Screening for proteins that interact with RPTPβ/ζ and potentially regulate PTN signaling, through mass spectrometry analysis, identified cyclin-dependent kinase 5 (CDK5) activator p35 among the proteins displaying high sequence coverage. Interaction of p35 with the serine/threonine kinase CDK5 leads to CDK5 activation, known to be implicated in cell migration. Protein immunoprecipitation and proximity ligation assays verified p35-RPTPβ/ζ interaction and revealed the molecular association of CDK5 and RPTPβ/ζ. In endothelial cells, PTN activates CDK5 in an RPTPβ/ζ- and phosphoinositide 3-kinase (PI3K)-dependent manner. On the other hand, c-Src, ανβ3 and ERK1/2 do not mediate the PTN-induced CDK5 activation. Pharmacological and genetic inhibition of CDK5 abolished PTN-induced endothelial cell migration, suggesting that CDK5 mediates PTN stimulatory effect. A new pyrrolocarbazole derivative previously identified as a CDK1 inhibitor, was found to suppress CDK5 activity and eliminate PTN stimulatory effect on cell migration, warranting its further evaluation as a new CDK5 inhibitor. Collectively, our data reveal that CDK5 is activated by PTN, in an RPTPβ/ζ-dependent manner, regulates PTN-induced cell migration and is an attractive target for the inhibition of PTN pro-angiogenic properties.Pleiotrophin (PTN) stimulates endothelial cell migration through binding to receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) and α PTN regulates angiogenesis directly, through stimulation of endothelial cells, and indirectly through its regulatory role on the angiogenic effects of vascular endothelial growth factor A3. RPTPβ/ζ initiates PTN signalling through cellular Src kinase (c-Src) dephosphorylation and activation that consequently leads to β3 Tyr773 phosphorylation and activation of phosphatidylinositol 3-kinase (PI3K)4. Moreover, other signalling molecules, such as focal adhesion and ERK1/2 kinases1, nitric oxide5 and xanthine oxidase6, have been shown to be activated down-stream of RPTPβ/ζ and required for PTN-induced endothelial cell migration. However, the cross-talk among all the identified signalling molecules involved, as well as novel unknown molecules that may mediate the RPTPβ/ζ migratory signalling pathways, are still to be investigated.Pleiotrophin (PTN) is a secreted growth factor that binds to receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) and α7. CDK5 is activated through binding to specific protein partners, p35, p39 and cyclin I, and its activity is determined by the amount of available binding partners9. Nowadays, it is known to be expressed in several extra neuronal cell types and tissues and has several pathophysiological roles, among which regulation of angiogenesis and cancer growth8. CDK5 has been recently implicated in the development and progression of a plethora of cancer types  and in important processes, such as angiogenesis13 and lymphangiogenesis14. At the cellular level, a major role of CDK5 is to control actin remodelling15, a process closely linked to cell migration and angiogenesis. However, it remains unclear how CDK5 is activated during angiogenesis.Cyclin-dependent kinases constitute a family of small serine-threonine kinases known for their major role in the progression of cell cycle. Among the members of the family, cyclin-dependent kinase 5 (CDK5) has no effect on cell cycle regulation and was initially reported as a neuronal kinase, expressed solely in the nervous systemIn the current work, mechanistic and functional studies were performed to elucidate in depth the molecular players involved in PTN-induced, RPTPβ/ζ-mediated, cell migration with an emphasis on CDK5.in situ proximity ligation (PLA) assays  lysates were immunoprecipitated with an anti-RPTPβ/ζ antibody and the co-immunoprecipitated proteins were identified through MALDI-TOF MS analysis. Among the proteins co-immunoprecipitated with RPTPβ/ζ, a protein band of about 70 kDa was found to contain peptides identical to p35  and NU2058 (a CDK 1 and 2 inhibitor) was tested. As shown in Fig. in vitro Histone H1 phosphorylation assays were employed. Maximum CDK5 activity was observed within 5 min, following PTN stimulation, and was sustained for up to 30 min. Total CDK5 was used as the loading control . As shown in Fig. Since CDK5 interacts with RPTPβ/ζ and the latter is required for PTN-induced migratory signalingνβ3 integrin and specifically the 177-184 cysteine loop of the β3 extracellular domain2. To investigate whether PTN induces CDK5 activation through ανβ3, we used a synthetic peptide 177CYDMKTTC184 (B3 peptide) that corresponds to the cysteine loop mentioned above. This exogenously added peptide acts as a decoy for PTN thus inhibiting its interaction with αvβ32. Peptide B3 had no effect on PTN-induced CDK5-p35 interaction  or ERK1/2 by the MEK inhibitor U0126, did not affect PTN-induced CDK5 activation, suggesting that PTN activates CDK5 in an SFK- and ERK1/2-independent manner. On the contrary, the broad spectrum PI3K inhibitor wortmannin abolished PTN-induced CDK5 activation, suggesting that PI3K lies upstream of CDK5 and is necessary for its activation 20 and topoisomerase I21 activity and endothelial cell proliferation21. The aim of the present work was to study the effect of the same pyrrolocarbazole derivatives  is a widely used 2. Interestingly, we observed that RPTPβ/ζ mainly interacts with a ~70 kDa protein identified by MALDI-TOF and Western blot analyses as p35, which is possibly a p35 dimer. The literature on the existence of p35 dimers is very limited, with only one study showing that p35 homodimerizes and induces F-actin bundles formation25. PTN has been shown to differentially affect actin cytoskeleton in HUVEC26. Through specific pharmacological inhibition, we were able to show that Rac1 activation, a major player in actin cytoskeleton rearrangements, is involved in PTN-induced migratory signaling. Whether and how RPTPβ/ζ is involved in Rac1 activation and consequently to actin filament rearrangements, is of great interest and a mechanism to be interrogated in future studies.In the current study we tried to characterize the endothelial cell signaling cascade involved in the pro-migratory effect of PTN. We mainly focused on CDK5 and its activator p35 based on the initial observation that p35 interacts with RPTPβ/ζ, the receptor through which PTN initiates the endothelial migratory signaling27. CDK5 regulates angiogenesis in vitro and in vivo in different settings28 and several roscovitine analogues elicit antiangiogenic effects31. This is the first study functionally linking CDK5 with PTN signaling. Previous studies indicate similarities between these two molecules in normal and pathological conditions, especially in the nervous system, where they are both over-expressed33. Both PTN35 and CDK537 are significant in brain growth and development through induction of neurite formation. PTN suppresses Long Term Potentiation (LTP) in hippocampus and plays a role in the regulation of learning-related behavior38. CDK5 activation is implicated in LTP inhibition39, while conditional knockout CDK5 mice exhibit increased LTP40. CDK5 and p35/p25 may play a role in the pathogenesis of Alzheimer’s disease(AD) leading to abnormal phosphorylation of substrates such as tau15. ΡΤΝ is overexpressed in the brains of patients with AD42, but its function remains unknown. Both PTN and CDK5 play important role in neuromuscular junction via their participation in the accumulation of acetylcholine receptors44. Finally, PTN is overexpressed in microvessels after acute ischemic brain injury45, while CDK5 shows increased expression in endothelial cells after ischemic brain injury46. The latter aligns with our results showing that CDK5 mediates PΤΝ-induced endothelial cell migration.Although CDK5 is mostly referred to as neuronal kinase, a possible role in angiogenesis has been further attributed to this molecule. The first report showed that CDK5 is expressed in proliferating and not quiescent bovine aortic endothelial cells; its expression is increased by basic fibroblast growth factor and mediates cell proliferationνβ3 as a key regulator of PTN-induced endothelial cell migration through the formation of a functional complex with RPTPβ/ζ2. In the present study, we found that ανβ3 is not involved in PTN-induced CDK5 activation and CDK5 is not upstream of β3Tyr773 phosphorylation. This suggests that CDK5 activation is part of an RPTPβ/ζ-mediated signalling pathway independent of ανβ3. However, the data showing that both ανβ32 and CDK5 (present study) are required for PTN-induced endothelial cell migration, indicate a possible cross-talk between the two pathways. One possibility would be through Rac1 activation downstream of CDK5. CDK5-mediated Rac1 activation in HUVEC has been previously shown to be significant for endothelial cell migration9 and this agrees with our data showing that Rac1 mediates PTN-induced migration, without being upstream of CDK5. Rac1 has been previously shown to be activated in endothelial cells and mediate cell migration, downstream of PI3K but independently of Akt and ERK1/247. Similarly, our data show that PTN does not activate Akt in endothelial cells1 and PTN-induced CDK5 and ERK1/2 activation are independent of each other (this study). Interestingly, Rac1 in Drosophila regulates the proper cellular localization of β-integrin Myospheroid48, and in mammalian cells integrin recycling and cell migration involve Rac118. It is tempting therefore to speculate that PTN through CDK5 activates Rac1, which then controls recycling of ανβ3 and thus, endothelial cell migration. Involvement of CDK5 in ανβ3-mediated cell migration is further supported by the observation that in C6 glioma cells, that do not express ανβ32, pharmacological inhibition of CDK5 by roscovitine has no effect in the inhibitory effect of PTN in the migration of these cells  neurons3, PTN stimulation of HUVEC had no effect on β-catenin tyrosine phosphorylation or cell membrane localization, suggesting that β-catenin is not implicated in the PTN pathway that leads to CDK5 activation. The reasons for the observed discrepancy related to β-catenin activation between HUVEC and the other types of cells are not known but could be due to the different set of receptors or co-receptors that participate in the effect of PTN in each cell type, or to the different ways of RPTPβ/ζ signaling initiation. It has been previously shown that PTN induces RPTPβ/ζ dimerization that results in inhibition of tyrosine phosphatase activity and increased tyrosine phosphorylation of several substrates, such as Fyn and β-catenin49. On the other hand, in HUVEC, we have previously shown that upon PTN binding, RPTPβ/ζ dephosphorylates Tyr527 of c-Src leading to its activation, subsequent tyrosine phosphorylation of ανβ3 and finally increased endothelial cell migration3. The differences between the two possible modes of signaling downstream of RPTPβ/ζ are being investigated.To further elucidate the signaling pathway that participates in PTN-induced CDK5 activation, we used pharmacological inhibitors of kinases that had been previously found to regulate PTN-induced endothelial cell migration down-stream of RPTPβ/ζ. The specific c-Src inhibitor SU6665 did not affect PTN-induced CDK5 activation. Although c-Src interacts with RPTPβ/ζ and is activated by PTN, down-stream of RPTPβ/ζ and upstream of α50, whereas PI3K inhibition leads to decreased CDK5/p35 activity51. This study may contradict our previous findings that PI3K lies down-stream of ανβ34. However, in all cases, we have used wortmannin a non-isoform specific PI3K inhibitor, which may inhibit different isoforms that function down-stream or upstream of ανβ3. Alternatively, the same PI3K isoform might participate in two parallel signaling pathways that are both downstream of RPTPβ/ζ.Similarly to SFKs, PTN-induced ERK1/2 activation is unrelated to CDK5 activation. On the other hand, PI3K was found to be upstream of CDK5 and affect both basal and PTN-induced CDK5 activity. These data are in line with previous observations that PI3K activation by brain-derived neurotrophin factor induces CDK5/p35 interaction and CDK5 activityα]carbazoles, we have identified one compound (1e) that inhibits CDK1 activity20, while the rest of them have a significant topoisomerase Ι inhibitory activity in vitro21. Besides topoisomerase I, compound 1e had no effect on the tyrosine kinase activity of several growth factor receptors20, suggesting selectivity for CDK1 and potentially other CDKs. In the present study, we show that 1e inhibits CDK5, which may be attributed to the high sequence homology of CDK5 with the mitotic CDK152 and as with previously developed inhibitors, including roscovitine, it inhibits both CDKs. The fact that compound 1e completely inhibited PTN-induced endothelial cell migration, further supports a central role for CDK5 in the effects of PTN on cell motility.Among a series of pyrroloATP , at 30°C for 30 min. The reaction was terminated by adding sample loading buffer and boiling for 5 min. Protein samples were electrophoresed in SDS-PAGE and transferred to PVDF membranes. Substrate phosphorylation was analyzed by autoradiography.CDK5 kinase assays were performed as previously describedAlternatively, a non-radioactive CDK5 activity assay was developed by using the ADP-Glo™ Kinase Assay kit (Promega Corporation) and 100 μg of total cell or tissue lysates immunoprecipitated with an antibody for CDK5.g for 30 min at 4 °C. The chorioallantoic membrane (CAM) from embryos of Leghorn fertilized eggs  at different developmental stages, was excised from the eggs, washed three times in PBS, homogenized in lysis buffer and centrifuged at 20,000 g for 30 min at 4 °C. Each experiment contained 4–5 eggs per data point. Equal total protein amounts of the supernatants from cells or CAMs were transferred to new Eppendorf tubes and incubated with primary antibodies for CDK5 , p35 , phosphorylated tyrosine  or RPTPβ/ζ , for 16 h at 4 °C under continuous agitation. IgG  was used as a negative control. Protein A- and protein G-agarose beads  were added, and samples were further incubated for 2 h at 4 °C. Beads and bound proteins were collected by centrifugation and washed twice with ice-cold PBS. The pellet was resuspended with 50 μl SDS loading buffer, heated to 95–100 °C for 5 min, and centrifuged. The supernatant was analysed by Western blot analysis.Cells were washed twice with ice-cold PBS and lysed with PBS containing 1% Triton X-100, 0.1% SDS, 20 nM sodium orthovanadate, 1 μg/ml aprotinin, 1 mM PMSF and 5 mM EDTA (lysis buffer). Cells were scraped off the plate, kept on ice for 30 min, and centrifuged at 20,000 3 (Tyr747) and integrin β3 , phospho-p44/42 MAPK and p44/p42 MAPK  and α-tubulin . Membranes were further washed three times with TBS-T and incubated in the corresponding HRP-conjugated secondary antibodies  for 1 h at room temperature under continuous agitation. Membranes were washed three times with TBS-T and twice with TBS. Detection of immunoreactive bands was performed using the ChemiLucentTM plus Western Blot enhancing kit , according to the manufacturer’s instructions. The pictures of the gels were digitized and the protein levels that corresponded to each immunoreactive band were quantified using the ImagePC image analysis software .Proteins were analysed by SDS-PAGE and transferred to PVDF membranes (EMD-Millipore). Blocking was performed by incubating membranes with Tris-buffered saline pH 7.4 containing Tween 20 (TBS-T), with 5% w/v nonfat dry milk for 1 h at room temperature. Membranes were further incubated in primary antibodies overnight at 4 °C under continuous agitation, as follows: mouse anti-RPTPβ/ζ , rabbit anti-CDK5 , goat anti-p35 , β-catenin , p-integrin βin situ PLA was performed. The components used  were as follows: anti-mouse PLA plus probe, anti-rabbit PLA minus probe, anti-goat PLA minus probe and Detection Reagents Orange. HUVEC were grown on chamber slides  till they reached 80% confluence. Following fixation and blocking, the cells were incubated with the primary antibodies: mouse anti-RPTPβ/ζ , rabbit anti-CDK5 , mouse anti-CDK5 , goat anti-p35 . Subsequently, the cells were incubated with secondary antibodies conjugated with oligonucleotides, after hybridization and ligation of which, the DNA was amplified resulting in red fluorescence signals. Nuclei were counterstained with Draq5; cells were mounted with Mowiol 4–88 and visualized with Leica SP5 confocal microscope. Estimation of nuclei and cytoplasm size was performed using the Duolink ImageTool software. To calculate the total number of spots per cell, an algorithmic procedure was used as previously described55.For detection of protein-protein interactions, The significance of variability between the results from each group and the corresponding controls was determined by unpaired t-test or ANOVA, as appropriate. Each experiment included triplicate measurements for each condition tested, unless otherwise indicated.All data generated or analysed during this study are included in this published article and its Supplementary information file.Dataset 1


Inhibition of BACE1 proteolytic activity has been confirmed to decrease Aβ generation and amyloid deposition, and thus specific inhibition of BACE1 by small molecules is a current focus for Alzheimer’s disease therapy. While BACE1 inhibitors are being tested in advanced clinical trials, knowledge regarding the properties and physiological functions of BACE is highly important and this review summarizes advancements in BACE1 research over the past several years. We and others have shown that BACE1 is not only a critical enzyme for testing the “Amyloid Hypothesis” associated with Alzheimer’s pathogenesis, but also important for various functions such as axon growth and pathfinding, astrogenesis, neurogenesis, hyperexcitation, and synaptic plasticity. BACE2 appears to play different roles such as glucose homeostasis and pigmentation. This knowledge regarding BACE1 functions is critical for monitoring the safe use of BACE1 inhibitors in humans.BACE1 was discovered as the β-secretase for initiating the cleavage of amyloid precursor protein (APP) at the β-secretase site, while its close homology BACE2 cleaves APP within the β-amyloid (Aβ) domain region and shows distinct cleavage preferences  For over 30 years, the study of β-amyloid (Aβ) peptides has been the largest field of research geared toward understanding Alzheimer’s pathogenesis and therapeutic intervention. After the molecular cloning of amyloid precursor protein APP; , it beca93TG and D289SG). Although not broadly cited, this enzyme was also named to be memapsin 2 based on the standard nomenclature for aspartic proteases . Both proteins share 59% identity and are two known aspartic proteases docked on the membrane through the type I TM domain  as a precursor protein having pre- (residues 1–21), pro- (residues 22–45), core protease- (residues 46–460), TM- (residues 461–477), and C-terminal domains (residues 478–501). BACE2 has 518 amino acids and almost identical structural organization (M domain . In the M domain  and can M domain . TherefoM domain . ConsistM domain . While iM domain . This is in vivo .N-acetylglucosamine (GlcNAc), a sugar-bisecting enzyme highly expressed in brain, regulates BACE1 stability  family were first shown to bind to BACE1 via the dileucine motif, and this binding impacts not only BACE1 endosomal trafficking but also cellular stability . DepletiIn neurons, BACE1 is also targeted to axons and presynaptic terminals  and its BACE1 cleaves many cellular substrates other than APP, so its biological functions will be affected by altered cleavage of these substrates. Various biochemical and proteomic approaches have been employed to search for BACE1 substrates. Initially, optimal BACE1 cleavage sites were explored , but thiTable 1 have gained the most attention and/or are fully validated.Using unbiased proteomic analysis of cultured media from cell lines with or without overexpression of BACE1, Selkoe and his colleagues reported 68 putative BACE1 substrates . By usinTable 1). It is highly important to understand the biological functions associated with BACE1 cleavage of these individual substrates. The following sections summarize studies on this topic that have been published in recent years.As outlined above, the list of BACE1 substrates has grown in recent years (A1050–A1051 site near the TM domain (BACE1 was found to cleave Jagged-1 (Jag1), a type I TM ligand for Notch receptors . BACE1 mM domain , and aboM domain . Notch iM domain . BACE1 aM domain . Hence, The role of BACE1 in the control of myelination during development and of remyelination in the adult appears to occur through its cleavage of Nrg1 . Nrg1, wActivated Nrg1 signaling, mainly initiated by type III Nrg1, is critical for optimal myelination: mice with reduced Nrg1 signaling activity exhibit hypomyelination of peripheral nerves during development . BACE1-nInterestingly, inhibition of BACE1 produces more dramatic suppression of myelination in a co-culture myelination system than pan inhibition of ADAM proteases . This isWhen peripheral nerves are severely injured, myelin on proximal segments of damaged axons can be removed due to Wallerian degeneration and regrowing axons will be remyelinated by Schwann cells via contacting regenerating axons in the proximal band of Büngner. BACE1 has been shown to be indispensable for remyelination in nerve crush experiments, as remyelinated axons remained hypomyelinated in BACE1-null sciatic nerves . In nervIn peripheral nerves, BACE1 cleavages of type I Nrg1 in Schwann cells and type III Nrg1 in axons contribute to normal remyelination. This conclusion is supported by mouse genetic studies using conditional deletion of Nrg1 isoforms or transgenic mice overexpressing either type I or type III Nrg1 . These sv1.2 protein was found to be elevated in BACE1-null hippocampal mossy fiber regions  and its family member Sez6L, was identified as BACE1 substrate through an unbiased proteomic approach and was recently validated as a strong substrate of BACE1 . Their lMuscle spindles, which are composed of specialized intrafusal muscle fibers, are innervated by afferent axons extending from sensory neurons . Nrg1 in1086 and E1087, 18 residues upstream of the TM domain , which is a type I membrane protein and a component of Sema3A receptors, is a natural BACE1 substrate with identified cleavage sites located between YM domain . After BM domain , while tM domain . BACE1-nM domain . It shouThe effects of BACE1 inhibition on APP- or Aβ-mediated synaptic functions has recently been summarized in a separate review . As mentBACE1-null mice also exhibit other synaptic dysfunctions. By electrophysiological recording of brain slices, it was demonstrated that hippocampal activity-dependent long-term potentiation at mossy fibers to CA3 is impaired, while long-term depression is increased , 2014. IThe role of BACE1 in retinal pathophysiology has gained increasing attention in recent years, as several BACE1 inhibitors have been found to cause retinal thinning, lipofuscin accumulation, and vascular dysfunction, which terminated clinical trials . BACE1 wOn the other hand, specific inhibition of BACE1 is likely to benefit retinal functions, as BACE1 activity in retina is elevated in response to stress conditions such as mitochondrial respiratory inhibition or oxidative stress . It has Table 2). In pancreatic MIN6 cells treated with a BACE2 inhibitor or siRNA, BACE2 was initially shown to mediate insulin receptor β-subunit (IRβ) expression and surface trafficking  in pancreatic β cells needs to co-secrete with insulin, and the formation of IAPP amyloid is a hallmark pathological feature of type 2 diabetes . BACE2 wOn the other hand, BACE2 inhibition can cause loss of pigmentation, as BACE2 cleaves the integral membrane form of PMEL within the juxtamembrane domain and exerts its role in melanosome biogenesis . AlthougIt is also demonstrated that Sez6L and Sez6L2 are effectively cleaved but in rate limiting proteolytic manner in pancreatic islet β-cells by BACE2 . AlthougInhibition of BACE1 is one the most promising therapeutic targets for treating AD, and five drugs have currently entered into clinical trials . While tThe author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human dendritic cell‐specific intercellular adhesion molecule‐1 grabbing nonintegrin, DC‐SIGN, and the sinusoidal endothelial cell receptor DC‐SIGNR or L‐SIGN, are closely related sugar‐binding receptors. DC‐SIGN acts both as a pathogen‐binding endocytic receptor and as a cell adhesion molecule, while DC‐SIGNR has only the pathogen‐binding function. In addition to differences in the sugar‐binding properties of the carbohydrate‐recognition domains in the two receptors, there are sequence differences in the adjacent neck domains, which are coiled‐coil tetramerization domains comprised largely of 23‐amino acid repeat units. A series of model polypeptides consisting of uniform repeat units have been characterized by gel filtration, differential scanning calorimetry and circular dichroism. The results demonstrate that two features characterize repeat units which form more stable tetramers: a leucine reside in the first position of the heptad pattern of hydrophobic residues that pack on the inside of the coiled coil and an arginine residue on the surface of the coiled coil that forms a salt bridge with a glutamic acid residue in the same polypeptide chain. In DC‐SIGNR from all primates, very stable repeat units predominate, so the carbohydrate‐recognition domains must be held relatively closely together. In contrast, stable repeat units are found only near the membrane in DC‐SIGN. The presence of residues that disrupt tetramer formation in repeat units near the carbohydrate‐recognition domains of DC‐SIGN would allow these domains to splay further apart. Thus, the neck domains of DC‐SIGN and DC‐SIGNR can contribute to the different functions of these receptors by presenting the sugar‐binding sites in different contexts. The spacing between sugar‐binding sites in receptors of the innate immune system contributes to the way that these receptors distinguish sugars on the surface of pathogens from those on host cells. This work describes how subtle differences in closely related receptors can change the way that sugar‐binding sites are held together, helping to explain why they interact differently with viruses that display similar types of sugars.x trisaccharide found on cell surface molecules, including ICAM‐1. Pathogens bound by the receptor include viruses and fungi, which bear high mannose oligosaccharides, as well as parasites that are rich in the Lewisx epitope. The dual binding specificity can be explained at least in part by accommodation of both Lewisx and high mannose oligosaccharides in the binding site of the C‐type carbohydrate recognition domain (CRD) that forms the ligand‐binding portion of the receptor.Human DC‐SIGN, the dendritic cell‐specific intercellular adhesion molecule‐1 grabbing nonintegrin, plays a dual role as an adhesion receptor for T cells and as a pathogen‐binding receptor.DC‐SIGN and DC‐SIGNR are similar in overall organization, consisting of tetramers that facilitate binding to multivalent targets, such as viral and cell surfaces. In both proteins, clusters of C‐terminal CRDs are projected from the cell surface by extended neck domains that consists largely of multiple 23‐amino acid repeat units Fig. . InformaTo facilitate analysis of the oligomer formation by DC‐SIGN and DC‐SIGNR, a strategy for purification of the neck domains has been developed, based on addition of the sequence Gly‐His‐His at the C‐terminus of the neck domain at the point where the CRD would normally be attached [Fig. Although similar in organization, the neck domains of DC‐SIGN and DC‐SIGNR differ in important respects. For instance, the neck domain of DC‐SIGNR is much more stable to thermal denaturation than the neck domain of DC‐SIGN.The goal of the present studies was to analyze the properties of engineered versions of the neck domains from DC‐SIGN and DC‐SIGNR in order to understand the effects of sequence variations in the neck domains, both between the two proteins and between the different polymorphic forms of DC‐SIGNR. The analysis suggests that differences in key residues can result in differences in the assembly and organization of the extracellular portions of the receptors.Two different crystal structures of the C‐terminal portion of DC‐SIGNR suggest that there are only limited interactions between the CRDs in the oligomer.When the purified neck domain and the extracellular fragment of DC‐SIGN were mixed, denatured and allowed to renature, fractionation of the resulting renatured protein on a mannose‐Sepharose affinity column results in both fragments being retained on the column and eluted with EDTA. Because the neck domain cannot bind to mannose‐Sepharose on its own, the results represent purification of mixed oligomers that contain both the neck domain and the full extracellular fragment Fig. . The facSome information about the effects of variations in the sequences of the neck repeat units can be derived from earlier studies. The N‐terminal portions of the extracellular portions of DC‐SIGN and DC‐SIGNR consist of a short region not related to the 23‐amino acid repeat units, followed by a half repeat unit that is significantly divergent from the other repeat units. In previous studies, truncation of the neck domain of DC‐SIGNR to remove the non‐repeat region at the N‐terminus plus the partial repeat unit at this end resulted in no change in the stability of the neck domain, since the observed denaturation temperature measured by differential scanning calorimetry remained at 82°C.The most divergent sequence is the C‐terminal repeat unit of DC‐SIGNR, adjacent to the CRD [Fig. Aside from this unusual C‐terminal repeat unit, a key point of variation between DC‐SIGN and DC‐SIGNR is at position 6 of the repeat units, which forms the first residue of the heptad pattern in the primary structure and the beginning of the α helix in the crystal structure. In all of the repeat units visible in the crystal structure of the DC‐SIGNR neck domain, corresponding to repeat units 2 through 7, this position is occupied by a leucine residue, which packs into the hydrophobic core of the coiled coil [Fig. To assess the effect of the residue at position 6 on the structure and stability of the neck domain, an engineered neck domain consisting of seven repeat units, each containing a leucine residue at position 6, was prepared. Gel filtration revealed that this version of the neck domain forms a stable tetramer at room temperature since it elutes at the same position as a natural fragment of the neck domain of DC‐SIGNR containing seven repeat units, which has been characterized as a tetramer [Fig. In addition to differences in stability reflecting minor variations in the sequences of the repeat units in the neck domains of DC‐SIGN and DC‐SIGNR, differences in the properties of the neck domains could arise from differences in the number of repeat units in polymorphic forms of DC‐SIGNR. To compare these factors, multiple versions of the neck domain were created with 3 to 7.5 copies of the 23‐amino acid repeat unit containing leucine at position 6 and arginine at position 15.Changes in the stability of the neck domain oligomers were demonstrated by gel filtration analysis, which reveals a single peak of tetramers for the longer constructs, but the appearance of increasing amounts of monomer for shorter fragments containing 5 and 4 repeat units, while a fragment containing only three repeat units appears entirely as monomer [Fig. The CD spectra of most of the constructs share the largely helical conformation of the natural neck domains of DC‐SIGN and DC‐SIGNR [Fig. A leucine residue is present at position 6 only in repeat units 2 through 4 of DC‐SIGN, with position 6 occupied by a glutamine residue in most of the remaining repeat units, although a methionine residue is present in repeat unit 5. To assess the effect of this variation at position 6 on the structure and stability of the neck domain, further engineered versions of the domain containing 7 repeat units with either glutamine or methionine at position 6 in each repeat unit were prepared.In contrast to the homogeneous neck domain containing leucine at position 6 of each repeat unit, otherwise identical neck domain in which glutamine is present at this position in each repeat unit fail to form stable oligomers. At room temperature, the all‐glutamine version of the neck domain elutes as a monomer on gel filtration [Fig. An analog of the neck domain containing methionine residues at position 6 of each repeat unit also did not show a defined denaturation peak by calorimetry (data not shown) and has even more diminished helical content [Fig. The combined results of the gel filtration, calorimetry and circular dichroism studies suggest that the presence of glutamine at position 6 of the neck domain repeat unit is compatible with formation of the tetrameric coiled coil, so that a repeat unit containing this sequence can have the same conformation as a repeat unit with leucine at position 6, although the glutamine form is less stable. In contrast, a methionine residue at this position disrupts the conformation.A final unusual feature of the DC‐SIGN neck domain is a tryptophan residue at position 15 in the fourth repeat unit. This substitution has little effect on the behavior of the neck domain, as shown by the fact that substitution of the first portion of the neck domain from DC‐SIGNR, including an arginine residue at this position, for the corresponding portion of DC‐SIGN results in removal of the tryptophan residue with no detectable effect on the stability of the neck domain.DC‐SIGN and its homologs are subject to rapid genetic change.The previously described crystal structure of the neck domain of DC‐SIGNR is based on an average from the different types of repeat units present. Each of these repeat units has a leucine residue at position 6, although they vary at position 15. The results can be assembled into a model for the full extracellular portion of DC‐SIGNR.Based on this combination of information, it is concluded that repeat units with leucine residues at position 6 and arginine residues at position 15 form particularly stable tetrameric coiled coils. Changing the arginine residue at position 15 results in loss of the salt bridge with the conserved glutamic acid residue at position 12, which causes some destabilization, while introduction of alternative residues at position 6 is more destabilizing. The summary of the sequences of DC‐SIGN and DC‐SIGNR molecules shown in Figure The loss of stability resulting from the presence of a glutamine residue at position 6 is somewhat surprising, since favorable packing and hydrogen bonding of glutamine residues has been observed in trimeric coiled coilsFor DC‐SIGNR, a key finding to emerge from these studies is the observation that the repeat units are mostly stabilized by leucine residues at position 6 and arginine residues at position 15. The presence of a phenylalanine residue at position 20 in the repeat unit adjacent to the CRD appears to cause a flaring apart of the CRDs, but the remainder of the neck domain is a stable tetrameric coiled coil. This arrangement of very stable repeat units in the neck domain, which is conserved in all of the DC‐SIGNR proteins in the primates, may be necessary to ensure that the neck domain remains tetrameric even though the final repeat units are splayed apart by the presence of the phenylalanine residue. The studies reported here suggest that the presence of stabilizing residues at positions 6 and 15 of the repeat units allows for the formation of stable tetramers even for shorter versions of the neck domain present in some individuals, which result from common genetic polymorphisms in the human population.In contrast to DC‐SIGNR, only the repeat units at the membrane‐proximal end of the neck domain in DC‐SIGN are stabilized by leucine residues at position 6. The presence of potentially less stable repeat units in the half of the DC‐SIGN neck domain further from the membrane, and the presence of repeat units containing the potentially disruptive methionine residues at position 6 in a repeat unit in the middle of the neck domain, raises the possibility that the C‐terminal portion of the neck domain might dissociate some of the time, so that the extracellular domain would be able to accommodate different spacings of sugar residues, as observed in recent studies with mannose‐coated particles.x epitope, which is bound by DC‐SIGN but not DC‐SIGNR.Some differences in the interactions of DC‐SIGN and DC‐SIGNR with glycans on pathogens may result from differences in the local sugar‐binding characteristics of the CRDs. For example, only DC‐SIGN binds to parasite glycoproteins because this binding results from recognition of the LewisEscherichia coli followed by purification, employing affinity chromatography on mannose‐Sepharose, were as previously described.2+ followed by anion exchange chromatography.Procedures for expression of the extracellular portions of human DC‐SIGN and DC‐SIGNR in Vectors encoding uniform neck domain repeat units with leucine at position 6 were created starting from the cDNA for the DC‐SIGNR neck.A similar strategy was used to generate a DNA sequence encoding three repeat units with glutamine at position 6, by substituting synthetic oligonucleotides for the portions of the DC‐SIGN cDNAFor creation of neck domain polypeptides containing methionine residues at position 6, three sets of synthetic oligonucleotides were assembled to generate a DNA fragment encoding two repeat units [Fig. 2 and elution buffer containing of 0.5 M NaCl, 25 mM Tris‐Cl, pH 7.8, and 2.5 mM EDTA. SDS‐polyacrylamide gel electrophoresis was performed in the buffer system of Laemmli.2 Superdex S200 column eluted with 100 mM NaCl, 10 mM Tris‐Cl, pH 7.8, 2.5 mM EDTA. The positions of marker proteins are indicated by the Stokes radius: cytochrome c, 17 Å; bovine erythrocyte carbonic anhydrase, 23.9 Å; bovine serum albumin, 35.5 Å; yeast alcohol dehydrogenase, 45.5 Å; β‐amylase, 51 Å; E. coli β‐galactosidase, 69 Å; and thyroglobulin, 85 Å. Protein concentrations were determined by ninhydrin assay following alkaline hydrolysis.Analytical affinity chromatography on mannose‐Sepharose2, degassed under vacuum and introduced into the sample loop of a Nano‐III calorimeter from Calorimetry Sciences Corporation. Repeated equilibration scans from 5 to 20°C were used to remove any remaining dissolved gas. Scans were performed at 1°min−1. Protein concentrations used were based on those previously employed for the native neck polypeptides, since these give signals that can be quantified accurately.Samples for calorimetry were dialyzed extensively against 125 mM NaCl, 25 HEPES, pH 7.8, 2.5 mM CaCl2. Analysis was performed on 200 μL samples in a 1‐mm cuvette in a Chirascan spectropolarimeter from Applied Photophysics, with data collected for 1 sec at 0.5 nm intervals followed by averaging of 10 scans. Comparisons were made between proteins within a three‐fold range of concentrations. In test experiments over this concentration range, the ratio of tetramers to monomer observed on gel filtration changed by less than 1.5‐fold for the fragments that undergo some dissociation into monomers, such as the version containing four repeat units with leucine at position 6.Proteins were prepared in 125 mM NaCl, 25 mM Tris–Cl, pH 7.8, 5 mM CaCl


SET is a conserved protein domain with methyltransferase activity. Several genome and transcriptome data in plant lineage (Archaeplastida) are available but status of SET domain proteins in most of the plant lineage is not comprehensively analysed.In this study phylogeny and domain organisation of 506 computationally identified SET domain proteins from 16 members of plant lineage (Archaeplastida) are presented. SET domain proteins of rice and Arabidopsis are used as references. This analysis revealed conserved as well as unique features of SET domain proteins in Archaeplastida. SET domain proteins of plant lineage can be categorised into five classes- E(z), Ash, Trx, Su(var) and Orphan. Orphan class of SET proteins contain unique domains predominantly in early Archaeplastida. Contrary to previous study, this study shows first appearance of several domains like SRA on SET domain proteins in chlorophyta instead of bryophyta.The present study is a framework to experimentally characterize SET domain proteins in plant lineage.The online version of this article (10.1186/s12870-017-1177-1) contains supplementary material, which is available to authorized users. Su(var)3–9 [Enhancer of zeste, E(z) [Trithorax (trx) [Epigenetic cellular memory is heritable during mitosis and/meiosis but is not encoded in the genetic material , 2. Hist(var)3–9 , Enhancete, E(z) , and Triax (trx)  having tax (trx) .Based on SET domain sequence similarity to Drosophila homologues, plant SET domain proteins are classified into 4 main classes , EnhanceHere we have taken a computational approach to analyse SET domain sequences of 16 different taxa of Archaeplastida. 506 SET proteins are identified that are classified into five classes. Our work will serve as a guideline for functional and biochemical characterization of SET proteins of plant lineage.Arabidopsis thaliana (At), Oryza sativa (Os), Picea abies (Pa), Selaginella meollendorffii (Sm), Physcomitrella patens (Pp), Micromonas pusila (Mpu), Micromonas RCC299 (Mr), Ostreococcus tauri (Ot), Ostreococcus lucimerinus (Ol), Chlorella vulgaris (Cv), Chlamydomonas reinhardtii (Cr) and Volvox carteri (Vc) are retrieved by BLAST search using SET domain (PF00856) as query from PFAM database Version 30.0 (http://pfam.xfam.org/family/PF00856) [http://www.uniprot.org/) and Phytozome v12.1  that are not available in PFAM. To better understand the evolutionary history and functionality of SET domain-containing proteins in plant lineages, we have use assembled transcriptome data from, Nitella mirabilis (Nm) (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA158153 158153), Klebsormidium flaccidum (Kf) (https://www.ncbi.nlm.nih.gov/bioproject/ PRJNA 51159). Cyanophora paradoxa (http://cyanophora.rutgers.edu/cyanophora/) and predicted proteome data for Marchantia polymorpha (Mp) (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA218052) and Gymnosperm, Picea abies (Pa) (http://marchantia.info/tom/blast/blast.html tom/blast/blast.html). In in silico proteome datasets, if two or more overlapping protein sequences arose from same genetic locus, we have selected the longest sequence. Transcript sequences obtained from NCBI website are transdecoded into predicted proteins using Transdecoder (https://transdecoder.github.io/) that gave the longest open reading frame. Local Blastp (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/. nih.gov/blast/executables/blast+/LATEST/) is carried out with these predicted proteins against 130–150 length SET domain sequences from Arabidopsis thaliana to filter the SET domain containing sequences. Thereafter, redundant SET domain amino acid sequences are removed from all the SET domain sequences obtained from the online search. The Arabidopsis thaliana and Oryza sativa SET domains protein sequences are used as queries because it is a representative of flowering plants and possess relatively complete annotated protein information.To uncover the SET proteins in Archaeplastida, the conserved SET domain amino acid sequences from PF00856) . The SEThttp://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi/) and NCBI-CD Hit (https://ww w.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).Presence or absence of specific domain and its organisation might provide an intimation to the functional divergence of the different protein groups. Additionally, it is conventional that proteins sharing identical domain may share close evolutionary relationship and may be functionally related. Predicted proteins from PFAM and local Blast were searched for the signature SET domain and their associated domain using NCBI Batch CD search tool , Ash, Trx and Su(var) group are included. SET domain containing proteins from the Orphan, SETD, and TPR are excluded due to low sequence similarity with E(z), Ash, Trx and Su(var). Multiple sequence alignments of SET domain sequences are performed using the ClustalW program. Evolutionary analyses are conducted in MEGA7.0 program . The evoArabidopsis and rice SET domain sequences using multalin program  with the default parameters. SET domain sequences sharing significant homology with Arabidopsis and rice. SET domain sequences are categorized to the corresponding group of the SET domain. Classification is also interpreted based on neighbouring domain organization.Multiple sequence alignment is performed for the unannotated conserved SET domain sequences of plant lineages with reference Arabidopsis thaliana and Oryza sativa homolog. If two or more proteins were equally close to a single Arabidopsis thaliana and Oryza sativa SET domain containing proteins, then the same name is used followed by the letters “a”, “b”, etc. The following nomenclature is adopted for classes and subclasses of the SET domain-containing proteins: For e.g. protein type, II-2C would belong to Class II, subclass 2 and subdivision of subclass C.SET domain proteins are named according to the nomenclature of annotated plant and animal SET domain histone methyltransferases with generic capitalized letter followed by species name in the lower case using the common specific protein name. SET domain proteins are named according to the name of the closest To get an insight into the SET domain proteins from plant lineage and their probable functional relationship, we performed a homology search from 16 Archaeplastida against SET domain sequences retrieved from PFAM and NCBI transcriptome assembly. A total of 506 candidate SET domain containing protein sequences are identified , Class II as Ash, Class III as Trx and Class IV as Su(var) and Class V. Class II, III and IV each has also a subclass called Orphan that has SET domain with significantly low homology to the conserved SET domain or absence of signature domains of the respective class. Class V includes Orphan, SETD, and TPR with low sequence similarity in the SET domains to other 4 groups. SETD protein family has a characteristic Rubisco domain. TPR has tetratricopeptide domain while Orphan family have either an interrupted SET domain or have SET domain with few associated domains Fig. . ClassifCyanophora paradoxa, (16) are about 3-fold lower compared to pteridophyta species, Selaginella meollendorffii (51). Nonetheless, small number of SET domain proteins detected in Cyanophora paradoxa may also be due to missing proteins in the currently available proteome. E(z) group of proteins is not detected in, glaucophyta and chlorophyta sp. Volvox carteri though they are present in another chlorophyta species, Chlamydomonas reinhardtii. Ash and Trx group of proteins are identified as early as in glaucophyta and there is a nonlinear increase in the number of Ash group with the increase in complexification of species. Among the four main subfamilies, Ash proteins have maintained nearly stable numbers among plant species. However, a noteworthy and surprising feature is the absence of Ash group of proteins in Micromonas pusila  group of proteins is first identified in chlorophyta, Chlamydomonas reinhardtii with their highest abundance in pteridophytes. SETD proteins are identified in chlorophyta but there is no proportional increase in number in various species of plant lineage. The absence of SETD in glaucophyta and charophyta remains controversial. Unlikely, Orphan proteins are identified since the emergence of glaucophyta. Orphan protein shows highest abundance in Micromonas RCC299, while Marchantia polymorpha possess a minimum number of Orphan family proteins. Sequence comparisons are performed among SET domain containing proteins from other Archeaplastida members and with reference species, Arabidopsis thaliana and Oryza sativa. Increased numbers and diversification of encoded SET domain proteins in embryophyta perhaps reflect increased diversity and complexification of multicellular programs. Characterization of these domains will be useful in understanding functions of SET domain proteins.The numbers of the SET domain containing proteins in unicellular glaucophyta species, aradoxa, 6 are aboTwo hundred and fifty one SET domain proteins belonging to E(z), Ash, Trx, and Su(var) are considered for phylogenetic clustering. Remaining SET domain candidate proteins from Orphan, SETD, and TPR are excluded from phylogenetic analysis due to low sequence similarity. The multiple sequence alignment of the 251 SET domain protein sequences are provided in Additional file Selaginella. A vanadium binding domain is present in Physcomitrella patens and Marchantia polymorpha. Cysteine/serine-rich nuclear protein, (CSR) and Male-specific lethal (MSL) domains are Ostreococcus specific.Cyanophora and Volvox lack this class of protein. Interestingly most members in plant lineage contain only one E(z) homolog whereas Marchantia and Arabidopsis contain three and Picea contains two homologs.E(z) is the catalytic component of Polycomb Repressive complex2 characterized in few members of plant lineage, catalysing the di- and tri-methylation of H3K27 . E(z) clChlorella vulgaris shows the initial presence of PostSET domain in Ash family . This class of proteins are likely to methylate multiple amino acids at different positions in histones.Ash class of proteins have diverse domain combinations. Based on domain combinations a novel classification of Ash proteins is presented here. Ash proteins are classified into 4 classes, Class II-1 to Class II-3 and II-Orphan  in this class of proteins. Variations in the number of PHD domain were noted among the subclass of Class III-1. The III-2 group usually contains one PWWP domain, two to three PHD domain, and one PostSET domain without FYR domain  at its N-terminus and only found in Marchantia polymorpha whereas Class IV-2D contains one Ubiquitin-binding WIYLD domain (WIYLD) at its N-terminus along with PreSET and SET, found in Arabidopsis thaliana, Oryza sativa, Picea abies, and Marchantia polymorpha  comprises of a large group of proteins characterized by the presence of PreSET, SET and PostSET domains. This class is designated here as Class IV Fig. . This clha Table . This doClass V category of proteins share a low level of sequence identity to conserved SET domain proteins, hence are grouped separately. Class V comprises of Orphan proteins, SETD and TPR. Diverse associated domains found in other groups are absent in Class V  domain is specifically associated with E(z) class of proteins. Glaucophyte neither have E(z) proteins nor TCR in SET domain proteins.Other interesting feature in the evolution of the E(z) protein is the presence of the Swi3, Ada2, N-Cor, and TFIIIB (SANT) domain only in rice is indicative of monocot specific function. Aldehyde dehydrogenase superfamily (ALDH-SF); Cysteine/serine-rich nuclear protein (CSR); Male-specific lethal (MSI) are biochemically uncharacterized domains associated with SET domains of E (z) members.Selaginella meollendorffii and Marchantia polymorpha [. This domain is also present in Trx class of proteins.Ash class of proteins have varied domain combinations, therefore we have newly classified them. Except in orphans, AWS domain is very tightly associated N terminally to the SET domain with Ash class of proteins. An interesting feature is the presence of single PHD domain in class II-2B whereas three of them on a protein in class II-2A. TUDOR domain is reported to be absent from plant lineage but we confirm its presence in Ash class proteins of lymorpha . This doIn general Trx class of proteins have larger number of domains. This is the trend towards SET domain proteins of animals. Predominantly PWWP and PHD domains are associated with Trx class of proteins. TUDOR domain has already appeared in SET domain protein in chlorophyta.Marchantia polymorpha). SRA domain emerged as early as chlorophyta and PostSET domain of Su(var)3–9 SET traced back to charophytes and then lost in some members during the subsequent evolution. 3–9 SET proteins of Archaeplastida into two classes is based on the presence or absence of SRA domains. Our analysis reveal WIYLD and Zinc Finger domains of Class IV to have originated in marchantiophyta , Ash, Trx, Su(var) and Orphan. Our work provided framework for experimental characterization of plant SET domain proteins.Additional file 1: Table S1.List of species considered in the present study with the corresponding number of SET domain containing protein. (PDF 10 kb)Additional file 2: Table S2.List of the SET domain containing proteins from species considered in the present study with their corresponding sequence id. (PDF 65 kb)Additional file 3: Figure S1.Multiple sequence alignment of the 251 SET domain protein sequences from E(z), Ash, Trx and Su(var) of the 16 Archeplastida species. (PDF 74 kb)Additional file 4: Figure S2.Introduction of the domains in the Ash SET protein in cryptogam plant lineages. The black arrow indicates the introduction of the indicated domain in the specifically mentioned Archaeplastida species. (PDF 212 kb)Additional file 5: Figure S3.Introduction of the domains in the Trx SET protein in plant lineages. The black arrow indicates the introduction of the indicated domain in the specifically mentioned Archaeplastida species. (PDF 221 kb)Additional file 6: Figure S4.Introduction of the domains in the Su(var) SET protein in plant lineages. The black arrow indicates the introduction of the indicated domain in the specifically mentioned Archaeplastida species. (PDF 85 kb)Additional file 7: Figure S5.Schematic diagrams showing the domain organization of Orphan proteins. (PDF 550 kb)Additional file 8: Figure S6.Domain architecture of Class V- A) SETD and B) TPR family. (PDF 507 kb)


Foraging fiddler crabs form a strict spatial relationship between their current positions and burrows, allowing them to run directly back to their burrows when startled even without visual contacts. Path integration (PI), the underlying mechanism, is a universal navigation strategy through which animals continuously integrate directions and distances of their movements. However, we report that fiddler crabs also use visual orientation during homing runs using burrow entrances as cues, with the prioritised mechanism  determined by the distance  between the goal, indicated by PI, and the visual cue. When we imposed homing errors using fake entrances  and masking their true burrows , we found that frightened fiddler crabs initially ran towards the true burrow following PI, then altered their behaviour depending on the distance between the fake entrance and masked true burrow: if the distance was large, they kept running until they reached the true burrow, ignoring the visual cue; however, if the distance was small, they altered the homing path and ran until they reached the fake entrance. This suggests that PI and visual mechanism in fiddler crabs are mutually mediated to achieve their homing behaviour. ThrouUca spp.) are short-range, central-place foragers, with males in particular using their burrows as a hub for foraging or attracting or visiting females in the neighbourhood. These crabs rely almost exclusively on PI to return to their burrows [Most fiddler crabs  on the true burrow entrance when the crab was inside its burrow and then moved the sheet once the crab had emerged from this superposed burrow, allowing us to create a situation where the fake entrance was shown while the true entrance was masked by the sheet. If the PI of fiddler crabs is strict and any error is compensated for after the straight homing path has been completed, we would expect frightened fiddler crabs to first arrive at the location of the masked true entrance and then search for the burrow. However, we found that they actually ran back towards the fake entrance without arriving at the location of the true burrow first, indicating that they could compensate for their homing errors by using burrow entrances as visual cues. Therefore, this raised the question of whether this compensation occurred before or during the homing runs, as well as over what distance the crabs can compensate for such errors.Therefore, we next conducted an experiment in which the fake entrance was shown and the true entrance was masked by an oblong structure, and the distance over which the oblong sheet was translocated was varied. With video analysis, we first evaluated the threshold value of the translocation distance, up to which the crabs can compensate for the mismatch between their true burrow location and the fake burrow entrance. We next determined whether the crabs compensate before or during the run when they move to the fake entrance: if they compensate for any error before starting their escape runs, they will follow a straight homing path and directly arrive at the fake entrance, and if they compensate for the errors in the middle of their homing runs, they will initially orient towards the true burrow at the starting point of the run but will finally run to the fake entrance without arriving at the location of the true burrow.2.Uca perplexa  at Funaura Bay on Iriomote Island, Okinawa Prefecture, Japan  during the day from 1.5 h before to 1.5 h after low tide in August 2016. We used male U. perplexa that show typical homing behaviour using PI. All experiments were recorded from above using a Panasonic HDC-TM700 camcorder  fitted with a Panasonic VW-W4907H-K wide conversion lens (0.75×) on a four-legged steel frame resulting in a recording area of 3 × 1.7 m. Immediately after the camera was set up, each experiment trial was conducted with a randomly chosen individual in the area.Field experiments were conducted in a large colony (approx. 15 × 20 m) of 2.1.We first tested whether the crabs responded to entrances as visual cues. A fake entrance was constructed by cutting a 1.5 cm diameter hole in the centre of a 10 × 10 cm cork sheet, to which a plastic sheet of the same size was attached for reinforcement . This fake entrance was then superposed on the true burrow entrance when the crab was inside its burrow. We observed that crabs emerged from this modified burrow entrance, foraged across the terrain, engaged in homing behaviour and defended the burrow from other intruding crabs, which is similar to behaviours that are observed under natural conditions.Two corners of the sheet were attached to a fishing line, allowing an experimenter, who was sitting on a chair 1.5 m away, to pull the sheet towards him. After the crab had emerged from its burrow, left the fake entrance and the sheet and moved away to feed, the sheet was translocated to allow either the fake entrance to be shown and the true entrance to be masked by the sheet; or the true entrance to be shown in addition by translocating the sheet a greater distance. The translocation of the sheet was performed slowly so as not to disturb the crab. Then the crab was frightened by the experimenter getting up from the chair. The latter situation was used to test whether the crabs responded to the fake entrance that visually corresponded to the entrance from which they had emerged but was not located at the end of the home vector they had formed during foraging, even though the true entrance, which visually differed from the entrance from which they had emerged, was located at the end of the vector. In total, 15 trials were conducted for each of the above conditions.2.2.a). In total, 40 trials were conducted for this experiment.Based on observations from the preliminary experiment (see Results), we conducted experiments using a fake entrance on an oblong sheet (10 × 30 cm) to determine when (before or during homing runs) the crabs were compensating for the mismatch between their true burrow location and the fake burrow entrance and how long they could do so for. These experiments were carried out in the same way as the preliminary experiment, except that the distance over which the oblong sheet was translocated varied over a distance that still allowed the true entrance to be masked a. In tot2.3.−1) from video images using image-processing software  as a single pixel whose side length was 1.58 mm. We considered the starting point of an escape run as a crab's position at a frame after being frightened and when it moved further than 3.33 mm in the 33.3 ms time interval preceding this frame and further than 10.0 mm over a three-frame interval (100 ms) starting at the previous frame, and the stopping point as a crab's position at a frame after the starting point and when it moved less than 3.33 mm in the 33.3 ms time interval preceding this frame and less than 10.0 mm over a three-frame interval starting at the previous frame. Note that during escape runs, crabs in 5 out of 40 trials were caught by the edge of the sheet and the stopping points were located here; therefore, we excluded these trials from the analysis. Where the stopping point was closer to the fake entrance than the location of the masked true entrance, we considered that the crab had run back to the fake entrance and called these trials ‘F events’. Conversely, when the stopping point was closer to the masked true entrance than the location of the fake entrance, we considered that the crab had moved to the true burrow and called these trials ‘T events’. To check the geometrical relation among the crab's starting point and both entrances, we calculated the relative angle between the direction of translocation (i.e. the direction from the true entrance to the fake entrance) and the direction from the crab's starting point to the true entrance (a).Time series of the crabs' positions in all trials of the main experiments using the oblong sheet were tracked frame by frame . The first model, F, was considered as a vector linking the starting point with the location of the fake entrance, i.e. a direct path to the fake entrance (which would be consistent with the situation where a home vector was updated before the crab was frightened). The second model, T, was considered as a direct path to the true burrow (which would be consistent with the crab using the home vector formed during foraging). For each model, we defined an initial error as the distance between the end of the model (entrance location) and the end of a vector whose direction corresponded to the crab's velocity at the starting point and whose length was scaled with the model. Thus, if a crab oriented towards the fake entrance from the beginning of a run, the error from the F model would be smaller than that from the T model; whereas if a crab initially oriented towards the true burrow , the error from the T model would be smaller than that from the F model. Therefore, we considered that the model was plausible for the initial direct path model if it had the smallest error value and assumed that the direction of the model represented the crab's initial orientation.To determine whether the crabs oriented towards the fake entrance or true burrow at the starting point, we used two direct path models b. The fiet al. [We also calculated direct path models and errors associated with them at each of a crab's positions during an escape run to observe whether and how the errors in both direct path models changed through time and compared the final errors (i.e. the error at the stopping point) between the models to visualize the transitions in them using an averaged error versus time curve. Since the escape runs of both events occurred over different lengths of time, they each had different numbers of data points. Therefore, we used an average curve to reflect the shape of the individual curves, not necessarily the precise timing. To calculate the averaged error versus time curve, individual curves were normalized to their maximum time , as performed by Layne et al. . The dat3.3.1.χ2 test: χ12 = 19.5, p < 0.0001). Therefore, if an entrance was located at the end of a crab's home vector that was formed during foraging, the crab moved there depending almost entirely on the home vector and ignored the fake entrance. However, if there was a mismatch between the location of an entrance and the end of the home vector, the crab compensated for this either before or in the middle of its homing run in this preliminary experiment.In the preliminary experiment, the frightened crabs ran rapidly and abruptly stopped running at the location of the fake entrance or the true burrow or entered the true entrance if it was visible. We found that when the true entrances were visible, all crabs ran to the true burrows. However, when the true entrances were masked, most crabs ran to the fake entrances  that is unexplained but may relate to whether crabs ran, finally having visual contact with the entrance during the runs. The translocation direction of the fake entrance relative to the direction from the crab's starting point to the true entrance did not differ significantly between both trials . Examples of the escape runs in T events and F events are shown in As observed in the preliminary experiment, in the main experiments, the frightened crabs ran rapidly and stopped running abruptly at either the location of the fake entrance or that of the masked true entrance. In total, we observed 14 T events and 21 F events. In the T events, the crabs ran in a straight line and stopped running at the location of the masked true entrance. Some crabs got close to the fake entrance for a few seconds or after a stereotypical crisscross search, while the others continued to search. In the F events, crabs ran towards the fake entrance without stopping at the location of the masked true entrance. Some crabs repeatedly attempted to enter the fake entrance regardless of the absence of a burrow and then searched in its vicinity. It should be noted that crabs in both events ran without stopping until they arrived at either entrance, and that there is significant but slight difference in running speeds during escape runs between the two events (Wilcoxon's rank-sum test: 3.3.a). The maximum translocation distance at which F events were observed was 53.8 mm. The distance at which F events had a 0.5 probability of occurrence was estimated to be 48.8 mm, while the probability that an F event would occur beyond 120 mm was estimated to be 0.0023. We also plotted F events and T events against the distances to the fake entrance and the true burrow and conducted a logistic regression .The distances to the fake entrance and the true burrow did not significantly affect the probability of occurrence of an F event, whereas the translocation distance did . Therefofigure 43.4.W = 170, p = 0.000523, n = 28; figure 5a), and the same was true for the final error . Furthermore, the averaged error from the T model versus time curve was always lower than that of the F model curve during escape runs (figure 5e). These findings indicate that crabs in the T event consistently ran along a direct path to the true burrow during escape runs. By contrast, during an F event, although the crab's initial error from the T model was once again smaller than that from the F model , the reverse was true for the final error . Furthermore, the averaged error from the T model versus time curve gradually became larger while that of the F model curve gradually became smaller, with the two intersecting in the middle of the escape run (figure 5f). These findings indicate that while crabs in the F event initially oriented to the location of the true burrow, they subsequently modified their homing path in the middle of the escape run to finally arrive at the fake entrance, depending on the translocation distance.During a T event, the crab's initial error from the T model was smaller than that from the F model (Wilcoxon's rank-sum test: a). A comparison of the absolute error values showed that the error value in an F event was larger than that in a T event , indicating that if the crab detected an error in PI by a visual cue (entrance) during the escape run, it no longer considered the length of the home vector calculated during foraging. Moreover, a comparison of the total path length in an F event with the sum of the length of a direct path to the true burrow and the distance between the true burrow and the fake entrance (i.e. the length of the shortest path to the fake entrance via the location of the true burrow) showed a significant difference, whereby we also confirmed that the crab ran to the fake entrance without going through the location of the true burrow .We then calculated the difference between the total path length during an escape run and the length of a direct path to the true burrow (i.e. the distance between the starting point and the true burrow) as a distance error a. A comp4.U. perplexa. Considering this, it should be noted again that there is a possibility that crabs in such trials perceived the fake entrance before being frightened. However, this would be unlikely for the following reasons; first, crabs that are actively feeding do not respond to the passive translocation, although the surroundings within the visual contact distance could be perceived to be moving; second, crabs in our experiment as well did not respond to the translocation of the platform; third, as we mentioned in the Results, our logistic regression analysis showed that even if the distance between a crab and the fake (or true) entrance at the starting point is within the visual contact distance it did not significantly affect the crab's burrow selection . Therefore, we conclude it unlikely that crabs perceive the fake entrance as the goal of their path before the run.Fiddler crabs use path integration to locate their burrows from distances at which they cannot see the burrow. When disturbed, fiddler crabs ‘play out’ the home vector and stop when it is exhausted, allowing them to rapidly and accurately return to their burrows via their predominant PI system. In this study, we examined when and how fiddler crabs compensate for an error that would inevitably occur at some stage during foraging. To investigate this, we imposed such an error by showing the crabs a fake entrance and masking the true entrance, i.e. a fake entrance was employed as a visual cue. Because we did not interfere with the crabs per se, they should have directly run to the location of the true burrow if escape runs are exclusively performed by the home vectors calculated during foraging. However, we observed that the crabs sometimes ran towards the fake entrance without stopping at the true burrow. Moreover, they should have oriented towards the fake entrance from the beginning of the run if they perceive it as the goal of their path before the run. However, we found that they always oriented towards the true burrow at the beginning of the escape run, while they finally arrived at the fake entrance. Therefore, we conclude that fiddler crabs can compensate for their homing errors during runs. Moreover, our logistic regression analysis suggested that this compensation is restricted by the distance between the location of the visual cue (entrance) and the end of their home vector. Note that, in some trials, the crab's starting point and the fake entrance might be within its visual contact distance that is approximately five times the eye height above ground ,31,32 anelection b,c. TherWhile crabs can measure the requisite distance by stride integration, we also observed that the total path length was changed if the error was detected using a visual cue. Such changes in distance during escape runs have previously been reported by Walls & Layne , who fouThese findings indicate that the PI system can be suppressed by the visual system in fiddler crabs. Such an interaction between the PI and visual mechanisms has also been found in other animals. For instance, desert ants can give priority to information from a landmark cue rather than from PI if there is a conflict between the two during their long journey, but then continue to follow the path given by PI if no further cue appears ; and honRegarding this point, our results suggest that the navigation system of a fiddler crab is also heterarchical, but whether it is performed in a weighted manner remains unclear. As mentioned previously, the escape runs of fiddler crabs depend almost exclusively on PI whether there is a landmark near to them or another crab's burrow at a closer distance . Moreove


C-terminal histidines of the T-domain. We also test the ability of these mutants to translocate their own N-terminus across lipid bilayers of model vesicles. The results of both experiments indicate that translocation activity does not correlate with previously published OCS activity. Finally, we determined the topology of TH5 helix in membrane-inserted T-domain using W281 fluorescence and its depth-dependent quenching by brominated lipids. Our results indicate that while TH5 becomes a transbilayer helix in a wild-type protein, it fails to insert in the case of the OCS-blocking mutant H322Q. We conclude that the formation of the OCS is not necessary for the functional translocation by the T-domain, at least in the histidine-replacement mutants, suggesting that the OCS is unlikely to constitute a translocation pathway for the cellular entry of diphtheria toxin in vivo.Cellular entry of diphtheria toxin is a multistage process involving receptor targeting, endocytosis, and translocation of the catalytic domain across the endosomal membrane into the cytosol. The latter is ensured by the translocation (T) domain of the toxin, capable of undergoing conformational refolding and membrane insertion in response to the acidification of the endosomal environment. While numerous now classical studies have demonstrated the formation of an ion-conducting conformation—the Open-Channel State (OCS)—as the final step of the refolding pathway, it remains unclear whether this channel constitutes an in vivo translocation pathway or is a byproduct of the translocation. To address this question, we measure functional activity of known OCS-blocking mutants with H-to-Q replacements of  C-domain), the translocation domain (T-domain), and the receptor-binding domain (R-domain)  leucine was measured.Protein Synthesis Inhibition Assay. This assay of physiological activity of diphtheria toxin utilizes a weakened strain of full-length diphtheria toxin carrying the E148S mutation, to reduce the toxic potency )  equipped with double-grating excitation and emission monochromators. The measurements were made at 25 °C in 2 × 10 mm cuvettes oriented perpendicular to the excitation beam. For tryptophan fluorescence measurement, excitation wavelength was 280 nm and emission spectra was recorded between 290 nm and 500 nm using excitation and emission spectral slits of 2 and 4 nm, respectively. Normally stock concentrations of vesicles and protein were mixed to achieve final concentration of 1 μM eviously . The speT-domain inserted into the vesicles containing lipids with bromine atoms attached at different positions [0, measured with 1:1 POPC:POPG mixture For quantitative analysis we used Distribution Analysis method, which fits the data to the following symmetrical twin Gaussian function [Depth-Dependent Fluorescence Quenching with Brominated Lipids: Quenching study is performed by taking a series of fluorescence measurements of the ositions ,52. Herem), dispersion of the transverse distribution of the probe (σ), and quenching efficiency (S). In this particular case, we fixed the value σ at 5.0 Å, which corresponds to a typical value observed in other systems, and used only two fitting parameters hm and S.The three parameters of this above distribution are: most probable distance of the fluorescent probe from the center of bilayer (h


Quality of life (QOL) is a universal, complex, and multidimensional concept used globally that encompasses the physical, psychological, social and spiritual dimensions of an individual’s current life circumstances . QOL shoCambodia is primarily a rural, agrarian country, with 80% of its 14.68 million inhabitants living in rural regions, in addition, almost half 45.9%) of the country’s population lives in poverty  approved the study protocol.P=0.036) or having over five members in the household (P=0.034) positively affected the physical domain . This was followed in descending order by the psychological (13.3), physical (12.8), and environmental (12.1) domains. Being 40 yr of age or over (l domain .P=0.002) and being female (P=0.038) or 40 yr old or over (P=0.020) were negatively associated with the psychological domain. Residents currently living with a spouse had better HRQL in the social relationships domain (P=0.010). Being female (P=0.002) was associated with a lower mean score in the environmental domain. Participants who had no diseases ranked higher in the global QOL score (P=0.041). Individuals who perceived their health as good or themselves as happy had a positive association with all domains except social relationships. The majority of the rural residents in Cambodia experience a moderate level of HRQL.Individuals never smoked had significantly higher mean scores than occasional smokers in the psychological domain  as well as targeting the enhancement of the individual’s subjective happiness in an agricultural community. This study informs health priorities and strategies for HRQL improvement in rural areas of Cambodia and other similar cultures.


Tetratricopeptide (TPR) domains are known protein interaction domains. We show that the TPR domain of FKBP8 selectively binds Hsp90, and interactions upstream of the conserved MEEVD motif are critical for tight binding. In contrast FKBP8 failed to bind intact Hsp70. The PPIase domain was not essential for the interaction with Hsp90 and binding was completely encompassed by the TPR domain alone. The conformation adopted by Hsp90 peptides, containing the conserved MEEVD motif, in the crystal structure were similar to that seen for the TPR domains of CHIP, AIP and Tah1. The carboxylate clamp interactions with bound Hsp90 peptide were a critical component of the interaction and mutation of Lys 307, involved in the carboxylate clamp, completely disrupted the interaction with Hsp90. FKBP8 binding to Hsp90 did not substantially influence its ATPase activity. Fig 1). Downstream of the TPR domain is a calmodulin binding- and a membrane anchor-region at the extreme C-terminus . Two isoforms of this immunophilin have been described, the full-length FKBP8 and a product of the truncated ORF of this gene known as FKBP38 [Immunophilins are a highly-conserved family of proteins that bind immune-suppressive drugs such as cyclosporin, rapamycin and FK506 –3. FKBP8s FKBP38 .FKBP8 belongs to the FK506 family of binding proteins, but unlike many members of this family it lacks a constitutive PPIase activity, which instead relies on regulatory control , 8. The Activated FKBP8 has been shown to bind Bcl-2 and block its interaction with the pro-apoptotic protein, Bad , an actiThe TPR domain of FKBP8 is homologous to other such domains including AIP, CHIP, HOP and Tah1p –24. Thes1-380) was cloned as an NdeI-HindIII fragment representing amino acids 1–380 and lacking the C-terminal trans-membrane helix into pDMXV4 with a non-cleavable C-terminal His6-Tag . The FKBP38 immunophilin domain, representing amino acids 92–207 (FKBP92-207), and a larger fragment containing both the immunophilin and TPR domain (FKBP92-380) were also cloned in pDMXV4 . The K307E mutation was generated in the FKBP892-380 construct by PCR mutagenesis. Full-length human Hsp90β, human Hsp70, two middle domain human fragments (Hsp90β295–689 and Hsp90β295–724) and a yeast C-terminal fragment of Hsp90 (yHsp90546-709) were expressed from pRSETA as His-tagged fusions. The Hsp90α464–732 construct was expressed from p2E , as a PreScission cleavable His-tagged fusion. Proteins were overexpressed in E.coli BL21 (DE3) and purified by Talon affinity chromatography  equilibrated in 20 mM Tris pH 7.5 containing 100 mM NaCl and eluted with the same buffer but containing 500 mM imidazole at pH 7.0. Protein was then concentrated using Vivaspin concentrators  and subjected, as appropriate, to Superdex 75, 200 or Sephacryl 300 HR gel-filtration chromatography equilibrated in 20 mM Tris pH 7.5 containing 500 mM NaCl, 1.0 mM EDTA and 0.5 mM TCEP. Proteins requiring further purification were subjected to Q-sepharose ion-exchange chromatography equilibrated in 20 mM Tris pH 7.5, 1 mM EDTA and 0.5 mM TCEP and eluted with a NaCl gradient in the same buffer. Pure protein was desalted in 20 mM Tris pH 7.5, 140 mM NaCl, 0.5 mM TCEP, concentrated and then stored frozen at −20°C.FKBP8 , with a cell volume of 200 μL, under the same buffer conditions. Typically, aliquots of 700 to 800 μM of FKBP1-380, FKBP92-380, FKBP92-380 K370E mutant protein, FKBP92-207 were injected into 50 μM of intact Hsp90β, Hsp90β295–689, Hsp90β295–724, Hsp90α464–732 or yeast Hsp90546-709. For peptide binding experiments aliquots of 2 mM of Hsp90β (EDASRMEEVD) or Hsp70 (GSGPTIEEVD) peptide were injected into 50 μM FKBP1-380. Heats of dilution were determined in a separate experiment by diluting protein into buffer, and the corrected data were fitted using a non-linear least-squares curve-fitting algorithm  with three floating variables: stoichiometry, binding constant and change in enthalpy of interaction.Proteins were dialysed overnight into 20 mM Tris, 300 mM NaCl, 1mM EDTA pH 7.5. The heat of interaction was measured on an ITC92-380 in complex with yHsp90 DTEMEEVD peptide were obtained by mixing FKBP92-380 with yHsp90546-709 in a 1:1 molar ratio at 18 mg ml-1 and crystalizing by using the sitting drop vapour diffusion technique. Crystals appeared in 0.02 M sodium potassium phosphate, 20% w/v PEG 3350 at 14°C. Data was collected from a single crystal to 2.2Å at the Diamond Synchrotron on beamline IO4_1 at wavelength 0.917Å. The dataset was processed using the DIALS [90-205, lacking the TPR domain) and 2FBN . Arp/Warp [Table 1. Structures and cartoons were displayed using PyMOl [Crystals of FKBPhe DIALS . Moleculhe DIALS , was conArp/Warp  and BuccArp/Warp  were theArp/Warp . Final rng PyMOl  and LigPng PyMOl .1-380, but lacking the transmembrane domain), showed that both peptides bound weakly to FKBP1-380  . In contrast to C-terminal peptides, full-length Hsp90β tightly bound FKBP1-380 (Kd of 2.8 ± 0.1 μM), while no binding interaction was seen for human Hsp70, suggesting that FKBP1-380 selectively binds Hsp90β and that binding may involve additional interactions outside those of the Hsp90β EDASRMEEVD peptide . The immunophilin domain alone (FKBP92-270) failed to bind full-length Hsp90β, indicating that it was not essential for binding to Hsp90β (. Using fragments of Hsp90 (Hsp90295-724 and Hsp90295-689) representing the middle and C-terminal domain of Hsp90β showed that sequences downstream of residue 689, that include the conserved MEEVD sequence, were essential for the interaction . Hsp90295-724 bound FKBP1-380 with a similar affinity  to that seen for intact Hsp90β 2.8 ± 0.1 μM; Fig 2C). The FKBP38 isoform (FKBP92-380) bound Hsp90β with similar affinity  to that seen with FKBP1-380 . The stoichiometry for binding full-length Hsp90β was 1:1 (2 FKBP molecules to 1 Hsp90 dimer) for both the FKBP8 (FKBP1-380) and FKBP38 (FKBP92-380) isoforms. Finally, we showed that FKBP1-380 bound the C-terminal domain of Hsp90α  and yeast , with similar affinities. Analytical gel-filtration chromatography showed that the C-terminal domain of yHsp90546-709 co-migrated with FKBP92-380 , thus confirming the ITC interaction data.TPR domains are known peptide interaction sites . While m92-380 yielded crystals of FKBP92-380 in complex with MEEVD containing peptides of the C-terminal domain of yeast Hsp90. . It was clear that during crystallization the C-terminal domain of Hsp90 had undergone proteolytic cleavage to leave the C-terminal MEEVD peptides bound to the TPR domain of FKBP92-380. Four molecules of FKBP92-380 were observed in the unit cell of the crystal and the structure was solved at 2.18 Å (PDB 5MGX) resolution (Table 1). The bound peptides found were DTEMEEVD, ATEMEEVD , EMEEVD and EMEE. For amino acid residues defined by visible electron density the conformation of the amino acid at similar positions was very similar in all cases .Attempts to crystallise the C-terminal domain of yeast Hsp90 with FKBP92-380 is similar to that solved by NMR, except in the crystal structure of FKBP92-380 a section in the middle of β-strand 3 of the immunophilin domain is restructured into a loop . Superimposition of FKBP92-380 with FKBP52 [Fig 5A). However, the DTEMEEVD peptide of the TPR domain of FKBP92-380 is bound in an opposite polarity to that observed for the MEEVD peptide bound to FKBP52 . In addition to the unexpected polarity, the conformation of the bound peptide for two molecules of FKBP52, found bound with peptide in the unit cell, was modelled differently . On closer inspection, the methionine and valine residues of the bound MEEVD peptide are unexpectedly exposed to solvent in the FKBP52 structure . In contrast, a comparison with the MEEVD binding TPR domain of CHIP [92-380 and CHIP is almost identical , and therefore similar to those also seen in AIP and Tah1 [Fig 6). It is evident that the peptide bound to FKBP52 is most likely not bound in the physiologically relevant state, or was modelled incorrectly.The immunophilin domain of FKBPh FKBP52  shows th of CHIP  shows thand Tah1 , 23. Becδ atom of arginine), but none-the-less access to the PPIase pocket is likely to remain restricted. For example, in an FKBP12 structure (PDB 1NSG) the equivalent atom is His 87 and lies 4.6 Å from the nearest atom of the bound rapamycin [92-380. Considering the rest of the Arg 184 atoms must protrude, at least partly, towards the bound rapamycin molecule, then it appears that access to the binding pocket of this PPIase domain would most likely be restricted.The side chain of Arg 184 . The C-terminal carboxylic acid makes direct hydrogen bonds to the amine nitrogen of Asn 229 and Asn 277 and a salt bridge interaction to one of the amine nitrogen atoms of Arg 225, as well as water mediated interactions to the amine group of Lys 273 . The main-chain amide group of the terminal aspartic acid residue is also hydrogen bonded to the carboxyl group of Asn 277. The aspartate group of the peptide makes a salt bridge interaction with the amine group of Lys 307 and a series of water mediated interactions with the amide groups of Asn 305, Asn 276 and Arg 311 . The other amine group of Arg 311 is directly bonded to the main-chain carbonyl of the last glutamate residue of the Hsp90 peptide (DTEMEEVD).The structure of the FKBPFig 6). The side chain of the peptide valine is itself bound in a hydrophobic environment formed by the side chains of Asn 229, Tyr 232, Glu 233, and Asn 277. While these are polar or charged amino acid residues, the aliphatic sections of their side-chains conspire to form the hydrophobic pocket. The penultimate glutamate of the peptide (DTEMEEVD) forms a salt bridge and a water mediated interaction with the amine group of Lys 314. The remaining interactions consist of hydrogen bonds between the main-chain peptide to the side-chain carbonyl, amide or hydroxyl groups, as appropriate, of Ser 388, Asn 339, and Glu 366. Finally, the methionine side chain is bound in a hydrophobic pocket lined by Ile 306, Lys 307, Phe 310, Thr 341, Ile 342 and Asn 339 . Once again, for the polar or charged residues the aliphatic sections of the side chain conspire to define the hydrophobic pocket.For the peptide valine, the main-chain amide group forms water mediated interactions to the carbonyl and amine side-chain groups of Gln 233 . This suggests that the carboxylate clamp of this TPR domain is similarly important in the binding of the conserved MEEVD motif of Hsp90 .The binding of the conserved MEEVD motif of Hsp90 is dependent on the carboxylate clamp interactions previously identified for this type of TPR domain . AlthougPrevious studies showed that the peptidyl-propyl isomerase, Cpr6, could weakly activate and that the TPR domain protein Sti1 (HOP in humans) could inhibit the ATPase activity of yeast Hsp90 , 42. In 92-380, and shown that this TPR domain is specific for Hsp90 binding over Hsp70. Specificity and high affinity binding to Hsp90 appears to include residues outside the terminal ten amino-acid residues of Hsp90 (EDASRMEEVD). While further structural work will be required to understand the complete interaction between FKBP8 and Hsp90, current attempts to crystallize larger C-terminal domain fragments of Hsp90 with TPR domains have so far failed to yield diffracting crystals.TPR domains are versatile protein interaction modules that have been found to bind the conserved MEEVD, IEEVD EDDVE motifs of Hsp90, Hsp70 and TOMM20, respectively. FKBP proteins represent a large group of proteins that can interact with Hsp90 using TPR domains . FKBPs a92-380 suggesting that its binding was completely disrupted. As with other TPR domains, Lys 307 is part of an extended hydrogen bond network called the carboxylate clamp, and is critical for the binding of MEEVD containing peptides [Yeast Hsp90 C-terminal domain failed to bind the K307E mutant of FKBPpeptides , 23, 27.peptides .92-380 are similar to immunophilin proteins such as FKBP52 [1-380 bound MEEVD containing peptides of Hsp90 were found bound in a similar conformation to that seen in other TPR domain containing proteins such as AIP, Tah1, and CHIP [Fig 5) [92-380 structure were bound within hydrophobic pockets as seen in other similar TPR domain structures, suggesting that the conformation seen in these structures is the correct physiological state [The TPR and PPIase domain of FKBPs FKBP52 . The FKBand CHIP –24. Our and CHIP , 23, 39.al state –24.1-380 did not affect the ATPase activity of Hsp90. In conclusion, we show that FKBP8 specifically binds Hsp90 over Hsp70, but doesn’t appear to regulate the ATPase activity of Hsp90.The regulation of the ATPase activity is a critical feature of the Hsp90 chaperone cycle which involves cycling between an open and closed state by dimerization of the N-terminal domains of Hsp90 –46. Sti1


Dear Editor,Proteins with large molecular weight exhibit increased number of NMR signals, and the resulting spectral overlap typically imposes limitations. Segmental isotope labeling of individual domains of multidomain protein facilitates spectral interpretation by reducing the number of signals and thus provides a solution to spectral overlap  that connects the two domains . Fig. S1C illustrates the generation of the tandem SH3-SH2  domain activated at its C terminus as α-thioester and the kinase domain containing a N-terminal cysteine residue. When combined under physiological conditions, the SH32 and kinase domains chemoselectively react through the well-established traditional chemical ligation  chemical shift changes among the peaks, which are indicative of changes in local environment, and (2) resonances that underwent significant line broadening, thus suggesting local changes caused by conformational exchange  and likely caused by the local effect of ligation. Other changes in chemical shifts occur in four regions, namely, K76–K86 (SH3-SH2 linker), V107-Y116 (βB-βC linker), Y129–S137 , and V172-A178 (SH2-kinase linker) regions, are all spatially close to the N-lobe of kinase domain . The largest of the observed chemical shift perturbations occurred at S78, L79, and M80, and the C-terminal tail of the SH2 domain, with the largest absolute change involving Y116. From the crystal structure of kinase, we can hypothesize that this arises from the close hydrophobic interaction among the SH2-kinase linker and the βBC, βC and two β-strands on kinase domain . These data show that interaction between the SH2 and kinase domain described above is present even in the absence of the SH3 domain.Superposition of the While chemical shift perturbations can be a useful means of mapping interfacial regions within Csk, they are not commonly indicative of the degree of conformational plasticity involved. This structure is considerably different from the active and inactive molecules in the SH2-kinase interface in the full-length Csk. The interactions between the βD-βE loop of the SH2 domain and β3-βC loop in the N-lobe of kinase domain are absent in putative inactive molecules . The Y116A mutant lost its ability to be activated by SH2 domain ligand almost completely, which suggests that the hydrophobic pocket formed by the side chains of Y116, Y133, L138 and L149 must be intact for the SH2 domain-mediated activation of Csk  with free Csk SH2 (with SH3-SH2 linker) crystal structure revealed that the βB- βC and βD-βE loops of the SH2 domain exhibit the largest conformational change upon complex formation. In addition to the Y116, which is the largest perturbation found in NMR, the Y112 conformation is changed significantly with and without the presence of kinase domain. In the lack of proper interacting partner, Y112 side chain folds back to SH2 domain. Overall, we suggest that phosphotyrosyl ligand binding causes conformational changes in the SH3-SH2 and βB- βC linkers, part of the βD, and βD-βE linker region, thereby allowing for modulation of kinase activity.The ligand to Csk SH2 is Cbp. Cbp has been implicated in various aspects of cancer cell biology Below is the link to the electronic supplementary material.