, full-length native SpaD protein was crystallized at a protein concentration of 77 mg mlet al., 2005In order to improve the data quality, the crystals were cryocooled under high pressure at the Cornell High Energy Synchrotron Source  and ambient pressure with an oscillation angle of 0.5° per image. Bragg diffraction spots were visible up to 2.1 Å, but anisotropic diffraction was observed beyond 2.8 Å. Therefore, the data set was cut off at 2.5 Å for structure determination and refinement.Diffraction data were collected from the high-pressure cryocooled SpaD crystals on beamline F1  at the Cornell High Energy Synchrotron Source (CHESS). All data were collected at 90 K (N2.4.w:w) ratio and incubating at 37°C for up to 20 h. Analysis by SDS–PAGE revealed a single strong band corresponding to a molecular weight smaller than the full-length SpaD. This was shown by mass spectrometry of the digested products to be a species with a molecular mass of 30 456.00 Da. This closely matches the theoretical mass (molecular mass of 30 457.10 Da) of residues 180–455 of SeMet SpaD, after accounting for the loss of 34 Da from two isopeptide bonds. For crystallization of this species, SeMet SpaDtryp, 1 ml of 7.0 mg ml−1 SeMet SpaD in SEC buffer was digested with 60 ng trypsin for 20 h, followed by concentration to 40 mg ml−1 using a 10 kDa molecular-weight cutoff concentrator. No further purification was carried out. Crystallization was then performed with the Cartesian dispensing robot using sitting drops comprising 100 nl protein solution and 100 nl reservoir solution (47% MPD and 2% t-butanol) at 18°C. The crystals were cryocooled by plunging them into liquid nitrogen without further cryoprotection and proved to be of high quality, diffracting to better than 2.0 Å resolution. An initial data set was collected in-house using a Rigaku MicroMax-007 HF generator with a rotating Cu anode (Rigaku) and a Mar345dtb image plate (MAR Research), covering a total rotation range of 200° with 0.5° oscillation. Following analysis, a second data set was collected using 1° oscillation images, covering a total oscillation of 864°.In order to identify a stable fragment of SpaD, limited proteolysis was performed by adding trypsin (Promega) to full-length SeMet-substituted SpaD in a 1:300. autoSHARP , final values of Rwork = 0.157 and Rfree = 0.190  were obtained. The final model of SeMet SpaDtryp comprised residues 184–455 of SeMet SpaD, which is consistent with the MS analysis of the digested product.Data sets collected from SeMet SpaDXDS . The structure was solved by molecular replacement with Phaser . Validation with MolProbity  was used.Native full-length SpaD diffraction data from the high-pressure crystals were processed using 2.6.BioAnalyst software (Applied Biosystems). For the MS analysis of protein crystals, the crystals were washed briefly in acetonitrile several times and then dissolved in deionized water. For identifying isopeptide bond-containing peptides, samples of purified SpaD pili or recombinant SpaD protein were digested and analysed in a similar way to that previously described for SpaA  and AspN endopeptidase (Roche) dissolved in 25 mM NH4HCO3 and 10% acetonitrile. The digested samples were analyzed using a Q-STAR XL Hybrid MS/MS system (Applied Biosystems) as described in Kang, Paterson, Gaspar et al. (2009The molecular masses of the intact proteins in solution were determined by infusion ESI-TOF mass spectrometry (MS) on a Q-STAR XL Hybrid MS/MS system (Applied Biosystems) in 50% acetonitrile and 0.1% formic acid. The raw MS data were deconvoluted using the Bayesian Protein Reconstruct tool in the  al. 2009.3.3.1.C. diphtheriae SpaD is a 490-residue protein containing a predicted N-terminal signal peptide of 32 residues and a sortase-recognition motif LPMTG encompassing residues 458–462. Post-expression processing yields a 429-residue mature protein comprising residues 33–461. After testing several different constructs of SpaD, we were able to obtain diffraction-quality crystals from a construct containing residues 27–455, comprising most of the mature full-length SpaD but lacking the C-terminal sortase motif residues. Both native and SeMet-substituted full-length SpaD were purified from E. coli and crystallized in space group P212121. Typical X-ray diffraction at synchrotron light sources was anisotropic with very high mosaicity, hindering data processing and precluding structure determination by SAD/MAD phasing.tryp) was crystallized in space group P212121 and its structure was solved by single-wavelength anomalous diffraction (SAD) methods from in-house data collected at a wavelength of 1.5418 Å, utilizing the signals from one Se atom, one Ca2+ ion and two S atoms (from a disulfide bond). The model, comprising residues 184–455, was refined using data to 1.87 Å resolution .We therefore sought to prepare a stable truncated fragment of SpaD that could be crystallized and used to solve the structure of the full-length protein by molecular replacement. This was achieved by partial trypsin digestion of the full-length selenomethionine-labelled form of SpaD (SeMet SpaD). Mass spectrometry confirmed the mass of the resulting product to be 30 456.00 Da, corresponding to residues 180–455. This truncated form of SpaD  as a search model. This full-length structure, which contains two SpaD molecules in the asymmetric unit, was refined at 2.5 Å resolution . In the final model, one protomer is complete apart from residues 27–41 (the N-terminus) and 437–439, whereas in the other a large loop between residues 63 and 76, smaller loops between residues 212 and 214 and residues 435 and 436, and the C-terminal residues 454–455 could not be modelled owing to insufficient density. Full refinement statistics are given in Table 1Improved crystals of the full-length native SpaD were then obtained by cryocooling the crystals under high pressure  has the typical CnaA fold , however. Aside from this, the two copies of SpaD in the asymmetric unit are very similar, with r.m.s.d.s of 0.28 Å between the two D1 domains over 126 Cα atoms and of 0.51 Å between the two D2/D3 domain pairs over 264 Cα atoms.The principal axis of the N-terminal D1 domain is angularly disposed with respect to that of the D2 domain that follows, with D1 positioned above the opening of the D2 β-barrel. In contrast, domains D2 and D3 are almost linearly connected, with strands β17 and β18 running through the length of both domains Fig. 1. The angC. diphtheriae . The major pilin FimP of Actinomyces oris . The calcium ion was identified by the electron density in conjunction with an average metal–ligand bond length of 2.27 Å for the main-chain carbonyls and 2.43 Å for the water and carboxyl group. The calcium ion was also used during phasing of SeMet SpaDtryp and gave rise to an anomalous signal consistent with that expected from calcium at an X-ray wavelength of 1.5418 Å. Although SpaA also contains a calcium-binding site, location is different from that in SpaD. The calcium site in SpaA . A disulfide bond is found at the analogous position in SpaA, but that in SpaD is fully formed as opposed to the partially formed bond observed in SpaA . In contrast, the cis isopeptide bond of the D2 domain forms two hydrogen bonds through its carbonyl and amido groups to the two carboxyl O atoms of Asp224 .Both isopeptide-bond stereoisomers are present in SpaD, with the D1 and D3 domain bonds having the 24 Fig. 2b.3.4.Each of the three SpaD domains contains the conserved Lys–Asn–Asp/Glu triad characteristic of isopeptide bond-forming domains of this type, and all three bonds are fully formed in the crystal structure. However, mass spectrometry of freshly purified SpaD revealed the presence of two partially overlapping charge envelopes Fig. 3. DeconvoThe intensities of the deconvoluted peaks were noticeably different; the protein with two isopeptide bonds was more abundant than that with three, with an approximate ratio of 3:1. When SpaD was incubated at room temperature or 37°C, however, the peak ratio changed and more of the three-isopeptide-bond species were observed Fig. 3. All thrC. diphtheriae, but have not been successful and are unable to confirm whether the polymerized SpaD subunits in native pili have all three isopeptide bonds fully formed.The most likely candidate for the slow-forming bond is that in the D1 domain, by analogy with other three-domain and four-domain major pilins, such as GBS80, SpaA, BcpA and RrgB, whose N-domains either display no isopeptide bond or lack isopeptide-forming residues. Consistent with this, we detected a mixture of peptides with and without the Lys58–Asn180 isopeptide cross-link in domain D1 when the full-length protein was analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) following a proteolytic digestion Fig. 3. In addi3.5.f; Gaspar & Ton-That, 2006a and 4b), as is seen in many other major pilin crystal structures, and as would be expected in the authentic biological assembly following polymerization. In the crystal structure, the carboxyl-terminus of one molecule, which precedes the sortase-recognition motif in the construct analysed here, is located close to the D1 domain of the following molecule, near the start of a groove that leads to Lys179 from the YPKN motif, with the distance between these residues being consistent with the number of C-terminal residues missing from the model .Like other most major pilins, SpaD also contains within its N-terminal D1 domain the canonical YPKN pilin motif that contains the lysine residue, Lys179, used to form the sortase-mediated intermolecular isopeptide bond during polymerization . It is also reflected in the higher average B factors for the D1 domains relative to the D2/D3 domains (56.4 versus 45.8 Å2).A feature of the SpaD structure is that the D1 domain, as in other major pilins in which the N-terminal domains have been modelled, is more flexible than the other domains. This is seen in the variable orientation of D1 relative to D2/D3 in the two SpaD molecules, and in comparison with the homologous SpaA Fig. 4d. It isB factors. This loop extends over Lys179, restricting external access to it . The equivalent loops in SpaA and FimP could not be modelled owing to poor density , RrgB and FimP, attest to the greater mobility of this domain, as does an NMR analysis of the D1 domain of RrgB . Moreover SpaA, a close homologue of SpaD, does not contain isopeptide bond-forming residues in its N-terminal domain, whereas most of its homologues do (Kang, Paterson, Gaspar et al., 2009et al., 2012There are clearly differences from one major pilin to another, since the D1 bond forms more readily in SpaD than in RrgB in the absence of any protein–protein interaction. RrgB can also be readily polymerized Finally, it is important to note that the intermolecular isopeptide-bond linkages between successive subunits in the pilus polymer involve a lysine that is in most cases close to the boundary between the N-terminal domain and the following D2 domain. This means that the load-bearing ‘spine’ of the pilus does not pass through the N-terminal domain, but extends through the following domains, all of which are strengthened with internal isopeptide-bond cross-links. The role of the N-terminal domain may be to provide the site for sortase action, and possibly contribute to pilus morphology, making the presence or absence of an isopeptide cross-link less important.SpaD, 4hsqPDB reference: 4hssPDB reference:


Eukaryotic cells have complicated membrane systems. The outermost plasma membrane contains various substructures, such as invaginations and protrusions, which are involved in endocytosis and cell migration. Moreover, the intracellular membrane compartments, such as autophagosomes and endosomes, are essential for cellular viability. The Bin-Amphiphysin-Rvs167 (BAR) domain superfamily proteins are important players in membrane remodeling through their structurally determined membrane binding surfaces. A variety of BAR domain superfamily proteins exist, and each family member appears to be involved in the formation of certain subcellular structures or intracellular membrane compartments. Most of the BAR domain superfamily proteins contain SH3 domains, which bind to the membrane scission molecule, dynamin, as well as the actin regulatory WASP/WAVE proteins and several signal transduction molecules, providing possible links between the membrane and the cytoskeleton or other machineries. In this review, we summarize the current information about each BAR superfamily protein with an SH3 domain(s). The involvement of BAR domain superfamily proteins in various diseases is also discussed. The relationship between cellular morphology and diseases, such as cancer, has been unclear. However, transformed cancer cells are often first recognized by changes in their morphology. The shapes of transformed cancer cells are apparently different from those of the normal parental cells, as observed microscopically. The shape of the eukaryotic plasma membrane changes during various processes, such as cell division, cell movement, and differentiation. Eukaryotic cells also contain various intracellular vesicles that synthesize, traffic, and degrade materials, such as proteins, during membrane remodeling, including the fission and fusion of vesicles . DynamicThe BAR proteins are evolutionarily conserved, from yeast to human ,2,3,4. TBAR proteins include subfamilies defined by their BAR, N-BAR/BAR (the Bin-Amphiphysin-Rvs167), F-BAR (extended Fes-CIP4 homology (EFC)/FCH-BAR), or I-BAR (IRSp53-MIM homology domain I-BAR/inverse-BAR) domains ,3,4. SH3The BAR subfamily contains the members that do not belong to the F-BAR and I-BAR subfamilies. The N-BAR domain is included in the BAR subfamily that contains amino acids that form an amphipathic helix upon membrane binding in front of the BAR domain fold. This helix is thought to enhance membrane-binding ability by its insertion into the membrane ,10. The 2]  . Further2] . The SH32] ,13. Amph2] . Recruit [et al. . The narIn addition to endocytosis, the N-BAR domain of amphiphysin is required for ruffle formation and phagocytosis, although its correspondence to the membrane curvature is unclear . Phagocyin vitro was originally reported by Takei et al. [amphiphysin gene in Drosophila disrupts the T-tubule network [2 binding is necessary to release the SH3 domain from the poly-basic region, enabling the interaction between the SH3 domain and DNM2. The existence of this intermolecular regulation was elucidated in cultured cells [The membrane tubulation activity of amphiphysin i et al. . Moreovei et al. . T-tubuli et al. . Interesi et al. . Amphiph network , and mis network . In addi network ,24. The  network . Indeed,ed cells .Endophilins are composed of an N-terminal BAR domain and a C-terminal SH3 domain. The BAR domain of endophilin A1 forms a wedge at the center of the BAR domain dimer, which is thought to be inserted into the membrane ,26,27. EThere are several possible links between diseases and endophilin A. Interestingly, it was recently reported that the SH3 domain of endophilin A binds with high affinity to Parkin, a protein linked to Parkinson’s disease , and alsIn contrast to endophilin A, endophilin B1, also known as Bif1 or SH3GLB1, interacts via its N-BAR domain with Beclin 1, the mammalian homologue of yeast Atg6 (autophagy-related gene 6), through the UVRAG protein (UV irradiation resistance-associated gene). This interaction regulates the formation of autophagosomes, by promoting the activation of phosphatidylinositol (3) kinase C3 (PI3 kinase C3) . Endophi2. The BAR and PX domains function as one unit with broad phosphoinositide specificity [Sorting nexins contain the BAR domain and the PX domain, but only SNX9 and 18 contain the SH3 domain. The BAR domain of the sorting nexin, SNX9, is in the proximity of the PX domain, which specifically recognizes phosphoinositides, such as PIPcificity ,37. The cificity . The BARcificity ,37,39. Scificity . The SH3cificity .SNX1 and SNX2 function in endosome trafficking. Although SNX1 and SNX2 lack the SH3 domain for N-WASP binding, another Arp2/3 complex activating protein, WASH, reportedly associates with SNX1 and SNX2 ,36. Tuba contains the BAR domain, four N-terminal SH3 domains, a DH domain and two C-terminal SH3 domains. The four N-terminal SH3 domains exhibit strong affinity for dynamin, the DH domain is a Cdc42 GEF, and the C-terminal SH3 domains bind directly to the N-WASP and Ena/VASP proteins. Tuba is localized at synapses and dorsal ruffles. The membrane deformation induced by the BAR domain of Tuba has not been elucidated yet . The rolAPPL1, Centaurin, and ASAP1 are composed of the BAR and PH domains. Among the three proteins, ASAP1 also contains an SH3 domain . The PH The BAR domain of APPL1 binds not only to lipids but also to small GTPases, using different surfaces. BAR domains of APPL1/2 interact with Rab5, which regulates endosomes maturation and fusion . In addi2, which stimulates the GAP activity [ASAP1 is an ArfGTPase activating protein (GAP) containing a BAR domain. The PH domain of ASAP1 binds to PIPactivity ,50,51. Tactivity  and CrkLactivity . These Sactivity . TherefoASAP3, which is closely related to ASAP1, is associated with focal adhesions and circular dorsal ruffles, but is not localized to podosomes . The redPICK1 contains a PDZ domain, but lacks an SH3 domain. The BAR domain of PICK1 reportedly interacts with the Arp2/3 complex to suppress the nucleation of actin filaments, thereby inhibiting the endocytosis of neurotransmitter receptors. The PDZ domain of PICK1 interacts directly with lipid membranes containing phosphoinositides, and the PDZ–lipid interaction is necessary for synaptic transmission ,59,60. TThe F-BAR-domain-containing proteins exist in all eukaryotes, except plants. These proteins are also known as Pombe/Cdc15 homology (PCH)-family proteins, from the founding member of this family .The F-BAR domain of FCHo2 also forms a crescent-shaped dimer, but the curvature of its membrane-binding, concave surface is smaller than those of the F-BAR domains of CIP4 and FBP17 . CorrespThe structures of the F-BAR domain from CIP4 and FBP17 were the first to be solved, among the F-BAR domains . The amiThe SH3 domains of FBP17 and CIP4 bind to dynamin, which antagonizes the tubulation activities of FBP17 and CIP4 ,68. The 2 may be required for the membrane localization of FBP17 as well as the WASP-WIP complex and dynamin-2, to determine where podosomes will form [FBP17, CIP4, and Toca-1 are involved in a variety of structures, in addition to clathrin-coated pits. The F-BAR and SH3 domains of FBP17 are essential for the formation of podosomes and phagocytic cups in macrophages . The micill form . et al. (2005) to involve disruption of Dynamin binding to Endophilin following SH3 domain phosphorylation by Src. This observation is consistent with CIP4 phosphorylation by Src to reduce MT1-MMP internalization [CIP4 localization to invadopodia was reported in breast cancer cells . Notablylization ,85.Although Toca-1 is thought to have a concave membrane-binding surface, the role of F-BAR domain-containing proteins, such as Toca-1, in filopodia formation has been suggested by reconstitution assay . This miSalmonella enterica serovar Typhimurium [Toca-1 promotes vesicle motility, filopodia and lamellipodia formation by recruiting N-WASP or Abi1, respectively ,91,92. Thimurium . The inthimurium . Moreovehimurium . In terms of their relationships to diseases, the SH3 domains of CIP4 bind to the huntingtin protein, which is mutated in patients with Huntington disease . CIP4 haThe syndapins (synaptic dynamin-associated proteins), also known as PACSINs (PKC and casein kinase substrate in neurons), have three isoforms. Whereas PACSIN1 is restricted to neurons, PACSIN2 is ubiquitously expressed and PACSIN3 is present in lung and muscle tissues . PACSINsStructurally, PACSINs have a concave membrane-binding surface, as in the other F-BAR domains, such as FBP17, CIP4, and FCHo2 ,103. TheHowever, overexpression of the full-length protein generates microspikes and lamellipodia-like structures, as well as cellular invaginations ,105. ForThe SH3 domain of PACSIN2/syndapin II also binds to N-WASP and dynamin . The SH3NOSTRIN contains an F-BAR domain and an SH3 domain, and is reportedly localized at caveolae. The SH3 domain of NOSTRIN binds to N-WASP and dynamin ,110. HowThe Fes/Fps and Fer proteins are a distinct family of non-receptor tyrosine kinases, with prominent roles in inflammation and immunity ,113,114.srgp-1  senses membrane invaginations through its BAR domain, and is involved in cell corpse clearance and sick-cell killing in C. elegans [The slit-robo GAP proteins, (srGAP)1–4, contain an F-BAR domain at the N-terminus, a RhoGAP domain in the middle and an SH3 domain at the C-terminus ,123. The elegans . srGAP2  elegans . srGAP3/ elegans . The ove elegans . The srG elegans .GAS7 contains the SH3 domain and the F-BAR domain. The SH3 domain is located at the N-terminus of the F-BAR domain, and this is the unique characteristic of GAS7. The structure of the F-BAR domain is unknown. The SH3 domain binds to N-WASP . GAS7 plPSTPIP1 was first identified as a tyrosine phosphorylated protein associated with F-actin . PSTPIP1In vitro, PSTPIP2 induces F-actin bundling, decreases the actin polymerization rate, and increases F-actin stability [PSTPIP2 shares about 41% sequence homology with its counterpart, PSTPIP . PSTPIP2tability . Moreovetability . In wholtability ,135. Mictability .Saccharomyces cerevisiae Cdc15, but lacks the long linker region between the F-BAR domain and the SH3 domain present in Cdc15. Cdc15 is localized at the contractile ring and is essential for cytokinesis [PSTPIP1 is localized to the cleavage furrow of cultured human cells . PSTPIP1okinesis ,139,140.okinesis . Cdc15 wokinesis . Cdc15 aokinesis . Saccharomyces cerevisiae proteins such as Cyk2/Hof1, Bzz1 and Rgd1–2 and Schizosaccharomyces pombe proteins such as Imp2, YB65 (pombe Bzz1p) and Rga7–9 [F-BAR domains are also found in various other d Rga7–9 ,145. Hofd Rga7–9 .The I-BAR domain binds to the membrane through its convex surface. The I-BAR domain, with the inverted geometry of the membrane-binding surface, as compared to most BAR and F-BAR domains, is involved in the plasma membrane protrusions of filopodia and lamellipodia. The structures of the I-BAR domains from IRSp53, MIM, and Pinkbar have been determined ,149,150 In vitro, the incubation of the I-BAR domain with liposomes induced membrane invaginations, which corresponded geometrically to cellular protrusions [2 and PIP3 was observed for the I-BAR of IRSp53 [trusions . The bintrusions . The I-Bf IRSp53 ,149,151.f IRSp53 ,153. Furf IRSp53 ,154. Whef IRSp53 ,156,157.f IRSp53 ,159. Thef IRSp53 ,148,149.f IRSp53 ,162,163.In cells, IRSp53 is involved in both filopodium and lamellipodium formation, as suggested from the localization and the binding of WAVE2, N-WASP, VASP, and Mena ,158,159.Dictyostelium discoideum, the IBARa protein (which contains an I-BAR domain) is involved in curvature sensing, and its SH3 domain recruits regulators of actin polymerization, including the Arp2/3 complex, at the neck of a particle during phagocytosis [The siRNA-mediated knockdown of IRSp53 also revealed its role in lamellipodia formation . Moreoveocytosis . 2, which it then deforms into tubular structures in vitro [MIM and ABBA lack an SH3 domain. Both MIM and ABBA are composed of a C-terminal actin-monomer binding WH2 (WASP homology 2) domain and an N-terminal I-BAR domain. The WH2 domain of MIM directly binds to actin ,169. In in vitro ,149,151.MIM is strongly expressed during development in muscles and postmitotic neurons, and in adult mice in the kidneys, liver, and Purkinje cells of the cerebellum ,170. Mou2-rich vesicles through electrostatic interactions. Due to its flat structure, the BAR domain of Pinkbar does not induce membrane protrusions or invaginations, and instead deforms phosphoinositide-rich membranes into planar structures [Pinkbar  contains an I-BAR domain and an SH3 domain. The I-BAR domain of Pinkbar is noticeably shorter (164 Å) than those of IRSp53 (182 Å)  and MIM ructures . The binPinkbar is expressed predominantly in intestinal and kidney epithelial cells, where it localizes to Rab13-positive vesicles and to the plasma membrane at the cell-cell junctions . The smaWe have summarized the current information available for the well-studied BAR proteins. More than 50 different types of BAR proteins are present in humans, and therefore we still lack a complete understanding of the membrane curvature generation and sensing mediated by BAR proteins. Several BAR proteins are associated with multiple structures, and thus the BAR proteins may simply function for the generation and recognition of membrane curvature, and might not have one-to-one relationships to subcellular structures. The various combinations of BAR proteins for each subcellular structure may provide its characteristic membrane shape.


Schistosomiasis is a chronic debilitating parasitic disease that afflicts more than 200 million individuals worldwide. Long-term administration of chemotherapy with the single available drug, praziquantel, has led to growing concerns about drug resistance. The PSD-95/Dlg/ZO-1 (PDZ) domain is an important module found in many scaffolding proteins, which has been recognized as promising targets for the development of novel drugs. However, the parasite-derived PDZ domains and their associated functions are still largely unknown.Schistosoma japonicum Scribble protein (SjScrib) was identified by homologous search with the S. mansoni Scrib sequence. By screening an arbitrary peptide library in yeast two-hybrid (Y2H) assays, we identified and confirmed the ligand binding specificity for each of the four PDZ domains of SjScrib. Both SjScrib-PDZ1 and SjScrib-PDZ3 recognize type I C-terminal PDZ-domain binding motifs (PBMs), which can be deduced as consensus sequences of -[Φ][x][E][TS][x][ILF] and -[x][RKx][ETS][T][WΦ][ILV], respectively. SjScrib-PDZ2 prefers stringent type II C-terminal PBMs, which significantly differs from that of its human ortholog. SjScrib-PDZ4 binds to typical II C-terminal PBMs with a consensus sequence -[x][FW][x][LI][x][LIV], in which the aromatic residue Phe is predominantly selected at position -4. The irregular and unconventional internal ligand binding specificities for the PDZ domains of SjScrib were confirmed by point mutations of the key amino acids within the ligand binding motifs. We also compared the differences in ligand specificities between SjScrib-PDZs and hScrib-PDZs, and explored the structural basis for the ligand binding properties of SjScrib-PDZs.The gene encoding the In this study, we characterized and confirmed the ligand binding specificities of all four PDZ domains of SjScrib for the first time. We denoted the differential ligand binding specificities between SjScrib-PDZs and hScrib-PDZs as well as the structural basis for these properties. This work may provide a fundamental basis for the rational design of novel anti-schistosomal drugs. S. japonicum, a panel of PDZ domains is conserved in this species. So far, however, little is known about ligand binding specificities and the molecular functions of parasite-derived PDZ domain-containing proteins. In this study, by yeast two-hybrid screening of a random library, we confirmed the ligand binding properties of a multiple PDZ domain-containing protein Scribble of S. japonicum for the first time. Divergent ligand specificities between the homologous PDZ domains of S. japonicum and human Scribble orthologs were revealed. Internal motif recognition and irregular ligand interaction models for the SjScrib-PDZ domains were identified. These results provide an important basis for the rational discovery of anti-schistosomal drugs.Schistosomiasis japonica remains a major public health problem in China and Southeast Asia. The long-term of treatments with the only available drug, praziquantel, has raised the concerns about drug resistance. Protein-protein interactions (PPIs), for highly discriminating specificities, are thought to be the innovative targets for a generation of new drugs. The PDZ domain is one of the most important modules for PPIs. A number of compounds screened based on binding specificities of PDZ domains have shown their potential therapeutic power in several disease models with less side effects. Although domain loss events are widespread in  Schistosoma, afflicts more than 200 million people living in the endemic areas of 77 countries worldwide, representing a major health and economic burden in tropical and developing nations Schistosomiasis, caused by members of the genus X-Φ*; Class II, -Φ-X-Φ*; Class III, -[D/E/K/R]-X-Φ*; and Class IV, -X-ψ-[D/E]*; where X is any amino acid, Φ is a hydrophobic residue, ψ is an aromatic residue, and * represents the stop codon of the peptide Protein-protein interactions (PPIs) mediate multiple biological processes and physiological functions in organisms, and offer a variety of opportunities for exploring potential drug targets Scribble is a member of the evolutionarily conserved LAP (LRR (leucine-rich repeats) and PDZ) protein family. The N-terminal LRR domain is necessary for targeting to the basolateral membrane of epithelial cells S. japonicum cell polarity protein Scribble (SjScrib). The binding specificity profiles were compared with those of human Scrib ortholog. The potential ligands of SjScrib were further predicted based on the ligand binding specificities of the PDZ domains, which were validated in the Y2H system. This work will facilitate the identification of novel drug targets against schistosome infection.In this study, by yeast two-hybrid (Y2H) screening of a random peptide library and introducing point mutations, we revealed and confirmed the ligand binding specificities, including the canonical C-terminal and non-canonical internal binding specificities for all four PDZ domains of the All procedures carried out on animals within this study were conducted following animal husbandry guidelines of the Chinese Academy of Medical Sciences and with permission from the Experimental Animal Committee of Chinese Academy of Medical Sciences with the Ethical Clearance Number IPB-2011-6.Schistosoma japonicum-infected Oncomelania hupensis were purchased from Jiangxi Institute of Parasitic Diseases, Nanchang, China. Cercariae were freshly shed from the infected snails. New Zealand White rabbits were percutaneously infected with ∼1,000 S. japonicum cercariae. Hepatic schistosomula and adult worms were recovered from the rabbits by hepatic-portal perfusion at 2 and 6 weeks post-infection (p.i.), respectively. Male and female adult worms were manually separated with the aid of a light microscope. Eggs were isolated from the liver tissues of the infected rabbits at 6 weeks p.i. by the sieving and enzymatic digestion method S. mansoni Scribble cDNA sequence (GenBank accession number: XM_002581163) as a bait. Forward (5′-GGCTCATAATGTTCAAGTGTTTGCCAATTATAGG-3′) and reverse (5′-CCAGAAGTGCTCCCATATTGTGCGTCATCG-3′) primers were designed based on these EST sequences and used to amplify the full-length ORF of SjScrib from adult worm pair cDNA templates with high fidelity Phusion DNA polymerase . The resulting PCR fragment was cloned into T-vector and sequenced. The amino acid sequence of SjScrib (GenBank accession number: AHC92618) was deduced from the obtained cDNA sequence (GenBank accession number: KF730248) for further bioinformatic analysis.Two EST sequences (GenBank accession number AY809835 and AY815909), respectively encoding the N-terminal and C-terminal fragment of SjScrib, were identified and retrieved directly from NCBI using the S. mansoni (GenBank accession number: CCD76510), Drosophila melanogaster (GenBank accession number: NP_733154) and Homo sapiens (GenBank accession number: AAL38976) were retrieved from GenBank. The domain boundaries of SjScrib and hScrib were analyzed using the following public domain tools: the NCBI CDD (http://www.ncbi.nlm.nih.gov/cdd) http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) http://www.pymol.org/). Free, open source Java software for visualizing ligand binding specificity profiles is available from http://baderlab.org/Software/LOLA.The amino acid sequences of the Scrib orthologs of S. japonicum at different developmental stages  were extracted using Trizol reagent . The possible contaminating genomic DNA was completely removed from RNA samples with TURBO DNA-free kit . RNA quantification and quality control was conducted by 1% agarose gel electrophoresis and the Nanodrop ND-1000 spectrophotometer . For each sample, 1 µg total RNA was reverse transcribed into first-strand cDNA using SuperScript III Reverse Transcriptase Kit (Invitrogen). The synthesis procedure was performed as follows: 25°C for 5 min, 50°C for 1 h, 70°C for 15 min. Each PCR reaction contained 12.5 µl of 2×Brilliant II SYBR Green QPCR Master Mix , 1 µl diluted cDNA (20×), 1 µl of the forward and reverse primer pair  −ΔΔCt method, according to the SDS 1.4 software.Total RNAs of mer pair , and 10.EcoR I and BamH I, and cloned into the EcoR I/BamH I sites of the GAL4 BD vector, pBridge (Clontech).Primer pairs were designed based on the boundaries of PDZ domains shown in . The DNAlacZ assays were performed to examine self-activation. The transformants were further spread on SD/-His/-Trp plates with different concentration of 3-amino-1,2,4-triazole for leakage test. A random peptide library (constructed with Tsp509I-digested human genomic DNA fragments) LacZ assays. After rescue, the potential positive plasmids were isolated and retransformed into the yeast strain CG1945 containing corresponding bait plasmid. Only the clones that were positive for all the reporter assays and confirmed by at least two independent tests were selected for specific interactions and sequenced Each GAL4 BD-fusion bait plasmid was transformed into the yeast strain CG1945 using the lithium acetate procedure. The transformants were grown on SD/-Trp plates and EcoR I and BamH I, and cloned into the GAL4 BD vector, pBridge. Each clone was subjected to sequencing to make sure that the mutation was correctly introduced. Each of the mutated plasmids was co-transformed with the corresponding PDZ domain bait plasmids into yeast strain CG1945, respectively. The interactions between the mutants and the corresponding PDZ domain were validated by Y2H assays as mentioned above.For each irregular ligand, a putative binding site was deduced from the pattern of regular C-terminal PDZ binding motifs. Point mutation was induced by PCR against key residues within the putative binding site. DNA fragments produced by self primer template PCR reactions or amplified from ligand plasmids were digested with S. japonicum predicted peptide sequences were downloaded from SDSPB (http://lifecenter.sgst.cn/schistosoma/cn/schdownload.do). The Tailfit software S. japonicum peptide dataset to retrieve the potential ligands whose carboxyl termini matched the consensus-binding sequences. These peptide sequences were then manually BLAST in NCBI to filter the truncated fragments lacking of the C-terminus. Further, the most promising candidates were selected based on biological information such as subcellular localization and potential molecular function, as well as the C-terminal homology between S. japonicum and S. mansoni orthologs The consensus C-terminal binding sequences for each SjScrib-PDZ domain were deduced from sequence alignments of positive clones from Y2H screening. The EcoR I and BamH I endonucleases, and cloned into the EcoR I/BamH I sites of GAL4 AD vector, pGADT7 followed by sequencing. Each constructed plasmid was co-transformed with the corresponding SjScrib-PDZ1 and SjScrib-PDZ4 bait plasmids into yeast strain CG1945, respectively. The positive interactions were selected by Y2H assays as mentioned above.The GAL4 AD plasmids expressing the carboxyl-terminus of the candidate ligands of SjScrib-PDZ1 and SjScrib-PDZ4 were first constructed. The self primer template PCR reactions were performed to produce DNA templates encoding the 15 amino acids of extreme carboxyl-terminus of each ligand candidate   . A leucie Scribs . It is oe Scribs . Among te Scribs . The pepe Scribs . The sece Scribs . TotallyTo assess the structural divergence between SjScrib and hScrib PDZ domains, we further superimposed the structures of SjScrib-PDZs with three available NMR structures of hScrib PDZ domains. Although the primary sequence identity of the second PDZ domain between SjScrib and hScrib is higher than that of the first and fourth PDZ domains , the strSjScrib at different developmental stages and between sexes of the parasite. As a result, we observed that the SjScrib gene was ubiquitously expressed during different developmental stages, but in a stage-biased pattern. The transcription levels of the SjScrib gene were similar in cercariae and schistosomula at a medium level. In adult worms, the transcriptional level was significantly higher in male than that in female worms. In the egg stage, the expression of SjScrib was relatively higher than that in any other stages detected , especially Ile (44.0%), Leu (36.0%), or Phe (20.0%). At P−1, no clear preference was observed. At P−2, only the polar amino acids Thr (80.0%) and Ser (20.0%) were selected. At P−3, Glu (100%) was absolutely preferred. At P−5, the hydrophobic residues, such as Leu (28.0%), Phe (24.0%) and Tyr (16.0%), along with a rare occurrence of other hydrophobic residues were selected  was observed, followed by other hydrophobic residues Val (13.3%), Phe (10.0%) and Tyr (10.0%). The P−2 site demonstrated an absolute preference for the polar residue Thr (100%). At P−3, Glu (60.0%) was predominantly selected, along with a rare selection of residues Thr (16.7%) and Ser (13.3%). While the positive charged residues, Arg (23.3%) and Lys (20.0%) were slightly prone to be at P−4 site. At P−5 site, no obvious preference was observed , Ile (26.7%), or Val (26.7%), were also predominantly preferred at Pobserved . Based o0, with a weak preference for the residues of Ala (11.1%) and Cys (9.3%). The P−2 site was also occupied by the hydrophobic residues of Ile (44.4%) and Leu (27.8%). At P−4, Phe (81.5%) was predominantly selected, with a less occurrence of Trp (14.8%). No clear preference was observed for the sites of P−1, P−3 and P−6 , Leu (25.9%), or Val (16.47%), were preferred at P and P−6 . Therefo0 specificity of ZO-1-PDZ1 and Erbin-PDZ. The PDZ domains that show a stringent preference for a Val0 side chain contain a Phe at position β2-1, whereas those that can accommodate a larger Leu/Ile at site 0 contain a Leu/Ile at the same position 0 specificity of LAP family PDZ domains ) and the β2:β3 loop (Pro (β2:β3-9)  . While iβ2:β3-9) . Howeveret al. have previously investigated the binding specificity profiles of a panel of PDZ domains of LAP proteins by phage display screening Φ][x][E][TS][x][ILF]) was found to be similar with that of hScrib-PDZ1 (-[Φ][Φ][E][TS][x][VIL]*), but not exactly the same. For example, hScrib-PDZ1 prefers a hydrophobic residue at P-4, whereas this is not the case for SjScrib-PDZ1. Except that both PDZ domains prefer the hydrophobic residues Leu and Ile at P0, SjScrib-PDZ1 exhibits a preference of the aromatic residue Phe; whereas hScrib-PDZ1 prefers an aliphatic residue Val at this site. The binding specificity pattern for SjScrib-PDZ3 (-[x][RKx][ETS][T][WΦ][ILV]) resembles that of hScrib-PDZ3 (-[Φ][Φ][EDQH][TS][x][LIV]*) −5. At P−4, unlike hScrib-PDZ3 that prefers hydrophobic residues, SjScrib-PDZ3 exhibits a preference for the positively charged residues, Arg and Lys, which may form electrostatic interactions with the negatively charged residue Asp at α2-2 or β2:β3-3  (the residue beyond the core binding motif) in these irregular and internal PBMs of SjScrib-PDZ1  and Ile (α2-9)  and the Asp+1 in the internal ligand of SjScrib-PDZ4 (P4-L4) can potentially interact with Arg (β1:β2-4)  displays a relatively strong preference for internal motif binding, followed by SjScrib-PDZ1 (3∶22); while SjScrib-PDZ4 (1∶55) shows a weak ability of internal motif recognition. Interestingly, preferences at P at β2-4  as previrib-PDZ1 . In cont or α2-9 . In the ion α2-9 , while te (α2-9) . Similarβ1:β2-4)  and Arg β1:β2-4) , respectDrosophila and zebra fish, it has been shown that some PDZ domains of Scribble can interact with ligands via internal recognition In S. japonicum predicted proteomic database (sjr2_protein.fasta) with less stringent core binding motifs as baits (W][x][IL][x][ILV]) of SjScrib-PDZ4 is just a “primary”, but not “tertiary” determinant of specificity. The characteristics, such as the charge and size of the residues at position -1 and -3, may also affect the ligand recognition. These residues must coordinate with the ones at position 0, -2 and -4 and function as a whole determinant for recognition. Vice verse, this fact may explain why some unusual PBMs  could interact with SjScrib-PDZ4, although they do not fit the typical consensus sequence well. As it has been suggested that each PDZ domain is capable of interacting with 17 partners on average in vivo.To identify the native ligand candidates of SjScrib-PDZs, the Tailfit program was employed to search potential ligands in the as baits . A panelas baits . HoweverS. japonicum. We defined the ligand binding specificities of the SjScrib-PDZ domains by screening a random peptide library in the Y2H system. The convergent and divergent ligand specificities between the SjScrib-PDZ domains and those of its human ortholog were denoted. The confirmation of internal ligand specificities and the identification of irregular ligands for the SjScrib-PDZ domains will assist in the rational design of novel drugs against the parasite. The strategy used here can also be generically applied to the determination of the ligand binding specificities of other parasite-derived PDZ domains.In summary, for the first time, we presented the molecular characterization of the PDZ domain-containing protein Scribble from Table S1Primer sequences used in this study.(XLS)Click here for additional data file.


The relationship among these theoretical and computational models shows that the regulation of Tau phosphorylation by PP2A and glycogen synthase kinase 3β (GSK3β) is essential under basal conditions and also describes the leading role of CDK5 under excitotoxic conditions, where silencing of CDK5 can generate changes in these enzymes to reverse a pathological condition that simulates AD.Alzheimer's disease (AD) is the most common cause of dementia worldwide. One of the main pathological changes that occurs in AD is the intracellular accumulation of hyperphosphorylated Tau protein in neurons. Cyclin-dependent kinase 5 (CDK5) is one of the major kinases involved in Tau phosphorylation, directly phosphorylating various residues and simultaneously regulating various substrates such as kinases and phosphatases that influence Tau phosphorylation in a synergistic and antagonistic way. It remains unknown how the interaction between CDK5 and its substrates promotes Tau phosphorylation, and systemic approaches are needed that allow an analysis of all the proteins involved. In this review, the role of the CDK5 signaling pathway in Tau hyperphosphorylation is described, an  Neurodegenerative diseases are a growing problem in the elderly population. On such diseases is Alzheimer's disease (AD), the most common cause of dementia in people over the age of 65. Sporadic AD affects approximately 20 million people worldwide, and in Colombia, there are approximately 1000 carriers of the E280A mutation that causes early-onset AD Lopera,  that havAD is characterized by two proteinopathies: amyloidopathy, a product of the abnormal cleavage of the amyloid precursor protein (APP), which leads to the accumulation of Aβ peptide in senile or amyloid plaques; and tauopathy, which is due to Tau hyperphosphorylation and aggregation. Tau is associated with microtubules (MTs) and, when it is abnormally phosphorylated, dissociates and aggregates, resulting in the formation of paired helical filaments (PHFs), which aggregate and form an insoluble conformation called NFTs. These two proteinopathies generate a loss of cell and tissue homeostasis during the neuropathological process of AD, which leads to progressive neuronal degeneration and the loss of the cognitive functions of the patient  and glycogen synthase kinase 3β (GSK3β) and the protein phosphatases 1 (PP1) and 2A (PP2A) in this process has been described, it is not known with certainty how they interact and how this interaction contributes to Tau hyperphosphorylation in different states of kinase activation. This review aims to describe and model in an integrated manner the CDK5 kinase pathway as related to Tau hyperphosphorylation. This strategy outlines and groups isolated experimental data of the molecular species, or proteins, involved in CDK5 and Tau phosphorylation, describing the behavior of the signaling pathway in a pathological process such as AD.Systems biology (SB) is a scientific research approach that brings together knowledge from various disciplines to the study of living organisms from a systems perspective that assumes the component parts as a whole and the interactions between the components as their functional dynamics. Thus, cellular organization is described at different functional levels, ranging from the more specific intracellular processes  to intercellular processes . SB addresses the living organisms as an integrated and interacting network of genes, proteins, lipids, carbohydrates, and biochemical reactions from which life arises and persists   and frontotemporal dementia associated with parkinsonism linked to chromosome 17, a phenomenon of abnormal Tau hyperphosphorylation or phosphorylation is responsible for a set of alterations, such as axonal transport and mitochondrial and lysosomal dysfunction, among other functions associated with MTs, that can lead to neuronal degeneration .In AD, the relationship of CDK5 with Tau has been described; the CDK5/p35 or CDK5/p39 complex regulates the normal function of Tau in the cytoskeleton. Upon activation, CDK5 phosphorylates its p35 activator, and possibly p39, to induce proteasome-dependent degradation by regulating the duration of kinase activation , making this an ever closer approximation of the phenomenon that helps in better understanding the regulation of DARPP-32.Some SB approaches have included CDK5 in mathematical modeling and have highlighted its importance in the function and regulation of other proteins. One model evaluated the regulation of phosphorylation sites of the dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32)  search terms, which allow a keyword search that is organized by subject headings, qualifiers (subheadings), definitions, synonyms and lists of closely related terms, were used to conduct this search. Of the retrieved documents, only original articles where the experimental design showed a direct biochemical connection with CDK5 and the substrate and that fit the description of a signaling pathway associated with Tau phosphorylation were selected. A description of the selected information is presented next.Brain disorders caused by chronic conditions such as AD or acute disorders such as cerebral ischemia, among other pathologies, promote excitotoxicity or an alteration of cellular homeostasis that triggers an increase in glutamate concentration; glutamate binds and activates glutamate-dependent calcium receptors such as NMDA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors. When activated, these channels allow an influx of calcium and, in turn, promote the activation of calcium-dependent signaling pathways  receptors; when activated by the binding of neuregulin, a member of the family of epidermal growth factor proteins, ErbB receptors induce the phosphorylation and activation of protein kinase B (PKB) and AKT, a serine/threonine kinase that is involved in cell survival pathways  and inhibitor-2 (I2), two regulators that bind to PP1 and inhibit its activity  was tested, and in silico silencing (SIS) of CDK5 and GSK3β was evaluated under different conditions.After coupling the equations describing the reactions, a set of 40 ordinary coupled differential equations was built that was dependent on 62 reaction parameters or constants and 40 initial concentrations. The model was run at an initial concentration of 0.1 μM baseline calcium, and then a series of simulations were performed that consisted of variations in the concentration and reaction speed of the different molecular species. Initially, variations were made in calcium levels, the enzymatic activity of phosphatases and kinases was changed, the effect of various knockouts The model and the simulations that were performed used calcium influx as input and the levels of unphosphorylated or phosphorylated Tau (pTau) as output, noting the ratio between the two. Specific Tau phosphorylation events were not taken into account because of difficulties with assigning constants that could account for a different phosphorylation pattern from normal; therefore, pTau is assumed to be an indicator of the level of phosphorylation, and increased pTau is regarded as a trend indicating hyperphosphorylation.Tau and pTau levels were studied at basal conditions; i.e., the model was run without any modification. It was found that Tau and pTau levels were balanced, indicating that approximately 50% of total Tau and pTau were constant Figure . This suTo examine the sensitivity of the model to variations in different molecular species, KOis were performed that consisted of completely suppressing the activity of the molecular species by reducing the K2 constant (Vmax) to a number very close to zero. It was observed that the model was sensitive to dramatic changes in GSK3β, which decreased pTau levels to 40.5%, and changes in PP2A, which increased pTau levels to 56%. The KOis of CDK5 showed a change in pTau of 13%, and the KOis of PP1 did not alter pTau significantly (Figure After evaluating Tau and pTau levels at basal conditions, calcium levels were increased by 2-, 10-, and 100-fold from an initial concentration of 0.1 μM. Thus, an unbalanced condition of calcium homeostasis or an excitotoxic process, which is characteristic of pathological processes, was simulated. With increasing calcium concentration, pTau increased, reaching even a 28% increase over the initial condition Figure . This inThe simulation of a 100-fold increase calcium added to SIS of GSK3β allowed the observation of the influence of this protein in a condition of excitotoxicity. A 75% decrease in the levels of GSK3β reduced pTau by 11%; although this is a significant reduction, we note that it is less than the 15% achieved by CDK5 reduction under the same conditions (Figure In AD, it has been described that the excitotoxic process, as well as the increase in p25-mediated CDK5 activity and a decrease in the activity of phosphatases, plays a key role in the development of the disease. To simulate a similar condition, calcium concentration was increased by 100-fold, and the activity of both phosphatases was decreased by 50% Figure . Under tThe SIS of CDK5 and GSK3β were assessed under the same conditions, simulating a pathological state. The SIS of CDK5 and GSK3β separately influenced pTau levels similarly, although CDK5 had a greater effect by decreasing pTau by 11.6%. In addition, the simultaneous silencing of both kinases produced a 28% decrease, reversing the effect caused by the pathological conditions (Figure This review shows the dynamic complexity of a cellular system that is in constant action. The scientific literature provides largely isolated experimental data combined with theoretical data capable of providing a specific cellular context. SB integrates experimental data and creates computational contexts limited by existing data but that is dynamic, inclusive and is able to create new questions to gain an understanding of biological phenomena. In our previous experimental results show a chronic disease model and the behavior of the molecular species after the decrease of CDK5 (Piedrahita et al., Our present computational model approximated the conditions reported in the literature; thus, it was confirmed that the deregulation of CDK5 induced by excitotoxicity is directly involved in Tau hyperphosphorylation and that the inhibition of this kinase in a murine model prevents Tau hyperphosphorylation and aggregation in association with the described pathway. The work presented here indicates that PP2A is more important than PP1 for Tau dephosphorylation and that its action regulates phosphorylation events mediated by different kinases. In this case, inhibition of CDK5 may increase phosphatase activity in the context of a chronic disease, facilitating Tau dephosphorylation and aiding in the disappearance of Tau aggregates. Together with phosphatases, the synergistic role of CDK5 and GSK3β appears to be essential for tauopathy, and decreased CDK5 inhibits GSK3β function, which affects the disappearance of tauopathy.Simulations using this model allowed the observation of the behavior of Tau and p-Tau at different calcium concentrations; however, it is important to describe in more detail the factors involved in the proposed signaling cascade to build a model that better approximates the chronic phenomenon of Tau hyperphosphorylation in AD. To this end, it is necessary to define in more detail the PP2A and GSK3β signaling pathways to improve the reproducibility of the model with other inputs. It is also essential to include new elements that may have a direct or indirect action on Tau phosphorylation and that are not associated with CDK5.John F. Castro-Alvarez, design and acquisition data, analysis and interpretation data, manuscript preparation; S. Alejandro Uribe-Arias, design and acquisition data, analysis and interpretation data, manuscript preparation; Daniel Mejía-Raigosa, design and acquisition data, analysis and interpretation data; Gloria P. Cardona-Gómez manuscript preparation and critical revision. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.