Self-quenching of NBD-PS was considerably increased when phosphatidylinositol-4,5-bisphosphate (PIP2) was present in the membrane. When PIP2 was the labeled phospholipid, in the form of TopFluor-PIP2, fluorescence self-quenching induced by the C2 domain was also observed, but this was dependent on the presence of phosphatidylserine. An independent indication of the phospholipid demixing effect given by the C2α domain was obtained by using 2H-NMR, since a shift of the transition temperature of deuterated phosphatidylcholine was observed as a consequence of the addition of the C2α domain, but only in the presence of PIP2. The demixing induced by the C2α domain may have a physiological significance since it means that the binding of PKCα to membranes is accompanied by the formation of domains enriched in activating lipids, like phosphatidylserine and PIP2. The formation of these domains may enhance the activation of the enzyme when it binds to membranes containing phosphatidylserine and PIP2.The C2 domain of PKCα (C2α) induces fluorescence self-quenching of NBD-PS in the presence of Ca The second group includes the isozymes called novel or new , which are also regulated by anionic phospholipids and diacylglycerols (or phorbol esters) but not by calcium. Finally there is another group which is relatively different, which includes the isoenzymes called atypical (ζ and ι/λ), which are not regulated neither by diacylglycerol nor by calcium Protein kinase C (PKC) is a family of related protein kinases that participate in cell control. The PKCs family includes eleven different mammalian isoenzymes, which are usually subdivided into three groups. Those in the first group are called classical or conventional  and they are activated by anionic phospholipids, diacylglycerol (or phorbol esters) and by Ca2+ acting at the C2 domain, which bridges with membrane phospholipids, especially phosphatidylserine 2, which also acts at the C2 domain 2  binds in the C2 domain of classical PKCs is a groove formed by strands β3 and β4, which is rich in lysine residues and is sometimes called the polylysine site and the three-dimensional structure of the domain bound to soluble PIP2 was determined in pioneering studies 2Classical PKCs are activated, at least, at three different sites; by diacylglycerols interacting with the C1 domain It has been observed that negatively charged lipids are recruited to some protein binding sites as a result of their affinity for the protein sn-phosphatidylserine) in a membrane in which POPC  was also present, an effect that is Ca2+-dependent. Surprisingly, the demixing of phosphatidylserine by the C2 domain was very much increased if PIP2 was also present. Similarly, the C2 domain may also induce the demixing of PIP2 from (POPC) when Ca2+ is present but this effect requires the presence of POPS. In addition 2H-NMR showed that POPC is demixed from a POPC/POPS/PIP2 membrane in the presence of Ca2+ and no demixing was detected in the absence of PIP2.We used fluorescent probes in this work to show that the C2 domain of PKCα may induce the demixing of POPS ; 1-palmitoyl (d31)-2-oleoyl-sn-glycero-3-phosphoserine (POPS-d31); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); 1-palmitoyl (d31)-2-oleoyl-sn- glycero-3-phosphocholine (POPC-d31), phosphatidylinositol 4,5-bisphosphate (PIP2); 1-oleoyl-2-[6-[ amino] hexanoyl]-sn-glycero-3-phosphoserine (NBD-PS); 1-oleoyl-2-[6-[4-(dipyrrometheneborondifluoride)butanoyl]amino]hexanoyl-sn-glycero-3-phosphoinositol-4,5-bisphosphate  were purchased from Avanti Polar Lipids . All other chemicals were highly pure and were obtained from Sigma Chemical Co. .1-Palmitoyl-2-oleoyl-PKCα cDNA was a kind gift from Drs. Nishizuka and Ono .The cDNA encoding the C2 domain of PKCα (residues 158–285) was amplified by PCR using the following primers:CAAGAATTCAAGAGGGGGCGGATTTACC2α:5′ CAAAAGCTTGTATTCACCCTCCTCTTGC2α:3′ The resulting 381 bp PCR fragment was subcloned into the HindIII and EcoRI sites of the bacterial expression vector pET28c(+), in which the insert is fused to a six-histidine tag. The construct was confirmed by DNA sequencing.Expression and purification of C2 domains were performed essentially as described in Protein concentrations were determined at 280 nm Lipid vesicles for FRET experiments were generated by mixing chloroform lipid solutions in the desired proportions and then dried from the organic solvent under a stream of oxygen-free nitrogen. The last traces of organic solvent were removed under vacuum for at least 3 h. Dried phospholipids mixtures were resuspended in the corresponding buffers by vigorous vortexing. Large unilamellar phosholipid vesicles (LUV) (100 nm in diameter) were generating by extruding the lipid emulsion through two stacked polycarbonate membranes  with a pore size of 100 nm (11 passes).2 using deuterium-depleted water. If the sample also included protein, a sufficient amount of lyophilized C2 domain protein to give the final lipid/protein molar ratio of 40∶1 was dissolved in the buffer prepared with water, or with deuterium-depleted water in the case of 2H-NMR, and added to the dried lipid to prepare multilamellar vesicles. The sample was then centrifuged to eliminate unbound protein and the pellet dispersed in 300 µl of buffer and transferred to NMR glass tubes.Samples for NMR were prepared using a total quantity of 15 mg of phospholipid, dissolving the desired amounts of phospholipids in chloroform before being mixed. Samples were then dried under a nitrogen stream and finally under vacuum. When the sample consisted of lipid only, this mixture was dispersed in 100 mM NaCl, 25 mM Hepes pH 7.4 buffer, 1 mM CaCl2 emission spectra was obtained using a 50 µM lipid suspension by scanning from 498 nm to 600 nm with the excitation wavelength set to 490 nm (4 nm excitation and 4 nm emission slit widths).The fluorescence decreases due to the change in volume produced by the addition of protein solution were corrected by subtracting the value obtained by adding the same volume of buffer without protein as a blank. The percent quenching was calculated according with the equation: pF and 0F are the fluorescence intensity in the presence or in the absence of protein respectively.Steady-state fluorescence measurements were carried out on a Fluoromax-3 fluorimeter  at 25°C in an assay buffer composed of 25 mM Hepes pH 7.4, 100 mM NaCl and 250 µM CaCl2. All measurements were performed at 25°C. Fluorescence intensities were determined according to Lakowicz 2H-NMR experiments were carried out on a Bruker Avance 600 instrument  at 92.123 MHz using the standard quadrupole echo sequence 1M, was calculated for each spectrum of the different samples and at each temperature using the following equation f(ω) is the spectral intensity, x is the frequency shift range (between −60 and 60 kHz) and A is defined as:2+ so that in its absence the level of quenching is about 3% only, approaching the experimental error  were noisier than those in the absence of PIP2 (2 (non-fluorescent) vesicles showed that there was a small increase in apparent fluorescence, due to scattering, which was lower of 5% of the signal even with the maximum concentration of C2  and this is interpreted as if PIP2 would facilitate aggregation of NBD-PS induced by C2 domain. It can be observed that this effect also depends on Ca2+ and the addition of EGTA produced a recovery of fluorescence, although a residual quenching of about 8% remains in the absence of Ca2+  was used  (Similar results were observed when the site-directed mutation  was intrr ratio)  membraner ratio) . These r2, a fluorescent analogue as TopFluor-PIP2 was used, labelled in one of the fatty acyl chains. BODIPY fluorescent probes have been also found to suffer self-quenching in lipid vesicles 2/TopFluor-PIP2 (95∶4.5∶0.5 molar ratio), 2/TopFluor-PIP2 (75∶20∶5∶0.5 molar ratio) it was observed that some quenching occurred, being about 19.5% when the molar ratio of total lipid to protein was 30.5  was used and in others the deuterated phospholipid was POPS (POPS-d31). Spectra were measured at different temperatures. When pure POPC, in a mixture POPC/POPCd31 (molar ratio 2.5∶1) was studied (cT) of the pure phospholipid, the spectra were powder patterns characteristic of a gel phase. At higher temperatures (0°C and higher), the spectra corresponded to a fluid-phase bilayer and were axially symmetric with some resolved quadrupole splittings arising from methylene segments in the acyl chains. In order to better discern the phase transition and other effects of the C2α domain on these membranes, the first spectral moment 1M was calculated for each spectrum. 1M measures the average spectral width, and since each phase has a distinct spectral width it will also have a characteristic 1M, which is proportional to the average order parameter. The variations in 1M with temperature can be used to characterize membrane phase transitions and the molecular order of a membrane. The phase transition has its onset at −8°C and it ends at −2°C , spectra that indicate gel state of the membrane were observed up to 8°C  was studied and a gel pattern of the spectra was observed until −4°C and a fluid pattern at higher temperatures  , the spectra showed a decrease in intensity as indicated by the appearance of the isotropic peak coming from the contaminating deuterated water   the patted water . This is31/PIP2 was studied , the effect of adding PIP2 was insignificant since the pattern of the spectra    shows a atio) (2 . Also thatio) (2 . Howeveratio) (2  were verure POPC  and in cn domain  it may b2, which are known to activate PKCα. We show using fluorescence quenching, that the C2α domain induces NBD-POPS demixing and that Ca2+ is indispensable for this effect. It is known that Ca2+ bridges the binding of the C2α domain to POPS 2+ neutralizes negatively charged residues present in the Ca2+-binding site facilitating the approximation of the protein to the membrane where negatively charged are present 2+ is so important, although the C2α domain also interacts through some amino acid residues directly with POPS 2 by annexin A2 has been described recently We have studied the effect of adding C2α to a membrane system composed of POPC plus anionic phospholipids like POPS and PIP2 with a high degree of affinity in another binding site located in a site rich in lysine residues 2The C2α domain is known to interact not only with phosphatidylserine through its calcium-binding site but it also binds one molecule of PIP2, little self-quenching was detected. However, in the presence of PIP2 the self-quenching of POPS-NBD was considerably higher. When TopFluor-PIP2 was used the C2α domain produced self-quenching but only when POPS is also present, indicating that the interaction of the domain with both POPS and PIP2 are necessary to produce anionic phospholipid demixing, although Ca2+ is also needed.The very interesting observation, made in this work is that, in the absence of PIP2+-binding site and the site rich in lysine residues are necessary for the self-quenching of POPS-NBD and the mutation of any of them almost abolish the lipid demixing. Therefore the fluorescence experiments indicate that demixing of anionic phospholipids is very much enhanced when both lipid binding sites of C2α are occupied, the Ca2+ binding site with this cation and phosphatidylserine and the site rich in lysine residues with PIP2. When POPC-NBD was used no self-quenching was detected, thus demonstrating that there is not a specific interaction of the NBD fluorophore moiety with the C2α domain and that the C2α domain establishes an electrostatic interaction with anionic lipids, which leads to their demixing and that an amphoteric lipid like POPC is not affected by this protein. When pure POPC was studied by 2H-NMR, C2α did not affect the phase transition, confirming that the domain does not interact with POPC. However it interacts with POPS as seen in the experiment in which it is added to pure phosphatidylserine, widening the phase transition.The site-directed mutagenesis of the C2α domain showed that both the Ca31 and POPC/POPSd31/PIP2 were studied it was observed that the addition of the C2α domain reduced the intensity of the spectra as indicated by the isotropic peak produced by the contaminating deuterated water, due to the widening of the signal corresponding to POPS molecules interacting with the protein. The 2H-NMR experiments indicated that POPC is demixed with respect to the anionic lipids, and shows a phase transition at the same temperature than pure POPC-d31. This can be explained if most PIP2 molecules are also excluded from POPC and form part of a lipid domain rich in POPS and PIP2, where the C2α domain molecules are bound.When the samples containing POPC/POPSd31/PIP2, it was observed that the presence of the C2α domain does not apparently affect the transition temperature of this phospholipid. We interpret this as competing influences on the transition temperature, since although the segregation of POPC should increase this temperature, PIP2 segregated with POPS should decrease it. The result is that no significant change was observed in the mixture deuterated POPS/POPC and PIP2 in the presence of C2α domain.In the case of the sample with POPC/POPSd31 and POPS are mixed, but in the absence of PIP2, the NMR experiments did not allow to observe any demixing, although the fluorescence experiments showed that this may happens although in a limited extent. This is probably due to the lower sensitivity of the NMR technique for this type of event.When POPCd2 could be due to the great increase in the protein binding to the membrane that is observed when PIP2 is added to a POPC/POPS membrane 2, which has been shown to change with respect to a membrane without this phosphoinositide, by both ATR-infrared spectroscopy We hypothesize that the reason why POPS demixing by the addition of C2α was greatly increased by the presence of PIP2 has been described to be around 1 mol% of the total lipid of plasma membranes 2 may take place in the plasma membrane induced by the presence of C2α domain.The physiological significance of the observed anionic lipid demixing can be suggested, since the concentrations of phospholipids used in these experiments are close to the concentrations found in animal plasma membranes. It is known at this respect that the concentration of POPS in the inner monolayer of eukaryotic plasma membranes, such as in erythrocyte or platelet cells, is roughly 20 mol% 2 to induce the demixing of anionic phospholipids; this can be explained, at least partially, by a recent demostration that shows that the C2A domain of sypnatotagmin 1 does not conserve a lysine residue critical for the interaction with PIP22H-NMR results show (see 1M values of POPC/POPS-d31 decreased in the presence of C2α at temperatures above the phase transition, when they are compared with the sample of just lipid. Nevertheless the decrease in the case of C2α is low and in some temperatures not significant.It has recently been described that the C2 domains of synaptotagmin 1 are capable of segregating POPS within complex membranes show see  that C2α2.In the case of synaptotagmin 1, the observed demixing may serve to facilitate membrane fusion. In the case of the C2α, the physiological implications are different and are probably linked to the activation of the enzyme, an effect supported by the establishment of domains rich in POPS and PIP


The atomistic deformation defects which are closely related to the local dilation volumes or excess volumes and unstable bonding have been systematically analyzed. It has been found that the icosahedral structures are relatively stable under shear deformation until fracture occurs. Plastic flow is indicated by interruption of percolating icosahedral structures, caused by unstable Fe-Si bonding of p-s hybridization in nature.Density functional theory has been employed to investigate the deformation behaviors of glassy Fe-Si-B model systems prepared by  Metallic glasses are non-crystalline alloys with many attractive mechanical properties such as high fracture toughness, exceptionally high tensile strength, and outstanding super-plasticity below their glass transition temperatures . CompareIt is convenient to define the loosely packed atomic clusters that are responsible for the plastic deformation as deformation defects in metallic glasses. In fact, the concepts of deformation defects have been proposed and developed by Spaepen as the free-volume zones  and by Aab initio molecular dynamics which is primarily based on DFT. Several types of stable glassy systems with different characteristic excess volumes or dilatation volumes, i.e., glassy alloys under ambient pressure and different hydrostatic pressures, were prepared for shear deformation. The responses of the free-volume zones and the ideal glassy structure in these systems under shear deformation were determined. It was thought such investigations could determine the relation between mechanical properties of metallic glass and its deformation defects from first principles.The iron-based metallic glasses are superior to their metallic glass counterparts because theoretically they possess greater strength and because their main content iron is relatively cheap . However80Si11B9 supercell consisting of 200 atoms was chosen for the simulation which was performed using first-principles simulation code VASP [ab initio MD simulation the system was first melted at T = 1500 K for 5 ps and is then quenched to T = 650 K with a cooling rate of 2 × 1014 K/s. The system with a density of 7.02 kg/m3 was maintained as undercooled liquid for 1.5 ps, followed by quenching to T = 300 K with a quenching rate of 4 × 1014 K/s. All the ab initio MD simulations were performed in the canonical (NVT) ensemble and a Nose thermostat was used for temperature control. A stable glassy Fe80Si11B9 system was finally obtained by relaxations of atomic coordinates and supercell volume using the conjugate gradient algorithm at T = 0 K and at a hydrostatic pressure of P = 100 kPa. Fe80Si11B9 glassy alloys with different densities which are subjected to hydrostatic pressures of P = 22.02 GPa, −13.13 GPa and −19.50 GPa were prepared using the same procedures. The tolerances of 1.0 meV for maximal change in total energy and 10 MPa for maximal change in stress components were used to determine a stable glassy system.Fe3 was performed by applying shear strain γxz at T = 0 K. Atoms are relaxed in the micro-canonical (NVE) ensemble until the fluctuations of the total energy and stress components of the system are within the tolerances.Deformation of the glassy alloys whose dimensions are about 1 × 1 × 2 nm80Si11B9 systems at ambient pressure (P = 100 kPa) and at T = 0 K, suggesting the alloy systems are fully amorphous. Voronoi tessellation can be used to extract local geometric structures among neighboring atoms [3, I4, I5, I6) where Ii is the number of faces of i edges in the Voronoi polyhedron. It has been revealed that the Voronoi icosahedron indexed as  has the prevailing short range order (SRO) in metallic glasses, and it is generally considered as a unit cell of an ideal glassy system [sure P = 0 kPa andab initio MD simulation is Fe80Si11B9 under ambient pressure since its free energy is the smallest compared with those of Fe80Si11B9 glassy alloys under different hydrostatic pressures. v of atoms which are defined as the volumes of Voronoi cells subtracted from the average volume of the icosahedrons. For those Voronoi polyhedrons with their volumes larger than the average volume of the icosahedrons (v > 0), the histogram can be fitted with an exponential probability function P(v) = (A/vf)exp(−Av/vf) with the fitting parameters A and vf. The fitting parameter vf = 1.15 × 10−3 nm3 can be defined as the average excess volume that an atom can be squeezed into during the mechanical deformation process.The histogram plots of the coordination numbers of atoms are shown in the inset of 80Si11B9 metallic glasses at different hydrostatic pressures are shown in σF = 4.06 GPa at a shear strain γF = 0.0845, beyond which the system is unstable toward fracture at a fracture strain of 0.12. The shear modulus is given by G = 62.47 GPa which is comparable with the experimental one [80Si11B9 glassy alloy subjected to hydrostatic pressure of P = 22.02 GPa shows higher shear modulus, flow strain and flow stress than those of the system at ambient pressure. While these three important mechanical properties of the glassy alloys subjected to hydrostatic pressures of P = −13.13 GPa and −19.50 GPa are significantly lower than those of the system at ambient pressure. The dependences of σF/G and γF on the average excess volumes vf of the glassy alloys are shown in the inset of σF/G and the flow strain γF increase linearly with decreasing vf, suggesting that vf could be used as a meaningful measure of the atomistic deformation defect whose size determines the mechanical properties of the glassy alloy.The stress-strain relations of Fev on the mechanical properties can be elucidated by the analyses on the histogram distributions of excess volumes in different glassy alloys under elastic shear strains, as shown in 80Si11B9 metallic glass subjected to shear strains of γ = 0–0.01 at ambient pressure are shown in vf = 1.25 × 10−3 nm3, manifesting itself by a peak in the distribution function, which does not change with the applied elastic strains, but such a characteristic peak or the value of excess volume changes with applied plastic strains as shown in vf = 1.25 × 10−3 nm3 are vulnerable to plastic deformation. Hence it is reasonable to consider those atomic sites as deformation defects which lead to plastic flow. For Fe80Si11B9 glassy alloy at a hydrostatic pressure of P = 22.02 GPa, there is no obvious peak in the distribution function of excess volumes, as shown in 80Si11B9 glassy alloy at hydrostatic pressures of P = −13.13 GPa and −19.50 GPa, there is more than one peak in their distribution functions of excess volumes, as shown in 80Si11B9 glassy alloys subjected to various hydrostatic pressures show a decrease in the shear strength σF/G and the flow strain γF when they have more vulnerable sites or deformation defects. This provides an atomistic explanation of the mechanical properties of the glassy alloys as shown in the inset of The effect of excess volume Although it is difficult to determine the details of the atomic structure of the deformation defects defined from the above analyses, basically the deformation defects should not be the atomic sites that possess Voronoi icosahedrons or zero excess volume. This could be demonstrated in 80Si11B9 glassy alloy at ambient pressure under a shear strain of γ = 0.075. Compared with that of the un-deformed system (γ = 0), the majority-spin DOS of the system under γ = 0.075 reduces at 0.4–3.0 eV below the Fermi level EF. The close view on such reduction is shown in p-s hybridized and is unstable against large shear strains.Besides the structural and volumetric details of the deformation defects, it is important to see how the electronic structure of the glassy alloy responds to mechanical deformation. γ = 0.075 at E-EF = 0.4–3.0 eV might be related to the changes of T2g and Eg symmetry states in body center cubic Fe, since the electrons of the Fe atoms bonding with other atoms contribute mostly to the total DOS in this energy range. The changes could be caused by the significant elongation of Fe-B bonds in the icosahedron clusters under shear strain, as shown in the insets of σF, while other bonds remain almost unchanged. The icosahedrons are forced apart if the Fe-B bond length is larger than 0.3 nm.The reduction of the majority-spin total DOS of the system under a shear strain of p-s hybridization in nature. The breaking of Fe-B bonds in the percolating icosahedral clusters of atoms could result in fractures of glassy alloys. The density functional theory thus describes accurately the details of the atomic structures, energetics and electronic structures of the modeled metallic glasses under mechanical deformation, which cannot be provided by experiments and other simulation methods.Density functional theory has been employed to investigate the deformation behaviors of glassy Fe-Si-B model systems. The atomic sites that are vulnerable to plastic deformation show a common excess volume, and are considered as flow defects in glassy alloys. Using the excess volume of atomic sites as a measure to characterize the deformation defects, the mechanical properties of glassy alloys subjected to various hydrostatic pressures were found to be successfully connected to the number of deformation defects and their characteristic volume. It was found that the structures of icosahedral clusters of atoms are relatively stable under shear deformation until fracture occurs. Plastic flow is indicated by interruption of percolating icosahedral clusters of atoms, caused by unstable Fe-Si bonding of


Src homology 2 (SH2) domain is a conserved module involved in various biological processes. Tensin family member was reported to be involved in tumor suppression by interacting with DLC-1 (deleted-in-liver-cancer-1) via its SH2 domain. We explore here the important questions that what the structure of tensin2 SH2 domain is, and how it binds to DLC-1, which might reveal a novel binding mode.Tensin2 SH2 domain adopts a conserved SH2 fold that mainly consists of five β-strands flanked by two α-helices. Most SH2 domains recognize phosphorylated ligands specifically. However, tensin2 SH2 domain was identified to interact with nonphosphorylated ligand (DLC-1) as well as phosphorylated ligand.We determined the solution structure of tensin2 SH2 domain using NMR spectroscopy, and revealed the interactions between tensin2 SH2 domain and its ligands in a phosphotyrosine-independent manner. It is a conserved domain of approximately 100 residues and shared by a number of cytoplasmic tyrosine kinase Src homology 2 (SH2) domain was firstly identified from N-terminal noncatalytic region of fujinami sarcoma virus P130Post-translational modification by phosphate group is a highly dynamic process, which is critical for regulation of various cellular events. Especially, phosphorylation/dephosphorylation of tyrosine residues of intracellular proteins is a critical signal for multiple biological events, e.g. DNA replication and repair, chromosome recombination and segregation, motility, metabolism, and gene transcription Tensin is a family of proteins localized to focal adhesions of cells 442DNVPG” of DLC-1 A previous work has revealed a relationship between tensin3 and tumorigenesis by showing a role of tensin3 in maintaining the transformed properties of cell lines from advanced cancers 442DNVPG” of DLC-1. Similar to cten, the interactions between the SH2 domain of tensin2 and DLC-1 were phosphortyrosine independent. The solution structure of tensin2 SH2 domain was determined by Nuclear Magnetic Resonance (NMR) spectroscopy. Tensin2 SH2 domain adopted a typical SH2 fold. Furthermore, chemical shift perturbation indicated a similar manner of tensin2 SH2 domain in binding to nonphosphorylated and phosphorylated ligands.In this study, we identified the interactions between the SH2 domain of tensin2, one member of tensin family, and the fragment “CSRLSIYhttp://smart.embl-heidelberg.de/). Accession number of the proteins used for alignments are listed as follows: Swiss-Prot, O60880, Homo sapiens SH2_SAP; Swiss-Port, P16277, Mus musculus SH2_BIK; Swiss-Port, Q56XZ1, Arabidopsis thaliana SH2_SHB; Swiss-Prot, P23615.1, Saccharomyces cerevisiae SH2_SPT6; Swiss-Prot, Q63HR2.2, Homo sapiens SH2_tensin2; Swiss-Prot, Q9HBL0, Homo sapiens SH2_tensin1; Swisst-Prot, Q68CZ2, Homo sapiens SH2_tensin3; and Swiss-Prot, Q8IZW8, Homo sapiens SH2_cten.Sequence alignments were performed using ClustalW2 CATATGGATACATCCAAGTTCTGGTAC-3′5′-CCG and reverse primer CTCGAGTTTGCTGGGAATGCGCAGGCAGCAfoG-3′5′-TT (restriction endonuclease sites underlined). The recombinant tensin2 SH2 domain contained a C-terminal tag of six histidine residues. The recombinant protein was expressed in Escherichia coli strain BL21 Gold (DE3) and purified according to previous procedure 15N-labeled and 13C, 15N-labeled tensin2 SH2 domain were prepared in the same way, except that super broth was replaced by M9 medium containing 0.5 g/L 15N-labeled ammonium chloride and 2.5 g/L 13C-labeled glucose as the sole nitrogen source and carbon source, respectively. The purified protein was dialyzed with buffer containing 20 mM NaH2PO4, 150 mM NaCl, 50 mM arginine, 50 mM glutamine and 2 mM EDTA, pH 6.8, and concentrated to 0.6 mM for further NMR experiments.Sequence encoding the SH2 domain of human tensin2 was amplified and cloned into pET-22b (Novagen). Following primers were used for polymerase chain reaction (PCR): forward primer 5′- CATTCATTCCAAGGAGCTTCTGGGCTGGCCCTCAAG -3′, reverse primer, 5′- GCCACCTTGAGGGCCAGCCCAGAAGCTCCTTGGAATG -3′. The PCR products were digested with DpnI (TaKaRa) for 10 hours, and then transformed into E. coli BL21. The plasmids containing mutated gene were sequenced to confirm the Y41S mutagenesis. The tensin2 SH2 (Y41S) were prepared as described above for further study.The site-directed mutagenesis (Y41S) was performed by PCR with the previousely constructed plasmid containing gene of Tensin2 SH2. The following primer sets were used: forward primer, 442DNVPG” and “CSRLSI pY442DNVPG” (residues 436–447 of DLC-1 with Y442 nonphosphorylated/phosphorylated) were synthesized by GL Biochem (shanghai). The peptides were purified by HPLC (purity of peptides >95%), and molecular weight of peptides were confirmed by MS analysis.Peptides “CSRLSIY2PO4, 150 mM NaCl, 50 mM arginine, 50 mM glutamine and 2 mM EDTA, pH 6.8). The kinetic analysis of the interactions between phosphorylated peptide and tensin2 SH2 domain was performed at 6-step concentration of tensin2 SH2 domain  at a flow rate of 30 µL/min for 2 min, whereas the kinetic analysis of the interactions between nonphosphorylated peptide and tensin2 SH2 domain was performed at 6-step concentration of tensin2 SH2 domain . The kinetic analysis of the interactions between phosphorylated peptide and mutated tensin2 SH2 (Y41S) domain was performed at 6-step concentration of mutated tensin2 SH2 (Y41S) domain  at a flow rate of 30 µL/min for 2 min, whereas the kinetic analysis of the interactions between nonphosphorylated peptide and mutated tensin2 SH2 domain (Y41S) was performed at 6-step concentration of mutated tensin2 SH2 domain (Y41S) . All analyses were performed three times. To overcome refractive index changes, data of control surface were subtracted from that of peptide-immobilized surface. Interactions between peptide and Albumin Bovine V  were measured as a negative control. Kinetic analyses of SPR data were performed using BIAevaluation 4.1 (Biacore Inc.). Curves were fitted with 1∶1 (langmuir) binding model. The equilibrium dissociation constant (KD) was derived from kinetic analysis.Real-time interactions between tyrosine-phosphorylated or nonphosphorylated peptide and tensin2 SH2 domain or mutated tensin2 SH2 domain (Y41S) were measured by surface plasmon resonance (SPR) on a Biacore 3000 (Biacore Inc.). SPR experiments were performed on sensor chips CM5 . Chip was treated as described previously 2PO4, 150 mM NaCl, 50 mM arginine, 50 mM glutamine and 2 mM EDTA, pH 6.8) to a concentration of ∼0.6 mg/mL. Different molar ratios of SH2/peptide  were prepared in the same buffer. Measurements were taken in a 1 mm path-length quartz cuvette at a rate of 50 nm/min and data pitch of 1 nm. CD spectra of the peptides at corresponding concentrations were measured as control and subtracted. Three successive scans were recorded and averaged. The analyses of CD spectra were performed using Jascow32 software .CD experiments were performed on a Jasco-810 spectrophotometer  over the wavelength range from 200 to 260 nm at 20°C. Tensin2 SH2 domain was dissolved in buffer CO, CBCA(CO)NH, CBCANH, H(CC)ONH, HBHA(CO)NH, HC(CO)NH. 3D 15N-edited and 13C-edited NOESY were acquired with mixing times of 100 and 130 ms, respectively. To identify the slowly exchanging amides, a 15N-labeled sample was lyophilized and dissolved in 99.96% D2O. Immediately, a series of 1H-15N HSQC were recorded to monitor the attenuation of NH signals. NMR data were processed using NMRPipe and NMRDraw NMR samples were dissolved in buffer containing 20 mM NaH15N-edited and 13C-edited NOESY spectra were used to determine the NMR distance restraints for structure calculations. Backbone torsion angle restraints were predicted from chemical shifts of five types of nuclei: 13Cα, 13Cβ, 13CO, 1Hα, and 15NH by using TALOS+ http://nihserver.mbi.ucla.edu/SAVES_3/).3D 15N-labeled protein were titrated with unlabeled tyrosine-phosphorylated or nonphosphorylated peptide to different molar ratios . 1H-15N HSQC spectrum was acquired for analysis. Peptide stock solutions in identical buffer were titrated stepwise with a sample dilution of less then 10%. Combined chemical shift perturfobation was calculated using the following equation: NH and ΔδN are the chemical shift variation in the proton and nitrogen dimensions, respectively. The threshold values used as a criterion for significance were 60% for intensity reduction and 0.1 ppm for chemical shift perturbation.To identify the residues responsible for binding to tyrosine-phosphorylated or nonphosphorylated DLC-1 peptide in tensin2 SH2 domain, 0.5 mM The SH2 domains have been found in hundreds of proteins involved in a variety of cellular processes. The SH2 domains from different proteins share low sequence identity . For exaRecently, the SH2 domain of tensin family protein cten that is able to bind DLC-1 peptides in a pTyr independent manner has been reported To investigate the secondary structure of tensin2 SH2 domain and its change upon binding to ligand, CD experiments were performed. The CD spectra of tensin2 SH2 domain in complex with non/phosphorylated DLC-1 peptides in different ratios indicated that there was no significant change in the secondary structure of tensin2 SH2 domain after binding to the ligands .Tensin2 SH2 domain, including residues from 1135 to 1248 of the protein, was recombinantly expressed and purified. The recombinant product exhibited good solubility and stability. The solution structure of tensin2 SH2 domain was calculated based on a series of NMR spectra. The NMR data used for structure calculations are summarized in As the SH2 domains of tensin2 and SAP both interact with ligands in a pTyr-independent way, the structure of free SAP was used to perform structural compare. The alignment of SAP and the lowest-energy structure of tensin2 SH2 domain was shown in 1H-15N HSQC experiments were recorded for 15N-labeled tensin2 SH2 domain before and after addition unlabeled nonphosphorylated or phosphorylated peptides to different molar ratios of SH2/peptide. The spectral changes that occurred after peptide addition were characterized by resonance intensity reductions and chemical shift changes. The residues of tensin2 SH2 domain interacting with nonphosphorylated ligand were signified by the obviously reduced peak intensity. Compared with it, the residues of tensin2 SH2 domain interacting with phosphorylated ligand were signified by the substantial resonance shift  Residues binding to DLC-1 ligands regardless of the phosphorylation status are located in the central anti-parallel β-sheets and the loops between βB and βC, including Leu30, Ile31, Arg32, Asp33, Ser34, Ser36, Phe37, Gln38, Gly39, Ala40, Tyr41, Gly42, Leu43, Ala44, Leu45 and Lys46. These residues might form the pTyr or Tyr binding site. Notably, Arg32, in βB strand of tensin2 SH2 domain, which is also present in other SH2 domains, is essential for recognition of the Tyr residue of ligand. (2) Residues  located in βE, βF, the loop between βE and βF, and C-terminal coiled coil that correspond to so-called “specificity determine region” might be responsible for the interactions between tensin2 SH2 domain and C-terminus of ligand. Additionally, the third group of residues, located in βA and αB, including Lys9, Leu12, Arg14, Ala17 and Ile18, were identified. Similar as those of SAP SH2 domain, these residues may participate in recognizing the N-terminus of ligand and be required for its specific phosphorylation-independent binding.In summary, we determined the solution structure of tensin2 SH2 domain and identified the pTyr-independent interactions between tensin2 SH2 domain and DLC-1 fragment. Our data imply the SH2 domains of tensin family might have conserved structure, common mechanism of ligand recognition in a pTyr-independent way.Figure S1Kinetic analyses of interactions between mutated tensin2 SH2 domain and nonphosphorylated/phosphorylated peptides by SPR. Kinetic analyses of interactions between peptides and mutated tensin2 SH2 domain were performed at 6 steps of concentration of recombinant SH2 domain at a flow rate of 30 µL/min for 2 mins. A. SPR spectra of the mutated SH2 domain (Y41S) binding to nonphosphorylated peptide. B. SPR spectra of the mutated SH2 domain (Y41S) binding to phosphorylated peptide. Analyses were performed three times at each step of concentration. KD of the SH2 domain binding different peptide was derived from kinetic analysis.(TIF)Click here for additional data file.


Klebsiella pneumoniae can be found in environmental habitats as well as in hospital settings where it is commonly associated with nosocomial infections. One of the factors that contribute to virulence is its capacity to form biofilms on diverse biotic and abiotic surfaces. The second messenger Bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) is a ubiquitous signal in bacteria that controls biofilm formation as well as several other cellular processes. The cellular levels of this messenger are controlled by c-di-GMP synthesis and degradation catalyzed by diguanylate cyclase (DGC) and phophodiesterase (PDE) enzymes, respectively. Many bacteria contain multiple copies of these proteins with diverse organizational structure that highlight the complex regulatory mechanisms of this signaling network. This work was undertaken to identify DGCs and PDEs and analyze the domain structure of these proteins in K. pneumoniae.K. pneumoniae genomes showed that there were multiple copies of GGDEF and EAL containing proteins. Both single domain and hybrid GGDEF proteins were identified: 21 in K. pneumoniae Kp342, 18 in K. pneumoniae MGH 78578 and 17 in K. pneumoniae NTUH-K2044. The majority had only the GGDEF domain, most with the GGEEF motif, and hybrid proteins containing both GGDEF and EAL domains were also found. The I site for allosteric control was identified only in single GGDEF domain proteins and not in hybrid proteins. EAL-only proteins, containing either intact or degenerate domains, were also identified: 15 in Kp342, 15 in MGH 78578 and 10 in NTUH-K2044. Several input sensory domains and transmembrane segments were identified, which together indicate complex regulatory circuits that in many cases can be membrane associated.A search for conserved GGDEF and EAL domains in three sequenced K. pneumoniae showed that most copies were shared among the three strains and that some were unique to a particular strain. The multiplicity of these proteins and the diversity of structural characteristics suggest that the c-di-GMP network in this enteric bacterium is highly complex and reflects the importance of having diverse mechanisms to control cellular processes in environments as diverse as soils or plants and clinical settings.The comparative analysis of proteins containing GGDEF/EAL domains in  Klebsiella pneumoniae, an opportunistic pathogen responsible for a wide range of nosocomial infections that include pneumonia, bacteremia and urinary tract infections, is estimated to cause approximately 8% of hospital acquired infections  and 40 K. pneumoniae PDEs identified here. The alignments were done using MUSCLE [The logo sequences were generated using WebLogo 3.0 . For DGCg MUSCLE .DGC, diguanylate cyclase; PDE, phosphodiesterase; c-di-GMP, Bis-(3’-5’)-cyclic dimeric GMP; REC, receiving domain with phosphoacceptor site; CACHE, Ca2+ channels and chemotaxis receptors domain; CHASE, cyclase/histidine kinases-associated sensing extracellular domain; MASE, Membrane-associated sensor domain; PAS, PER, ARNT and SIM domain; HAMP, Histidine kinases, Adenylyl cyclases, Methyl binding proteins, Phosphatases domain; GAF, cGMP phosphodiesterase, adenylyl cyclase domain; BLUF, Sensing blue-light using FAD.The authors declare that they have no competing interest.The bioinformatics analysis was carried out by DC, analysis of results and discussions were done by DC, MH, ML, LZ and MMZ, the manuscript was prepared by DC, MH, ML, LZ and MMZ. All authors read and approved the final manuscript.Title: Inventory of GGDEF proteins inK. pneumoniae342, MGH 78578 and NTUH-K2044.Click here for fileTitle: Inventory of EAL proteins inK. pneumoniae342, MGH 78578 and NTUH-K2044.Click here for file


PDZ  domains are protein interaction domains that bind to the carboxy-terminal amino acids of binding partners, heterodimerize with other PDZ domains, and also bind phosphoinositides. PDZ domain containing proteins are frequently involved in the assembly of multi-protein complexes and clustering of transmembrane proteins. LNX1  is a RING  domain-containing E3 ubiquitin ligase that also includes four PDZ domains suggesting it functions as a scaffold for a multi-protein complex. Here we use a human protein array to identify direct LNX1 PDZ domain binding partners. Screening of 8,000 human proteins with isolated PDZ domains identified 53 potential LNX1 binding partners. We combined this set with LNX1 interacting proteins identified by other methods to assemble a list of 220 LNX1 interacting proteins. Bioinformatic analysis of this protein list was used to select interactions of interest for future studies. Using this approach we identify and confirm six novel LNX1 binding partners: KCNA4, PAK6, PLEKHG5, PKC-alpha1, TYK2 and PBK, and suggest that LNX1 functions as a signalling scaffold. PDZ domains are protein interaction domains often found in molecules involved in signal transduction, cell polarity and synaptic transmission. PDZ domains were first found to recognize the extreme carboxy-terminal tail of target molecules by recognizing the last few amino acids as well as the terminal carboxylate residue Lnx1 mRNA is expressed in brain, heart, lung, skeletal muscle and kidney LNX1 mRNA is similarly expressed in brain, heart, kidney as well as placenta and pancreas Lnx2 is more widely expressed and mRNA is detected in liver, spleen, lung, heart, kidney, thymus, skeletal muscle and brain LNX1 and LNX2 are RING finger domain-containing E3 ubiquitin ligases that each contain four PDZ domains in vitro and targets specific Numb isoforms for degradation in vivoLNX1 was first identified as a binding partner of the cell-fate determinant Numb. The Numb phosphotyrosine binding (PTB) domain binds specifically to an NPXY motif found between the RING domain and the first PDZ domain  [8]. LNXThe other PDZ domains of LNX1 have been shown to bind the cytoplasmic tail of transmembrane proteins including CAR (Coxsackievirus and Adenovirus Receptor), a tight junction protein that is important for viral binding to target cells In vitro, both PDZ1 and PDZ3 were able to interact with Src A number of cytosolic proteins have also been identified as LNX1 PDZ binding proteins. A yeast two-hybrid screen to identify molecular partners of the pre-synaptic active zone protein CAST identified LNX1 The first PDZ domain of LNX1 is known to recognize four cytoplasmic proteins, Numb, SKIP, RhoC and Src, two of which (Numb and Src) have also been identified as LNX1 ubiquitination substrates The arrangement of modular domains in LNX1 and their ability to recognize a wide variety of target proteins suggest that it acts as a scaffold molecule for a multiprotein signalling complex. In order to further elucidate the activities of LNX1 in the cell, we performed a protein array screen using the individual PDZ domains of LNX1 and identified 53 LNX1 interactors. These potential interactors were combined with the LNX1 interactors described above and predicted interactions mined from other high-throughput screens. Further bioinformatic and experimental analyses suggest that LNX1 functions as a membrane targeted scaffold protein involved in the assembly of signalling complexes regulated by ubiquitylation.The individual PDZ domains of hLNX1 were subcloned by PCR to create pEXP15-hLNX1 PDZ1 (aa 222–380), pEXP15-hLNX1 PDZ2 (aa 373–466), pEXP15-hLNX1 PDZ3 (aa 498–597), and pEXP15-hLNX1 PDZ4Δ3C (with last 3 amino acids deleted to remove a PDZ binding motif) (aa 630–725). Each pEXP15-hLNX1 PDZ construct was transformed into BL21-AI cells  and induced to express GST fusion protein by the addition of 0.2% arabinose. Optimal expression was found under the following induction conditions: for pEXP15-hLNX1 PDZ1 3 h at 30°C; for pEXP15-hLNX1 PDZ2 3 h at 37°C, for pEXP15-hLNX1 PDZ3 and pEXP15-hLNX1 PDZ4Δ3C 1 h at 30°C followed by overnight at 4°C (no shaking). After induction, cells were pelleted and resuspended in PBS++++ ) at 10 mL of buffer/50 mL of culture. Resuspended cells were sonicated three times at 4°C for 30 s using 1 s on/1 s off pulsing. Sonicated cells were centrifuged for 10 minutes at 10,000 rpm in a Beckmann centrifuge rotor JA25.5. Supernatant was incubated with glutathione sepharose beads for 30–60 minutes at 4°C with rotation. Beads were washed 5 times with PBS++++. After washing, beads were resuspended in an equal volume of PBS++++.As needed, GST fusion proteins were eluted from glutathione sepharose beads using 3 incubations with gentle mixing at 4°C with 10 mM glutathione in 50 mM Tris buffer pH 8.0. The concentration of fusion protein in each elution was estimated by running a 10 uL sample in 12.5% SDS-PAGE and comparing to known quantities of bovine serum albumin (BSA). Elutions containing sufficient amounts of protein were combined and dialysed overnight at 4°C against 1× PBS using Thermo Scientific Dialysis Cassettes (Slide-a-lyzers) with 10,000 molecular weight cut-off . The final concentration of eluted, dialyzed fusion protein was estimated by running a 10 uL sample in 12.5% SDS-PAGE and comparison to known quantities of BSA.The Molecular Probes MP30009 Alexa Fluor 647 Microscale Protein Labelling Kit from Invitrogen  was used to fluorescently label approximately 20 ug of eluted and dialyzed fusion proteins GST-hLNX1 PDZ2, GST-hLNX1 PDZ3 and GST-hLNX1-PDZ4Δ3C according to the manufacturer's instructions . The amoIn order to confirm that LNX1 PDZ domains were able to recognize known interactors in a protein array format, custom arrays were printed and screened. Regions of Numb (Entrez Gene ID 18222), either containing (PTB+) or lacking (PTB−) an 11 amino acid insert in the PTB region, were utilized. GST, GST-Numb PTB+ and GST-Numb PTB- were printed in rows at the following concentrations (ng/uL): 250, 125, 63, 32, 16, 8, 4. Each row also contained a blank spot identified as 0 ng/uL. This pattern of immobilized fusion proteins was repeated to create 12 sub-arrays per slide. We tested 0.05 ug, 0.10 ug and 0.20 ug of GST-hLNX1 PDZ2, GST-hLNX1 PDZ3 and GST-hLNX1 PDZ4Δ3C each individually and a combination of 0.05 ug, 0.10 ug and 0.20 ug of each of GST-hLNX1 PDZ2, GST-hLNX1 PDZ3 and GST-hLNX1 PDZ4Δ3C mixed together each on their own sub-array.2, 1% Triton-X-100, 1% glycerol, 0.33 ug/mL Alexa647-labelled hLNX1 PDZ2, 0.50 ug/mL Alexa647-labelled hLNX1 PDZ3, 0.20 ug/mL Alexa647-labelled hLNX1 PDZ4Δ3C) was pipetted over slides and incubated for 90 minutes at room temperature. Slides were washed three times for 15 minutes each time with wash buffer . Slides were left to dry overnight in the dark. Fluorescence was visualized using a PE ProScanArrayHT with 633 nm laser at 10 uM resolution. Spot fluorescence was corrected for local background and normalized using LOWESS normalization as available in the ProScanArray Express software (Perkin Elmer). Quantitated spot fluorescence was adjusted for the concentration of protein at each spot using ProtoArray Prospector® (Invitrogen). Every protein is spotted twice on the human ProtoArray® v4. A protein was considered a hit on a given slide if both spots have an average Z-score of greater than three and the coefficient of variation for the signals from the two replicates is less than 0.5.Optimized conditions were used to incubate the mixture of GST-hLNX1 PDZ2, PDZ3 and PDZ4Δ3C with duplicate Invitrogen human ProtoArray® v4 slides. Briefly, slides were rinsed with PBS+0.1% Tween 20 then blocked with blocking buffer  for 2 hours at room temperature. Then 750 uL of reaction mixture 8-lacZ) was confirmed by western blot of transformed yeast cell lysate. The yeast two-hybrid screen was performed as previously described Expression of GAL4-DNA binding domain-mLNX fusion protein in L40 yeast cells We assembled a list of other LNX1 interactors by searching published literature and interaction databases GRID . The lisThe set of 220 mammalian LNX1 interactors were clustered by Gene Ontology Annotation (GOA) as previously described Murine LNX1, LNX1C45A 293T cells were maintained in 10% FBS in DMEM (Dulbecco's Modified Eagle Medium). Cells were transfected with 2 µg of each plasmid and transfected at 50–60% confluency with 5–10 ug of pFLAG-LNX1, pFLAG-LNX2 with or without N-myc tagged cDNAs as described above, using OPTI-MEM and lipofectamine 2000 (GibcoBRL). After 24 hours, cells were lysed with HNTG-ZE Transfected 293T cell lysate (50–100 uL) was mixed with 3 ug of purified GST fusion protein immobilized on glutathione sepharose beads in a total of 1 mL PLC lysis buffer Binding of GST-LNX1 and individual GST-LNX PDZ domains to immobilized PIP lipids was performed according to manufacturer's protocol with some modifications. Briefly, PIP Strip membranes (Echelon Biosciences) were blocked with TBS containing 3% (w/v) Bovine Serum Albumin (BSA). Blocked membranes were incubated with GST-LNX1 (0.025 µg/ml) and GST-LNX1 PDZ domains (1 µg/ml) in TBS with 3% BSA, at 4°C overnight. Membranes were washed three times with TBS containing 0.1% Tween-20 and then incubated with rabbit anti-GST (2 µg/ml) in blocking solution for one hour at room temperature. After washing three times with TBS with 0.1% Tween-20, membranes were incubated with anti-rabbit HRP (Horseradish Peroxidase) in blocking solution, washed, developed using ECL  and exposed to film.A recent study demonstrated that the binding specificity for a given PDZ domain can be reliably predicted from that of another PDZ domain with known specificity if there exists sufficiently high identity in the binding site sequence, as represented by 17 residues that have contact with the ligand In order to identify novel LNX1 PDZ domain binding targets we screened Invitrogen ProtoArray® v.4 slides with the isolated PDZ domains of LNX1 expressed as GST fusion proteins. We used custom printed protein arrays to optimize conditions for LNX1 PDZ domain binding to immobilized fusion proteins. Test protein arrays were printed with positive and negative control proteins to confirm that Alexa 647-labelled GST-LNX1 PDZ domains were able to bind to immobilized GST fusion proteins but not GST alone. The fusion proteins GST, GST- Numb PTB+ and GST-Numb PTB- were printed on PATH slides at eight concentrations ranging from 0–250 ng/uL. We found that the individual hLNX PDZ domains bound to proteins on the protein array with differing efficiency, therefore, different amounts of each were used in the screen. Specifically, GST-hLNX PDZ4Δ3C binding to positive controls was detectable at a lower concentration than binding by GST-hLNX PDZ3. Therefore, the final screen utilized 0.33 µg/mL GST-hLNX PDZ2, 0.50 µg/mL GST-hLNX PDZ3 and 0.20 µg/mL GST-hLNX PDZ4Δ3C.Duplicate Invitrogen ProtoArray® v.4 slides were treated identically and probed with a mixture containing Alexa 647-labelled GST-PDZ2, -PDZ3 and -PDZ4Δ3C . Slides Of the 62 proteins identified as LNX1 PDZ domain binding partners, four proteins corresponded to 3′ untranslated regions of cDNA, three proteins corresponded to cDNA that has been removed from NCBI's GenBank database due to standard genome annotation, one protein corresponded to a cDNA that is permanently suppressed in NCBI's GenBank database as it is now thought that this gene does not encode a protein and one protein corresponded to a cDNA that is permanently suppressed in NCBI's GenBank database as it is a non-sense mediated decay candidate. The 53 remaining proteins corresponded to annotated proteins.As it has been shown that some PDZ domains are able to recognize and bind other PDZ domains, we searched the 53 hits for proteins containing PDZ domains. We found that only one protein, DEPDC6, that contains two DEP domains and one PDZ domain. The very low number of hits containing PDZ domains could indicate that LNX1 PDZ domains do not recognize other PDZ domains, that the LNX1 PDZ domains do not recognize other PDZ domains when expressed individually or that LNX1 PDZ domains are not able to effectively recognize other PDZ domains in this assay format.Among the 53 newly identified LNX1 PDZ binding proteins, 11 proteins were found to contain a kinase domain: DAPK1, PAK1, CDK2, PLK3, AURKB, AURKC, MAPKAPK3, DYRK3, EPHA8, EPHB3 and TRIB1 . To deteInspection of the disease relationships of the 53 hits revealed 11 proteins that have previously been linked to tumours and/or cancer . The proWe also identified three proteins, KCNA4 (Kv1.4), KCNAB1 and KCNAB2 that are voltage-gated potassium channel subunits while another hit, ARPP-19, is able to act as a potassium channel regulator. In addition, SCLT1 is known to be a sodium channel subunit. This is in keeping with the observation that a number of PDZ domain containing proteins are known to associate with and regulate activity of transmembrane proteins and channels. Finally, nine of the 53 hits are uncharacterized proteins containing either no known domains or domains of unknown function.C. elegans and humans have been shown to preferentially recognize motifs with a carboxy terminal cysteine We examined the carboxy-terminal tails of all 62 proteins for information about the binding selectivity of the LNX1 PDZ domains. Approximately one-third of the hits (21) had carboxy terminal tails that conform to an expanded list of PDZ domain binding motifs based on Tonikian et al., To further investigate the binding specificity of LNX1 PDZ2 we superimposed the structure of LNX PDZ2 (PDB id: 2VWR) with that of PSD-95 PDZ3 bound to a Class I PDZ ligand (PDB id: 1TP3), and modeled mutagenesis of the ligand's carboxy terminal valine to a cysteine residue using Coot 6 transformants. Of the 77 colonies that were selected and restreaked from the library plates, 44 clones exhibited ß-galactosidase activity. After eliminating false positives, out of frame fusions and fusions with non-coding regions, we found six clones that interacted with an individual LNX1 PDZ domain  and MF4–Serine/threonine kinase activity (containing 30 proteins). To determine whether interactions identified in the protein array and other high-throughput screens could be verified by co-immunoprecipitation, we obtained cDNAs corresponding to 14 proteins for further study. We selected three proteins from MF1, seven proteins from MF4 (protein serine/threonine kinase activity), as well as one protein from MF7 (voltage-gated ion channel activity), one protein from MF9 (receptor activity) and two proteins that did not have GOA . These cWe then investigated which of the LNX1 PDZ domains were able to recognize the coimmunoprecipitated proteins described above by performing GST-fusion pull downs with isolated LNX1 PDZ domains . GST-PDZNext we used the carboxy terminal residues of the proteins recognized by individual LNX1 PDZ domains to search the list of 220 LNX1 interactors for other proteins that have similar carboxy termini . AllowinAs PDZ domains have also recently been shown to interact with membrane phosphoinositides (PI) In this study, protein array and yeast two-hybrid screens were performed to identify binding partners for the PDZ domains of the E3 ubiquitin ligase LNX1. We combined our data with that of published reports to develop a comprehensive list of potential LNX1 binding partners. Computational analysis of this large data set has further defined the LNX1 PDZ domain specificities, and cluster analysis based on Gene Ontology annotation has revealed potential new biological roles for LNX1.In addition to the protein array screen performed here, two other published accounts used LNX1 as bait for high-throughput screens of defined libraries. In Rual et al. a high-throughput yeast two-hybrid screen, 7194 human Entrez Gene IDs were provided for the defined library + channel Previously, LNX1 PDZ1 has been shown to bind the intracellular proteins SKIP LNX1 PDZ2 has been found to bind both intracellular and transmembrane targets The first published binding partner for LNX1 PDZ3 was the proto-oncogene c-Src, a cytoplasmic protein tyrosine kinase Previously, no ligands have been identified for LNX1 PDZ4. Here we identify two protein ligands, PKC-alpha1 and PAK6, as well as multiple PIPs as ligands for PDZ4. Both PKC-alpha1 and PAK6 also interact with PDZ2. Only PDZ4 was able to bind to PIPs, suggesting that an important function of PDZ4 is to interact with membrane phospholipids that may influence LNX1 subcellular localization. Interestingly, a similar binding specificity has been observed for PTP-BL (Protein Tyrosine Phosphatase - Basophil Like) PDZ5, i.e., very few protein interactors and the ability to bind phospholipids http://www.reactome.org) highlighted two pathways that are statistically over represented. This analysis suggests a role for LNX1 in tight junction organization through association with claudin family members as well as a potential role for LNX1 in GAP junction trafficking and regulation. Future work to identify the specific molecules present in the LNX1 multi-protein signalling complex in different cell types will be needed to elucidate LNX1 functions.Our work has shown that each PDZ domain of LNX1 contributes a specific element to the overall function of LNX1 as a signal transduction complex scaffold. We also considered that as components of a scaffold protein, individual PDZ domains of LNX may interact with distinct elements of a single pathway. Analysis of the list of 220 LNX interacting proteins using Reactome Pathway Analysis Click here for additional data file.Figure S2Comparison of the amino acid sequences of 35 PDZ domains, 29 type I domains as classified by Tonikian et al. , four PDZ domains shown to bind peptides with cysteine in the 0 position including PDZ-RhoGEF , PTP-BL  and SITAC , as well as LNX1 PDZ2 and LNX2 PDZ2. Cys-peptide binding PDZ domains are highlighted in blue. ClustalW generated a guidetree from the distance matrix of the alignments, visualized using Dendroscope (TIFF)Click here for additional data file.Table S1List of proteins that was positive for LNX1 PDZ domain binding on both ProtoArray® slides.(XLS)Click here for additional data file.Table S2Comprehensive list of LNX1 PDZ domain interacting proteins assembled from ProtoArray® and yeast two-hybrid screens as well as those previously identified in three separate high-throughput screens .(XLS)Click here for additional data file.Table S3The carboxy terminal sequences of confirmed LNX PDZ domain interacting proteins was used to search the complete list of interactors in  Twenty-eight proteins with similar carboxy terminal sequences were identified.(XLS)Click here for additional data file.


Protein-protein interactions (PPIs) are essential events to play important roles in a series of biological processes. There are probably more ways of PPIs than we currently realized. Structural and functional investigations of weak PPIs have lagged behind those of strong PPIs due to technical difficulties. Weak PPIs are often short-lived, which may result in more dynamic signals with important biological roles within and/or between cells. For example, the characteristics of PSD-95/Dlg/ZO-1 (PDZ) domain binding to internal sequences, which are primarily weak interactions, have not yet been systematically explored. In the present study, we constructed a nearly random octapeptide yeast two-hybrid library. A total of 24 PDZ domains were used as baits for screening the library. Fourteen of these domains were able to bind internal PDZ-domain binding motifs (PBMs), and PBMs screened for nine PDZ domains exhibited strong preferences. Among 11 PDZ domains that have not been reported their internal PBM binding ability, six were confirmed to bind internal PBMs. The first PDZ domain of LNX2, which has not been reported to bind C-terminal PBMs, was found to bind internal PBMs. These results suggest that the internal PBMs binding ability of PDZ domains may have been underestimated. The data provided diverse internal binding properties for several PDZ domains that may help identify their novel binding partners. Caenorhabditis elegans , which once led to the speculation that alternative splicing may occur more frequently in humans than invertebrates. Further investigation revealed no major differences in the frequency of alternatively spliced genes among humans and any of the other animals tested, including C. elegansThe human genome encodes 20,000∼25,000 protein-coding genes, which is much lower than previous estimates  Protein domains, such as SH2 (Src Homology 2), SH3 (Src Homology 3), PDZ (PSD-95/Dlg/ZO-1), and WW domains, are abundant PPI modules that mediate a variety of biological and cellular functions Most PDZ domains are involved in the recognition of the extreme C-terminal PDZ-domain binding motifs (PBMs) of target ligands; however, accumulating evidences have revealed that some of them can also bind internal PBMs of partner proteins . Efforts8, , which will not encode any of the three stop codons: TAA, TAG, and TGA. Further, we symmetrically explore the potential internal sequence binding properties of a panel of PDZ domains by screening this special library in the Y2H assays.For some PDZ domains, no C-terminal ligands were screened out in the C-terminal phage-display peptide system Escherichia coli and positive clones were selected for DNA sequencing.The information of 24 PDZ domains from 18 proteins was shown in GAATTC GCT5′-TAG GGG  (NNY)8 TAC GGGATCCAT CGA-3′ , encoding 8 random codons in the middle, which flanked by EcoR I and BamH I restriction sites, was synthesized to construct a random peptide library. PCR was performed with 240 pmol of the oligonucleotide and 240 pmol of the primer 5′-TCGATGGATCCCGTA-3′ in a total volume of 120 µl. The complementary chain of the template was synthesized by program included denaturation at 94°C for 1 min; followed by annealing at 42°C for 10 min, and extension at 50°C for 10 min. After double digestion with EcoR I and BamH I, the products were recovered by short DNA fragment rapid recovery kit and ligated into pGADT7 AD vector, which predigested with the same enzymes. A negative control, in which no insert DNA fragments were added to the ligation reaction, was performed to monitor vector self-ligation. The overnight ligation products were desalted and transformed into electrocompetent cells at 1.8 kv with E. coli Pulser Apparatus (BioRad). The transformation product was recovered in 1 mL of LB broth in 37°C for 1 h at 200 rpm. Transformation product (1 µl) was diluted 1∶1000 times. The diluted product was spread on a pre-warmed LB-Amp plate and incubated at 37°C overnight until colonies appeared. The numbers of the colonies were counted and the transformed clones calculated with: Transformed clones  =  (Colony number/Volume plated (µl)) ×dilute times×1000 µl/ml 5′-CTATTCGATGATGAAGATACCCCA-3′, and reverse primer, 5′-CTTGCGGGGTTTTTCAGTATCTAC-3′). The plasmids extracted from these clones were further sequenced.An oligonucleotide 7+Leu+Trp transformants were selected on plates with SD -Trp-Leu-His medium in the primary screening and then tested by the improved LacZ assay in the second screening. After rescue, the potential positive plasmids were isolated and retransformed into the yeast strain CG1945 containing corresponding bait plasmid. Only the clones that were positive for all the reporter assays and confirmed by at least two independent tests were selected for specific interactions and sequenced Each GAL4 BD-fusion bait plasmid was transformed into the yeast strain CG1945 using the lithium acetate protocol. The transformants were grown on SD/-Trp plates and lacZ assays were performed to examine self-activation. The transformants that had no background growth or had background growth but could be inhibited by 3-amino-1,2,4-triazole and were also negative for the LacZ assay were selected for the subsequent screening. The random octapeptide library was screened following the MATCHMAKER Two-Hybrid System protocol (Clontech).Approximate 10Internal PBMs for each positive PDZ domain were aligned with ClustalX1.83, respectively, and further refined by GeneDoc software. Consensus sequences were deduced from sequence alignments.7 transformed clones. We evaluated the recombination frequency and diversity of our library. Fifty clones were randomly selected from the library and identified by colony PCR for further sequencing. The library may have a negligible number of empty vectors; however, in some clones, the insertion sequence was elongated due to multiple DNA fragments self-ligation  0) of the C-terminal motifs of PDZ domains 0 with a less buried surface. For C-terminal PBMs of ZO1-1, both W and Y were preferred at P−1, which was shown to interact with the Asp in position β3-5 (a) ZO1-PDZ1: ZO-1 is a scaffold protein of the tight junction complex that is known to bind α-actinin-4 requency . The mosS[K/R][I/L]*), cadherin 23 (-ITEL*), hFrizzled 8 (-LSQV*), and neuroligin (-STTRV*) (* represents the stop codon of the peptide) via its PDZ domain T-C-[FL]-C-[SH]-R-C-  GOPC-PDZ: GOPC has been reported as a canonical class I PDZ protein that can bind to SSTR5  RIMS2-PDZ: RIMS2 (Rab-3-interacting molecule 2) is a scaffold protein of the pre-synaptic active zone, which is involved in exocytosis and synaptic plasticity. It has previously been suggested that the hScrib-4, ZO1-1, and RIMS2-1 PDZ domains can recognize internal sequences due to their larger flexible carboxylate-binding loop ino acid . The intZ domain .VTI]-L-Y-[SG]-S-Φ (L-X-[HD]-X-X-D-H-I ((d) Dvl2-PDZ: Previous studies have revealed that the Dvl2-PDZ binding site is inherently flexible and can accommodate different types of ligands (e) LNX2-PDZ1: Ligand of numb protein-X2 (LNX2) protein contains four PDZ domains that might target specific substrates to mediate ubiquitin ligase activity (f) HtrA2-PDZ: The serine protease HtrA2/Omi helps maintain mitochondrial function by handling misfolded proteins in the intermembrane space. The PDZ domain within the protein recognizes both C-terminal and internal PBMs of extended, hydrophobic tri-peptides in the phage-display system A-I-X-[ALV]-[IV]  Syntenin1-PDZ2: Syntenin is a multifunctional intracellular adaptor protein with two adjacent PDZ domains. The PDZ domains were not restricted to binding a unique sequence, but were capable of binding multiple peptide motifs EXLV/I-COOH) and internal (-XEXAVDX-) motifs E may be important for both recognition events. However, this amino acid was not encoded by internal fragment in our system. Nevertheless, the internal PBMs were detected for Par6A-PDZ, which contains extensively hydrophobic tri-peptides that end with the conserved aromatic amino acid F ((h) Par6A: Par6 is a cell polarity protein containing a single PDZ domain that has been shown to bind both C-terminal , which is also a class I motif T-[FLV]-I- is the main interface for the internal recognition.(i) Whirlin PDZ3: Whirlin is a multi-PDZ scaffold protein, and defects in this protein have been shown to be involved in deafness and short cochlear hair cell stereocilia in mice and recessive deafness (DFNB31) in humans -[FLV]-I , which c(j) Harmonin PDZ1: The harmonin-1 PDZ domain binds the C-terminal PBM (-LTFF*) of harmonin and an internal PBM of cadherin 23 (k) Only one internal sequence was screened out from the library when the PDZ domains NHERF1-1, NHERF1-2, DlG5-3, and Par3L-1 were used as bait sequences, respectively . For theAccording to the intrinsic properties of the internal PBMs, they can be divided into three major categories: cryptic class I, such as those from ZO1-1, GOPC-1, harmonin-1, and whirlin-3, in which obvious motifs similar to C-terminal Class I PBMs could be found; cryptic class II, such as those from RIMS2-PDZ, HtrA2-PDZ, and syntenin1-2 in which the composition of the sequences were almost hydrophobic amino acids, or similar to those from LNX2-1 in which the hydrophobic amino acids were separated by hydrophilic residues ; cryptic class III, such as those from DVL2-1 and Par6A-1, in which diverse internal PBMs were detected.C-terminal PBMs were probed against several PDZ domains  due to tDCP from the Pals1-PDZ domain, in which the negatively charged amino acid Asp forms salt bridge with the positively charged Lys residue from the carboxylate binding loop of Par-6-PDZ, and the salt bridges induces a conformational change of the carboxylate binding loop, resulting in the interaction DCP.The proportion of hydrophobic residues in the internal PBMs was higher than that of hydrophilic ones, indicating that, for internal recognition, hydrophobic amino acids may be preferred by the binding sites of PDZ domains. In contrast, some charged amino acids  are usually located at the N-terminus or C-terminus of the internal PBMs, such as those from the ZO1-1, syntenin-1, and harmonin-1 PDZ domains. The presence of these amino acids may be consistent with the Par-6-PDZ domain recognizing the internal sequence HREMAV219EMYYS223COOH) belongs to a Class 3a motif −1 and P−5, indicating that the interactions with some the binding sites within the pocket of PDZ domains were highly specific. The C-terminal binding properties of the Cask-PDZ domain (WXΩFDV-COOH) were determined in the phage display system, in which the P−5 site was ready to accommodate the amino acid W. Thus, the absence of particular amino acids, such as W, may be an obstacle for probing the internal PBMs for these domains. Another reason may be that these PDZ domains may prefer longer internal PBMs, so a library with internal peptides of 10 or 12 residues may suit for screening these PDZ domains.No internal motifs were probed for some PDZ domains, which could be caused by the deficiency of five amino acids in our system as mentioned above. The Scrib-PDZ4 domain has shown an ability to bind to TBEV-NS5 via a predominant internal motif, Although the C-terminal binding properties of two PDZ domains tend to be similar, such as ZO1-1 and HtrA2-1, or GOPC-PDZ and harmonin-1, their internal binding properties may be different in regards to several aspects, which indicates that the diversity in PDZ domain binding properties can be more easily revealed by studying their preferred internal sequences.SP) score was used to measure the position specificities of C-terminal ligands in a previous study SP score ranged from least specific  to most specific . The SP score could be an indication of the flexibility of the binding groove of the PDZ domain. The lower SP score suggests higher plasticity of the PDZ pocket, which accommodates more kinds of ligand residues. We introduced the SP score for C-terminal PBMs determined in the phage-display system SP score for human PDZ domains  at the P0 and P−1 positions of their C-terminal PBMs  score has potential to predict the internal PBMs binding ability of PDZ domain.Here, we focused on the interaction patterns between PDZ domains and internal peptide motifs of their ligands. A systematic strategy based on Y2H screening of a special octapeptide library was developed for characterization of the internal sequences binding properties of a panel of PDZ domains. Among them, 14 PDZ domains were confirmed to bind to the internal sequences, most of them exhibited obvious consensus against one particular PDZ domain, and showed that the internal binding properties were diverse among different PDZ domains. These data suggest that the internal PBMs binding ability of PDZ domains may have been underestimated. The specificity potential Click here for additional data file.Table S1General information for 24 PDZ domains.(XLS)Click here for additional data file.Table S2Primers used for molecular cloning in this study.(XLS)Click here for additional data file.Table S3Detailed sequence information of the C-terminal and internal PBMs screened from the special peptide library against 14 PDZ domains.(XLSX)Click here for additional data file.Table S4SP values for C-terminal ligand subsite for some PDZ domains determined in the phage display system .(XLS)Click here for additional data file.


The two prevalent isoforms in humans  are time-tested targets for drugs that release nitric oxide (NO) and new compounds that either sensitize the enzyme for activation by NO or activate the enzyme independently of NO. Homologous NO-GC subunits consist of an amino-terminal HNOX domain followed by a PAS- and a coiled coil domain and the carboxy-terminal catalytic domain.NO-sensitive guanylyl cyclases (NO-GC’s) that catalyze the reaction of GTP to the second messenger molecule cGMP are heterodimeric enzymes consisting of an α and a β1 carboxy-terminus leading to a monomeric enzyme complex. Surprisingly but in accordance with the FRET results these conjoined NO-GC’s  showed specific enzyme activity and stimulation by NO and various modulators of GC activity.Measurement of FRET between fluorescent proteins tagged to the amino- and carboxy-terminus of the subunits indicated close proximity between the amino-terminal HNOX domains and the carboxy-terminal catalytic domains of the enzyme. On the basis of these results we constructed conjoined NO-GC’s by fusion of the α amino-terminus to the βThese obligate enzyme variants faithfully reproduced the pharmacological properties of the heterodimeric enzymes including an isoform specific differential activation of the NO-independent drug cinaciguat. Novel applications of conjoined NO-GC’s including adenoviral gene transfer will be discussed.


SCOP is a hierarchical domain classification system for proteins of known structure. The superfamily level has a clear definition: Protein domains belong to the same superfamily if there is structural, functional and sequence evidence for a common evolutionary ancestor. Superfamilies are sub-classified into families, however, there is not such a clear basis for the family level groupings. Do SCOP families group together domains with sequence similarity, do they group domains with similar structure or by common function? It is these questions we answer, but most importantly, whether each family represents a distinct phylogenetic group within a superfamily.Several phylogenetic trees were generated for each superfamily: one derived from a multiple sequence alignment, one based on structural distances, and the final two from presence/absence of GO terms or EC numbers assigned to domains. The topologies of the resulting trees and confidence values were compared to the SCOP family classification.We show that SCOP family groupings are evolutionarily consistent to a very high degree with respect to classical sequence phylogenetics. The trees built from  structural distances correlate well, but are not always consistent with SCOP (hand annotated) groupings. Trees derived from functional data are less consistent with the family level than those from structure or sequence, though the majority still agree. Much of GO and EC annotation applies directly to one family or subset of the family; relatively few terms apply at the superfamily level. Maximum sequence diversity within a family is on average 22% but close to zero for superfamilies. Proteins are made up of domains. Protein domains in this context can be regarded as the building blocks of proteins, and the smallest units of protein evolution. A small protein may consist of a single domain, larger proteins maybe contain multiple domains. A domain can be defined as a protein unit which is seen in nature either on its own or in combination with other different domains.Detecting the evolutionary relationship between two or more domains using sequence information alone is often not possible, as sequences often diverge beyond the point of detection by comparison methods. Lack of sequence information does not necessarily show that there is no relationship between domains. If the three dimensional structure of the domains is known, evolutionary relationships can usually be recognised. The Structural Classification of Proteins (SCOP)-3, is a Levels of classification in SCOP from the top down are: class, fold, superfamily, family. A class is just a convenient grouping, e.g. domains containing only alpha-helices. Folds and superfamilies have a clear and precise definition of what they are supposed to represent: a fold groups together domains which have the same topological arrangement of secondary structure; a superfamily groups together domains which share a common evolutionary ancestor. The family level sub-groups domains within a superfamily, but unlike the other levels lacks a precise definition. The first SCOP paper states 3Many projects have been based on the SCOP classification leading to several thousand citations-8. Most We also draw on protein functional information taken from gene ontology (GO) terms. GO is aTo understand the meaning of a family, we compared the groupings of domains in SCOP to determine the similarity to automatically generated groupings based independently on the three aspects we wished to investigate: sequence, structure and function. Since we begin without a pre-conceived idea of the granularity or size/depth of the groupings it is necessary to generate the automatic groupings at every possible level. This is represented by a tree which is the result of hierarchical clustering of the domains based on one of the three sources of information: sequence similarity, structural similarity, functional labels (in the forms of Gene Ontology and Enzyme Classification). The level of agreement between one type of information and the grouping of a SCOP family can be assessed by asking whether each edge in the tree divides domains into family groups, or splits a family, grouping together domains from different families.The ROC curve FigureWithin the literature there is variation in suggested levels for the minimum informative bootstrap confidence,12, withThe top 13 edges which conflicted with the sequence trees were examined. These are shown in a table in FigureA potential factor which contributes to the disagreements seen in trees calculated from sequence data compared to those from the other data sources is also worth noting. Diverse superfamilies with very low sequence identity between member domains may provide an unreliable multiple sequence alignment thereby creating a result tree with limited accuracy. Anomalies introduced from this effect are more likely to be seen in very large superfamilies with a great deal of structural variation.The trees built from automatically generated structural distances largely agree, but are not always consistent with SCOP’s hand annotated groupings. The hand classification of structures in SCOP at the superfamily and fold levels is often referred to as the gold standard in the field, and clearly surpasses any fully automatic method. Since detectable structural similarity remains long after sequences have diverged beyond the point of recognition, the structurally-derived trees are able to resolve deeper edges of the tree with higher confidence than the sequence-based ones , but the granularity at which to divide the members of a superfamily is decided by function. I.e. domains are not grouped based on their function, but the number of groups relates to the number of functions.Sequence information contributes to the classification of domains into families, but alone is not enough. To classify a family evolutionarily: it must be consistent with sequence phylogenetics, will likely draw on structural distance, and will often coincide with a particular function. Sequence diversity between families (within a superfamily) is considerably greater than within a family. Sequence phylogenetics do not give a strong enough signal at the superfamily level to classify families, but where there is a signal it is consistent with the SCOP classification. Structural information is necessary for identifying evolutionary relationships of families in a superfamily where sequence identity is low. We see that although function does not determine the relationships, i.e. edges, it is used to guide the level at which the tree is cut to make a family, i.e. the choice of node from which to derive a clade (granularity).The families in SCOP represent a level at which sequence, structure, function plus other information on a shared peculiarity must all be taken into account. A balance of the strengths of signals available is used to establish the evolutionary relationships and resolve the groupings.http://supfam2.cs.bris.ac.uk/pethica/scopresults. The data may be ranked on each of the confidence scores separately or together. For every superfamily there are tree images for sequence, structure and function annotated with the PDB domain and SCOP family ID as shown in FigureThe data for all trees used to generate FigureDomain sequences for SCOP version 1.73, filtered to 95% sequence identity were obtained from ASTRAL. The comFor each superfamily in the classification the sequences of assigned domains were used to produce an alignment using MUSCLE. AlignmeA second set was also produced where domain sequences were padded with homologue sequences from the SUPERFAMILY database. These were aligned, and trees created as with the original set. A script was used to remove the homologues from the trees leaving only the original domain sequences, but preserving all phylogenetic relationships. The dataset calculated without homologues, with 300 replicates was chosen as very little difference was seen between the two replicate sets, and the addition of homologues sequences created larger alignments which were handled badly by the phylogenetic algorithms.PDB style protein three dimensional structures for the same filtered SCOP 1.73 set of domains were taken from ASTRAL. The same filtered set of SCOP 1.73 domains as for sequence was used. Structal was usedGene ontology (GO) data from EBI GOA was usedPhylogenetic trees of domains in each superfamily produced by each method could then be compared with the groupings at the SCOP family level. An algorithm was produced to traverse the trees and identify if a particular edge agreed, disagreed or was uninformative with regard to SCOP families:An edge of the tree is said to agree with SCOP if one side contains the full set of domains for a certain family and no members of another family.An edge disagrees with SCOP when domains from a certain family are found on both sides along with domains from a different family.A neutral or uninformative edge is where one side contains only members from a certain family, but not the complete set. i.e. more are found on the other side of the edge.An overview of the algorithm used is shown in FigureSequences for domains in SCOP 1.73 superfamilies were acquired from ASTRAL. Superfamilies containing a single domain only were removed. For each superfamily grouping, sequence identities were sequentially calculated with Washington University BLAST, the higFor each GO term in the EBI GOA dataset a list of single domain proteins with the particular annotation was generated. The sequence identity of the two most distant sequences in the set was determined. The distribution of domains across the SCOP classification and level in the hierarchy for a specific functional annotation was also calculated. e.g. All domains contained within a specific family or superfamily.The author(s) declare that they have no competing interests.Both JG and RP designed the study, ML advised at a later stage of the study. RP carried out most of the work, ML carried out modifications to STRUCTAL. RP and JG were involved in the preparation of the manuscript. All authors read and approved the final manuscript. ML is supported by NIH GM063817 and is the RW and VKC Professor of Cancer Research. JG is supported by BBSRC grant (BB/G022771/1).· Sub Family: GO terms are found in some, but not all members of a family. · Family Equivalent: Exactly fits a family, i.e. found in all members of a family, but not in other families. · Multi Family: Found in all members of more than one family, but not in all families in the superfamily. · Partial Family: Completes one or more families, is absent from one or more families and is incomplete from exactly one family of a specific superfamily. · Scattered Families: May or may not complete one family, more than one incomplete family, and at least one empty family per superfamily. · Scattered in Superfamily: Present in, but does not complete, all families in a superfamily. · Almost Superfamily: Present in all families, competes some of them. · Superfamily Equivalent: Present for every domain of just one superfamily. · Multi Superfamilies: Present in every domain in more than one superfamily. · Partial Superfamilies: Completes at least one superfamily; partially completes exactly one other superfamily. · Scattered Superfamilies: May or may not complete one superfamily; present but not completing at least one other superfamily.An Extended View of the Levels at which All Single Domain Proteins Associated With a Specific GO Term are Found in SCOP. Table shows an extended version of the level in SCOP at which all single domains associated with a particular GO term are found. The different levels shown are designed to illustrate the level at which GO terms fall, independently to the SCOP hierarchy. The distribution of GO terms assigned to domains is broken down into the following categories: Click here for fileStatistics for the number of domains used in the phylogenetic analysis. Statistics for domains in raw SCOP 1.73 and after filtering to 95% sequence identity and removal of trivially solvable cases.Click here for file