The Kelch-like (KLHL) gene family encodes a group of proteins that generally possess a BTB/POZ domain, a BACK domain, and five to six Kelch motifs. BTB domains facilitate protein binding and dimerization. The BACK domain has no known function yet is of functional importance since mutations in this domain are associated with disease. Kelch domains form a tertiary structure of β-propellers that have a role in extracellular functions, morphology, and binding to other proteins. Presently, 42 KLHL genes have been classified by the HUGO Gene Nomenclature Committee (HGNC), and they are found across multiple human chromosomes. The KLHL family is conserved throughout evolution. Phylogenetic analysis of KLHL family members suggests that it can be subdivided into three subgroups with KLHL11 as the oldest member and KLHL9 as the youngest. Several KLHL proteins bind to the E3 ligase cullin 3 and are known to be involved in ubiquitination. KLHL genes are responsible for several Mendelian diseases and have been associated with cancer. Further investigation of this family of proteins will likely provide valuable insights into basic biology and human disease. Drosophila melanogaster bric à brac 1, tramtrack, and broad-complex proteins (BTB) [Drosophila to humans and mice with more than 49 family members in the latter two [The KLHL (Kelch-like) gene family encodes proteins that constitute a subgroup at the intersection between the BTB/POZ domain and Kelch domain superfamilies. First identified in the vaccinia virus , the BTBns (BTB) . BTB domtter two . The POZtter two  as revietter two . Functiotter two , ion chatter two , transcrtter two ,11.Drosophila melanogaster to Homo sapiens[The Kelch repeat or domain is also an evolutionarily conserved structure that can be found from o sapiens. Based oo sapiens. The Kelo sapiens. Kelch-co sapiens. KLHL faIn addition to BTB/POZ and Kelch domains, the KLHL family members contain a BACK domain, first described as a 130-residue region of conservation observed amongst BTB-Kelch proteins -19.The BTB superfamily includes KLHL, KBTBD, and KLHDC subfamilies, which encompass structurally related molecules that differ in the types and numbers of their protein domains. In general, KLHL proteins contain one BTB/POZ domain, one BACK domain, and five to six Kelch motifs . KBTBD proteins have one BTB/POZ domain, occasionally a BACK domain, and two to four Kelch motifs , 2/4 Kelch). Most KLHDC family members contain only three to seven Kelch motifs and usually do not have BTB or BACK domains (3/7 Kelch). Herein, we describe the evolutionary structure of the human KLHL family, discuss examples of KLHL family member biology, and present an overview of KLHL protein relationship to human disease.There are nine KBTBD genes in the human genome, and their encoded proteins typically possess a BTB and BACK domain and two to four Kelch motifs. Ten KLHDC genes have been defined by the HUGO Gene Nomenclature Committee (HGNC) in the human genome. KLHDC proteins generally do not have BTB or BACK domains and have three to seven Kelch motifs. These subfamilies will not be discussed further in this review.KLHL proteins generally have a BTB/POZ domain, a BACK domain, and five to six Kelch domains. However, the domain composition for this family can appear to vary depending on the protein domain prediction program used to examine their protein sequences. The KLHL family protein structure is shown in Figure Homo sapiens and Mus musculus). ClustalO and phylogenetic analyses of KLHL family members suggest that they can be subdivided into multiple subgroups  interacting protein. NRF2 is a transcription factor that is essential for the cellular reaction to electrophiles and binds to the antioxidant response element (ARE) present in promoters of genes involved in phase II detoxifying and oxidative stress enzyme response [The response . KEAP1 wresponse . Some ofresponse ). NRF2 iresponse ).gstp) induction was tested in the absence and presence of KEAP1. NRF2-induced gstp expression was repressed when KEAP1 was also overexpressed [KEAP1's ability to repress NRF2's downstream target activation was demonstrated in zebrafish by gain-of-function and loss-of-function experiments in which transcriptional control by NRF2 of glutathione S-transferase gene  protein [KLHL2 RNA can be detected in developing and adult brain, heart, spleen, lung and liver, and adult kidney but most predominantly throughout the brain including the hippocampus, caudate nucleus, corpus callosum, and amygdala [KLHL2/Mayven is an actin-binding protein that shares 63% identity (77% similarity) with the  protein . The hum protein . KLHL2 Ramygdala . The KLHamygdala ,37. In damygdala , and it amygdala . KLHL2 oamygdala . KLHL2 camygdala . Upon deamygdala . The funNpcd gene in mouse which is a read-through between Cbx6 and Nptxr) and with cullin 3. In this case, KLHL2 appears to function as a specific adapter for NPCD ubquitination via cullin 3. Notably, overexpression of NPCD in hippocampal neurons leads to apoptosis [Recently, independent interactions with two different proteins have underscored KLHL2 involvement in ubiquitination. KLHL2 binds to nucleus accumbens-associated 1 (NACC1), a transcriptional repressor protein that interacts also with another KLHL family member, ectodermal-neural cortex 1 (ENC1/KLHL37). KLHL2 may form part of the ubiquitin complex through which NACC1 affects degradation of specific gene products and promotes proteasome activity and trafficking . Finallypoptosis .KLHL3 have been identified in patients with familial hyperkalemic hypertension. Molecular analyses have shown that most mutations increase the activity of the Na+Cl− symporter in the distal convoluted tubule portion of the nephron. The increased activity causes more Na+ and Cl- reabsorption, increasing blood pressure and culminating in hypertension. Although mutations were identified in this familial disease, screening for single nucleotide polymorphisms (SNPs) in KLHL3 did not reveal any significant association with blood pressure measurements in human subjects, indicating that common variants are not responsible for variations in blood pressure [KLHL members associated with inherited forms of human disease include KLHL3, KLHL7, KLHL9, KLHL12, and GAN (KLHL16). Mutations in pressure .KLHL7[KLHL7 patients differs because of its late onset and preserved rod function in older family members [Patients with autosomal dominant retinitis pigmentosa (adRP), a neurodegenerative disease that leads to loss of rod and cone photoreceptors, carry mutations in KLHL7. Early a members ,43. Rece members . KLHL7 a members . No sign members .KLHL9 leads to the development of distal myopathy in human patients. Distal myopathy is a degenerative disease that initially causes atrophy of distal limb muscles and subsequently extends to proximal limbs [A missense mutation in al limbs . Mutatioal limbs . Giant aal limbs . Compounal limbs .KLHL6 are recurrent in cases of chronic lymphocytic leukemia (CLL), a type of leukemia that is predominant in adults. Six mutations were identified within KLHL6 in CLL patients, and all disrupt KLHL6 function in germinal center B cell formation [KEAP1[Four KLHL family members are associated with cancer: KLHL6, KEAP1 (KLHL19), KLHL20, and ENC1 (KLHL37). Mutations in ormation ,49. Inseon [KEAP1,35 have on [KEAP1,50. KLHLon [KEAP1. Mutatioon [KEAP1. KLHL37 on [KEAP1.There are 42 KLHL family members encoded in the human genome, containing conserved BTB, BACK, and Kelch domains. KLHL proteins are known to be involved in the ubiquitination process, but the specific roles for each family member have not yet been elucidated. KLHL proteins will likely have multiple substrates. KEAP1 (KLHL19), for example, has at least three  as reviewed in Tian et al. . SimilarThe authors declare that they have no competing interests.BSD, TC, AGS, EAB, and JSF. drafted the manuscript. BSD, EAB, and JSF carried out sequence alignments and/or domain structure analyses. All authors read and approved the final manuscript.http://www.ncbi.nlm.nih.gov), HUGO Gene Nomenclature Committee , and Online Mendelian Inheritance in Man  databases.Human KLHL member summary as recorded in the National Center for Biotechnology Information (NCBI, Click here for file


Besides the well-accepted head-to-tail 90° uncharged domain-walls, we have identified not only head-to-head positively charged but also tail-to-tail negatively charged domain-walls. The widths, polarization distributions, and strains across these charged domain-walls are mapped quantitatively at atomic scale, where remarkable difference between these domain-walls is presented. This study is expected to provide fundamental information for understanding numerous novel domain-wall phenomena in ferroelectrics.The atomic-scale structural and electric parameters of the 90° domain-walls in tetragonal ferroelectrics are of technological importance for exploring the ferroelectric switching behaviors and various domain-wall-related novel functions. We have grown epitaxial PbTiO Ferroelectrics possess controllable polar states and electromechanical couplings, they were found extensive applications as high-density memories, thin-film capacitors and actuators as well as sensors1234578910113 TiO3 and Pb(Zr0.52Ti0.48)O3  and weak beam transmission electron microscopy1920212223151617181920212223326Tetragonal ferroelectrics generally exhibit two types of domain-walls: 90° and 180° domain-walls, which have dipoles across the domain-walls arranged as 90° (nearly) and 180° configurations, respectively789101112138101178910113 ref. , then 4–O3 refs. ,1718. Th3/SrTiO3 multilayer films and mapped atomic details and polarization distributions across the 90° domain-walls in an aberration-corrected STEM at high angle annular dark field (HAADF) mode. We have directly observed not only positively charged but also negatively charged 90° domain-walls at atomic scale.The atomic and electronic behaviors of ferroelectrics have become readily accessible through aberration-corrected scanning transmission electron microscopy (STEM)283014323335363/SrTiO3 multilayer films were prepared by pulsed laser deposition (PLD). The films were deposited on GdScO3 (GSO) substrate, which exert tensile strain on the epitaxial PbTiO3/SrTiO3 superlattices3940PbTiO3 (PTO) layer near the PbTiO3/SrTiO3 (PTO/STO) interface. The four insets are magnification of images overlying the respective areas. Yellow cycles denote Pb2+ columns and red cycles denote Ti4+ columns. O2− columns are invisible due to its weak scattering effects of electrons. As illustrated by the schematic diagrams in 4+ and O2− columns are shifted upward  towards the upper Pb2+ positions and away from the respective lower ones, but the O2− columns are shifted more strongly. As a consequence, a charge dipole forms in the PTO unit-cell because of the separation of negative (O2−) and positive (Ti4+ and Pb2+) charges, as seen by means of coherent high-resolution imaging with negative Cs techniquePs  measured in the atomic resolution HAADF image. A careful inspection reveals that in a = 0.389 nm, c = 0.414 nm, ref. a and c domains , thus domain A is shorter than domain B and C on both sides. This results in a complex strain state near the coherent PTO/STO interface which makes a bending of the STO lattice. The changing of one domain to another gives rise to the switching of polarization vector by 90°. Domain C has the same Ps direction as domain B. The position of these 90° domain-walls (DWs), indicated by the blue dotted lines, can be determined directly by mapping the δTi vectors of each PTO unit-cells. It is of interest to notice that the left 90° domain-wall terminates within the PTO matrix, this scenario is suggestive of some unusual dipole behaviors at the bottom left corner of Ps directions in domain A and B have to encounter each other. Thus, a 90° charged-DW (CDW) is identified, with a ‘head-to-head' arrangement of Ps vectors. Such a 90° CDW is actually a broad area  and thus is marked by two white dotted lines , which is generally thought to be the location of 90° DWs in tetragonal ferroelectrics2223Ps vectors are determined by corresponding δTi because the relationship between the δTi and the Ps is well-knowna = 0.5488 nm, ref. A careful observation indicates that, on both structural and electric level, the 90° PCDW is rather wider than the 90° UCDW. According the famous Kittel's law, the DW width is a crucial factor for determining the DW patterns and thus the properties such as nonlinear electro-opticsTi and Ps in area I, across the left white dotted line). Amazingly, the changing of both lattice and polarization is much slowly across the 90° PCDW. From domain B to area I, the out-of-plane lattice (cB) continuously changes from 0.42 nm to 0.395 nm without a sharp jump, while the in-plane lattice (aB) almost kept constant of about 0.39 nm  to area I (0)  and light red (lower segment near the PTO/STO interface) dotted lines. The 90° NCDW separates domain A and B in Ps directions pointing to left and top, respectively  across the 90° UCDWs makes obvious contrast in To directly visualize the 2D structural parameters and  ‘U.C.', . The uniPs angles of each unit-cell near the 90° PCDW and 90° NCDW are also mapped , which possess positive charges screening the negative bound charges induced by the 90° NCDW. This inference is constant with our observation because the 90° NCDW is less disturbed compared with the 90° PCDW, and the later probably deserved a depletion layer of Vo2+, which was repulsed by the positive bound charges2+ and electron holes, or negative, such as electrons), only one kind of CDW can be effectively screened423  observation where the DW width was absent from atomic-scale information limited by the AFM tip radius. In tetragonal PbTiO3 films of the present study, the difference between the 90° PCDW (carrier depletion) and NCDW (carrier accumulation) at atomic scale may qualitatively explain the different conduction behaviors between ‘head-to-head' and ‘tail-to-tail' 180° DWs in HoMnO3 (ref. Ps and elastic strain for the 90° DWs is much stronger than the 180° DWs8101148The charge carrier accumulation was hypothesized to be a cause for the increased conductivity at the CDWs in ferroelectrics3nO3 ref. . In addi0.2Ti0.8)O3 thin films formed by in-situ electric responseIn summary, by using aberration-corrected STEM, the unusual frustration of dipole arrangements and strain behaviors of 90° CDWs in PTO/STO multilayer films are identified on the atomic-scale, where the widths, polarization distributions, and strains across these charged domain walls are mapped quantitatively. “Glass-like” dipole behaviors are observed at the 90° CDWs. We anticipate the present atomic-scale investigations of the uncharged and charged 90° DWs may help to interpret the switching behaviors, the newly realized domain-wall functions, and the retention failure mechanism in ferroelectrics. Moreover, the present study is expected to clarify the long-standing argument about the width of the 90° DWs in tetragonal ferroelectrics. During the review stage of this paper, a research group in Michigan University reported an occurrence of 90° charged domain walls in Pb(Zr3/SrTiO3 thin films were deposited on GdScO3 substrates by pulsed laser deposition (PLD), using a Lambda Physik LPX 305i KrF (λ = 248 nm) excimer laser. The PbTiO3 targets were 3 mol% Pb-enriched sintered ceramics. The target-substrate distance was 40 mm. The background pressure was 10−5 Pa. During the growth of PbTiO3, the substrate temperature was kept at 650°C, with a laser energy density of 2 Jcm−2, a laser repetition rate of 5 Hz and under an oxygen pressure of 20 Pa. For the growth of SrTiO3 layers, the substrate temperature was also 650°C, with a laser energy density of 1 Jcm−2, a laser repetition rate of 2 Hz and under an oxygen pressure of 8 Pa. Before deposition, the GdScO3 substrate was pre-heated at 750°C for 5 min to clean the substrate surface and then cooled down to the growth temperature (10°C/min). The laser was focused on the ceramic target for 30 min pre-sputtering to clean the target surface. After deposition, the film was in-situ-annealed at 650°C in an oxygen pressure of 5 × 104 Pa for 10 min, and then cooled down to room temperature at a cooling rate of about 5°C/min. The samples for the STEM experiments were prepared by slicing, gluing, grinding, dimpling, and finally ion milling. A Gatan PIPS was used for the final ion milling.The PbTiO4+ shifts (δTi) were deduced. The atom positions were determined accurately by fitting them as 2D Gaussian peaks by using Matlab143334Ti were calculated as a vector between each Ti4+ and the center of mass of its four nearest A-site neighbor Pb2+. The Ps vectors were deduced by the δTi. The visualization of the 2D Ps vectors  fitted with a high-brightness field-emission gun (X-FEG) and double Cs correctors from CEOS, and a monochromator operating at 300 kV). The convergence angle of the electron beam is 25 mrad, yields a probe size of less than 0.10 nm. The determination of the atom coordinates in the HAADF-STEM images were carried out by fast Fourier transform (FFT) filtering the images using only a low-pass annular mask restricted slightly more than the resolution limit of the image, thus the lattice spacing and Ti vectors  was carrs angles  was carrThe project of interfacial STEM characterization in oxides was conceived by Y.L.Z. and X.L.M.; thin film growth, TEM specimen preparation and STEM observations were performed by Y.L.T.; Y.J.W. participated digital analysis of the HAADF-STEM images; W.Y.W., Y.B.X., W.J.R. and Z.D.Z. have contributions in thin film growth; Y.L.T., Y.L.Z. and X.L.M. jointly interpreted the data and wrote the paper.


The coal permeability is an important parameter in mine methane control and coal bed methane (CBM) exploitation, which determines the practicability of methane extraction. Permeability prediction in deep coal seam plays a significant role in evaluating the practicability of CBM exploitation. The coal permeability depends on the coal fractures controlled by strata stress, gas pressure, and strata temperature which change with depth. The effect of the strata stress, gas pressure, and strata temperature on the coal  under triaxial stress and strain conditions was studied. Then we got the change of coal porosity with strata stress, gas pressure, and strata temperature and established a coal permeability model under tri-axial stress and strain conditions. The permeability of the No. 3 coal seam of the Southern Qinshui Basin in China was predicted, which is consistent with that tested in the field. The effect of the sorption swelling on porosity (permeability) firstly increases rapidly and then slowly with the increase of depth. However, the effect of thermal expansion and effective stress compression on porosity (permeability) increases linearly with the increase of depth. The most effective way to improve the permeability in exploiting CBM or extracting methane is to reduce the effective stress. Coal bed methane (CBM) is a natural product in the coalification process , 2. CBM Coal permeability is an important parameter in mine methane control and CBM exploitation because it determines the practicability of methane extraction. The permeability determined in the laboratory is not the real value of coal seam tested in the field. The coal seam permeability is completed based on the well logs in the CBM exploitation and is calculated based on the gas flow theory of the hole in or through the coal seam during coal mining , 6. In tThe permeability of coal depends on the fracture characteristics, including the size, spacing, connectivity, width, mineral fill, and distribution , which aSeveral models have been proposed to explain the variability of coal permeability –19. The However, uniaxial strain conditions are a simplified homogenisation of the stress-strain states of coal during mining and exploitation and may be valid at the scale of a relatively large basin; the mechanical conditions at the local scale are expected to be much more complex in coal seams . And theα is assumed to be 1 [The effects of effective stress and matrix sorption deformation are considered by most coal permeability models , 11–19 e to be 1 , 16, 17. to be 1 , 21. The to be 1 , 23.Most models consider the matrix deformation to be equal to the fracture deformation. However, only part of the matrix deformation contributes to the fracture deformation . Liu andThe strata stress, gas pressure, and temperature of the coal seam change with the depth –29. As aThe coal has a natural dual porosity structure that consists of the coal matrix and the fracture in which there are numerous inorganic minerals, mainly kaolinite, pyrite, and illite, as shown in Coal is considered to be a dual continuous isotropic elastic medium even though the coal consists of coal matrix and fracture.The strain is elastic and infinitesimal, so the second and higher order terms can be ignored. Therefore, the strains induced by different factors can be added.The effect of methane on the coal is divided into normal effective stress effect of the gas like nonadsorptive gas and the effect of sorption deformation, which are added directly.Before analysis, we make the following assumptions.εe and Ve are the coal bulk strain and volume caused by the effective stress, respectively; εep and Vep are the pore strain and volume caused by the effective stress, respectively; K = E/3(1 − 2v) is the coal bulk modulus, MPa; Kp is the coal pore modulus, MPa; α = 1 − K/Km is Biot's coefficient; β = 1 − Kp/Km is the effective coefficient for the pore system; Km is the coal matrix modulus, MPa; E is the elastic modulus of the coal, MPa; and v is Poisson's ratio.According to the effective stress principle , the bulϕ is the porosity.Without the gas sorption effect, the volumetric change of the porous medium satisfies the Betti-Maxwell reciprocal theorem The coal matrix swells due to adsorb gas and temperature increase underground.(1) Thermal Deformation. The deformation of the coal matrix due to temperature change is expressed asεmt is the thermal deformation of the coal matrix; η is the coefficient of the thermal deformation, K−1; T is the temperature of the strata, K.(2) Sorption Deformation. The gas sorption capacity of coal increases with pressure and closely follows Langmuir type isotherm. Langmuir isotherm describes the amount of adsorbed gas on the coal as a function of pressure and has the following form [Vad is the adsorbed gas volume, m3/t; a is the Langmuir volume, m3/t; b is the adsorption coefficient, MPa−1.ing form :(5)Vad=H is the heat of sorption, J/mol; R is the gas universal constant, J/mol·K.Adsorption coefficient indicates coal's ability to adsorb gases and is exponentially increased with the energy of adsorption and decreased with the temperature of adsorption. Adsorption coefficient in a Langmuir-type isotherm can be estimated using the Arrhenius rate equation at equilibrium condition, in which affinity is a function of temperature and is given by the following equation :(6)b=b0This is to say that the adsorption capacity is independent of temperature, and as a result the heat of adsorption is a constant, independent of loading. Increase in the temperature will decrease the adsorbed amount at a given pressure. This is due to the greater energy acquired by the adsorbed molecule to evaporate.εs is the sorption deformation of the coal matrix; χ is the coefficient of sorption deformation, t/m3.It has been tested and verified that the coal swells it when adsorbing gas and the adsorption deformation is proportional to the adsorbed gas volume , 24, 34:εexp is the deformation measured in the experiment.However, the coal matrix is also compressed by the sorptive gas in addition to swelling due to adsorption , 21, 35.Therefore, the coal matrix deformation is the sum of adsorption deformation and thermal deformation:(3) Fracture Deformation Caused by the Coal Matrix. Deformation of the coal matrix can affect the deformation of both the bulk coal and the fractures in the coal [the coal , 14, 36.the coal , 11, 15.the coal , 17, 24.the coal  significThe coal is divided into the matrixes by the fracture, but there are bridges between the matrixes, as shown in fm, is introduced to measure the degree of influence of the coal matrix deformation on fracture deformation. The factor fm is a parameter of the coal structure and depends on the distribution of fractures, the filling characteristics of the fracture, and other factors. For a particular coal, fm is a constant between 0 and 1. If there is no fracture in the coal, the parameter fm is equal to 0. The parameter fm would be equal to 1 when two surfaces of the fracture are smooth and parallel.The effective coal matrix deformation factor, Vmf is the fracture volume deformation due to deformation of coal matrix; Vm is the volume of coal matrix.Thus, the fracture deformation due to the coal matrix deformation is expressed asV is composed of the matrix volume Vm and the pore volume Vp:As a porous medium, the coal bulk volume ϕ = Vp/V, we obtainBased on the definition of porosity, Vmv is the bulk volume deformation due to the matrix deformation.The bulk volume deformation of coal is equal to the sum of the deformation due to the effective stress and the coal matrix deformation due to adsorption and temperature change:Dividing both sides of  by the cSimilarly, from  and 10)10), we oBy substituting  and 15)15) into Kp = (ϕ/α)K and β = 1 − Kp/Km into (ϕ ≪ 1 (ϕ < 10%), we can rearrange and simplify the equation to obtainThen, substituting Km into  and consIntegrating  gives model , the Shifm, which ranges from 0 to 1, is introduced to measure the degree of influence of the coal matrix deformation on the fracture deformation in the ESMD model. The factor fm is a parameter of the coal structure and does not vary with the type of gas. The factor fm of the Sulcis coal sample is 0.1723 for both N2 and CO2. Biot's coefficient α of coal is less than 1, and α = 0.925 for the Sulcis coal sample, which has a bulk modulus of 778 MPa and a matrix modulus of 10,340 MPa.Only part of the matrix deformation contributes to the fracture deformation. The factor 2 but poorly for CO2. The decline rate of fracture compressibility with increasing effective stress θ for the R-C model is 2.65 × 10−14 MPa−1, which implies that the fracture compressibility does not vary with the effective stress. However, the decline rate θ varies between 2.45 × 10−2 MPa−1 and 2.61 × 10−1 MPa−1 [The three models poorly match the experimental data for two reasons: in all three models, Biot's coefficient is assumed to be 1 in the P-M model and the S-D model by assuming that the coal matrix is incompressible. Deformation of the coal matrix contributes to the fracture deformation entirely in the three models, which is an overestimation. As shown in −1 MPa−1 , 41.The SQB has become the China's first commercial CBM reservoir. CBM reservoir is an unconventional gas reservoir and located at shallow depths, compared to the conventional gas reservoir. The SQB refers to a region, including Changzhi, Gaoping, Jincheng, Yangcheng, Qinshui, and Anze in the southeast of Shanxi Province. It is the most important production base for high quality anthracite in China. Coal seams, generated in Carboniferous and Permian periods, contain abundant methane. Permeability in the coalbed reservoir is relatively high compared to other CBM reservoirs in China. The exploration and production tests in this field have been conducted since 1990s. The results show that the Qinshui Basin is a very promising coalbed methane reservoir with the most exploration wells, the best development prospect, and a higher commercialized production in China's CBM reservoirs. The No. 3 coal seam is the major coal seam mined and CBM reservoir in the SQB. The thickness of No. 3 coal seam in the SQB changes from 0.5 m to 7.0 m with an average value of 5.5 m. The maximum depth is about 2000 m. The coal is anthracite mainly. In this paper, we predicted the permeability of the No. 3 coal seam.There are two different directions of gas migration in the coal seam with an outcrop. For example, the generated methane migrates upward and the surface air migrates downward in the coal seam. Thus, there are two vertical zones of the CBM occurrence in the coal seam, which are the gas weathered zone and the methane zone . GeneralH is the depth of coal seam, m.The gas pressure of the No. 3 coal seam in the SQB is present in The gas pressure calculated by  at the dσV is the vertical stress, MPa; σH is the maximum horizontal stress, MPa; σh is the minimum horizontal stress, MPa.It has been practically verified that the strata stress increases linearly with depth , 43. MenThe temperature of the strata also increases linearly with the depth. Sun et al.  investig−6 K−1, which was used in the prediction.The parameters of the No. 3 coal seam in the SQB have been measured by many researchers , 44–47. The prediction of the permeability of the No. 3 coal seam in the SQB was conducted by using  and 20)20) to 2 based on(1) The statistics of the permeability of the No. 3 coal seam tested in the field was investigated by Meng et al.  and was (2) The average strata stress, the gas pressure, and the strata temperature of the No. 3 coal seam in the SQB increase linearly with the depth. But the gradient of the average strata stress is larger than that of the gas pressure . So the (3) There is a cubic relationship between the coal permeability and the coal porosity. The different effects of the effective stress, the adsorption deformation, and the thermal deformation of the coal seam on the permeability could be represented by different effects of them on the porosity which are calculated easily and simply. The different effects of the effective stress, the adsorption deformation, and the thermal deformation were calculated using , shown iThe effect of the adsorption swelling on the porosity increases rapidly at first slowly with the increase of depth. The effect of the thermal expansion and the effective stress compression increases linearly with the depth increase. The effect of the effective stress compression is larger than the others.As a result, the most effective way to improve the permeability in deep and low permeability coal bed for exploiting CBM or extracting methane is to reduce the effective stress. For example, the method of protective layer mining, which is widely used in China, is very effective and it reduces the stress of the overlying and underlying strata .(4) The temperature affects the sorption capacity of the coal. The sorption capacity decreases with the increase of the temperature. The adsorption volume increases with the increase of the depth to a maximum value at the depth of 1600 m, and then it decreases. However, if the effect of the temperature on the sorption capacity is neglected, the adsorption volume increases always with the increase of the depth. And the adsorption volume is overestimated . As a reIn the ESMD model proposed in this paper, only part of the matrix deformation contributes to the fracture deformation. If the matrix deformation contributes to the fracture deformation, the effect of the matrix deformation due to adsorption swelling and thermal expansion is overestimated significantly .The Biot's coefficient is less than 1 in the ESMD model, which is true for the coal. But in mangy coal permeability models, it is equal to 1 based on the assumption that the bulk modulus of the coal matrix is much larger than that of the coal, which overestimates the effect of the effective stress .The coal permeability is an important parameter in mine methane control and CBM exploitation, which determines the practicability of methane extraction. Permeability prediction in deep coal seam plays a significant role in evaluating the practicability of CBM exploitation.The coal permeability depends on the coal fractures controlled by strata stress, gas pressure, and strata temperature which change with depth. The effect of the strata stress, gas pressure, and strata temperature on the coal  under tri-axial stress and strain conditions was studied. Then we got the change of coal porosity with strata stress, gas pressure, and strata temperature and established a coal permeability model (ESMD model) under tri-axial stress and strain conditions. The coal permeability is controlled by the effective stress, sorption deformation, and thermal expansion. The sorption capacity decreases with the increase of the temperature and thus the sorption deformation is affected by temperature. And only part of the matrix deformation contributes to the fracture deformation. The Biot's coefficient is less than 1 in the ESMD model, which is true for the coal.The permeability of the No. 3 coal seam of the SQB in China was predicted, which is consistent with that tested in the field.The effect of the sorption deformation on porosity (permeability) firstly increases rapidly and then slowly with the increase of depth. However, the effect of thermal expansion and effective stress compression on porosity (permeability) increases linearly with the increase of depth. When exploiting CBM or extracting methane, the most effective way to improve the permeability in CBM exploitation or mine methane control is to reduce the effective stress. For example, the method of protective layer mining, which is widely used in China, is very effective, which reduces the stress of the overlying and underlying strata.


In the traditional 2° structure, the center of the 23S rRNA is an extended single strand, which in 3D is seen to be compact and double helical. Accurately assigning nucleotides to helices compels a revision of the 23S rRNA 2° structure. Unlike the traditional 2° structure, the revised 2° structure of the 23S rRNA shows architectural similarity with the 16S rRNA. The revised 2° structure also reveals a clear relationship with the 3D structure and is generalizable to rRNAs of other species from all three domains of life. The 2° structure revision required us to reconsider the domain architecture. We partitioned the 23S rRNA into domains through analysis of molecular interactions, calculations of 2D folding propensities and compactness. The best domain model for the 23S rRNA contains seven domains, not six as previously ascribed. Domain 0 forms the core of the 23S rRNA, to which the other six domains are rooted. Editable 2° structures mapped with various data are provided .We present a  The ribosome, a macromolecular assembly of ribosomal RNAs (rRNAs) and ribosomal proteins (rProteins), synthesizes coded proteins in every cell of every organism. The ribosome comprises of large and small subunits that catalyze peptide bond formation (LSU) and decode mRNA (SSU). A key advance in understanding the ribosome was the determination of rRNA secondary structures (2° structures) by Brimacombe , BranlanRNA 2° structures, with symbolic representations of base pairs, double-helices, loops, bulges and single-strands, provide frameworks for understanding structure, folding and function and for organizing a wide variety of information. RNA 2° structures reveal how local 2° elements are organized into quasi-independent domains, which can be used to infer mechanisms of global assembly and evolution. The depiction of the 23S rRNA 2° structure in A second key advance in understanding the ribosome was the determination of high-resolution 3D structures  from allHowever, critical differences distinguish the 2° structure of the 23S rRNA from 3D structures. The most significant discrepancy is in the heart of the 23S rRNA, which is represented by extended single-strands in the traditional 2° structure a. By conde novo re-determination of the 2° structures of the 23S and 5S RNAs, using high resolution 3D structures as the input data. The goal is to establish 2° structures that are fully consistent with 3D structures and are as accurate and useful as possible. We seek to obtain 2° representations that allow facile conceptualization of vast available structural, functional and phylogenetic data. Previous revisions of the 23S rRNA 2° structure  forms the essential core of the 23S rRNA, to which the other six domains of the 23S rRNA are rooted. The 5S rRNA is positioned and oriented as an adjunct to Domain 2, based on its location and interactions in the 3D structure. The 2° structure3D lacks the central single-stranded region of 2° structurephylo.We refer to our revision as 2° structurehttp://apollo.chemistry.gatech.edu/RibosomeGallery so that others can evaluate and hopefully exploit and extend the revisions. We provide high-resolution editable versions of both 2° structure3D and 2° structurephylo mapped with a variety of data related to molecular interactions and geometry, phylogeny and evolution and partitioning of rRNA into helices and domains. These representations are available for Escherichia coli, Thermus thermophilus, Haloarcula marismortui and Saccharomyces cerevisiae. The work presented in this article is based on the bacterial 23S rRNA of E. coli, although the findings are applicable to the 23S and 28S rRNAs of other species.Representations of both LSU and SSU rRNAs are available online at phylo  and finalized with Adobe Illustrator. Atomic coordinates of the 23S rRNA of E. coli were obtained from the Nucleic Acid Databank (PDB ID: 3R8S) (http://apollo.chemistry.gatech.edu/RiboVision. Images of 3D structures were generated with PyMOL (The 2° structurephylo a was obtD: 3R8S) . Base-paD: 3R8S)  and confth PyMOL .phylo, we left unaltered the incorrectly assigned portions of rRNA in 2° structure3D if they did not impact the domain model. For example, new helix 49b remains represented by a loop in structure3D, as it is in structurephylo. We incorporated new Helices 25a, 26a, 49a and 49b.To build the best 2° structural model of the 23S rRNA, we defined helices by specific geometric and interaction criteria. Helices contain paired bases. Optimum helical definitions maximize intra-helical base stacking and minimize inter-helical base stacking. For base pairing, we used the geometric criteria of Leontis and Westhof . We assuSupplementary Table S1.Sequence conservation across all phylogeny was used to evaluate the generality of various helix and domain models. We aligned 23S rRNA/28S sequences from 122 organisms  intendedi  in each position along the sequence. The probability (ip) of a nucleotide type at a given position was approximated by its fractional occupancy at that position in the aligned sequences. The Shannon Entropy (H) at each position was calculated from the probabilities ip according to A multiple sequence alignment of the 23S/28S rRNAs was used to calculate the fraction of nucleotide type The Shannon Entropy ranges from 0 to 2. The minimum value indicates that the nucleotide type at that position is universally conserved. The maximum value corresponds to equivalent populations of all four nucleotide types.2+–RNA interactions. First-shell Mg2+–RNA interactions are defined by Mg2+–RNA distance of <2.6 Å . Domain boundaries were adjusted to minimize interactions between domains and to maximize interactions within domains. Each nucleotide belongs uniquely to one domain.in silico, the RNA fragments of Domain 0 were linked at the strand termini. Because Mfold cannot predict folds with non-canonical base pairs, Helix 26a was replaced with a canonical helix composed of Watson–Crick base pairs. The sequence of the substituted helix in the Mfold calculation is (5′GUAUAUGC3′:5′GCAUAUAC3′). The substitution is not expected to affect the energetics of the predicted folds, as the loop E motif and the substitute duplex have similar folding energies  and 2° structure3D with seven domains (a 7×7 matrix). The diagonal elements of these matrices  describe the number of the intra-domain interactions for Domain i, whereas the off diagonal elements  contain the total number of interactions between Domain i and Domain j. To account for the difference in the numbers of nucleotides in various domains, the elements were scaled: the diagonal elements  were normalized to the number of nucleotides iN in Domain i, and the off-diagonal elements  were divided by an average number of nucleotides (iN+jN)/2 in Domains i and j.We quantified molecular interactions of all of the rRNA, both helical and non-helical. RNA–RNA interaction frequencies within domains and between domains were estimated as a sum of base–base, phosphate–RNA and RNA–Mganalysis . Local sphylo as proposed in 1981 . Helix 26a forms a total of one base pair, three base-phosphate or sugar-phosphate and one RNA–Mg2+–RNA interactions with Helices 26, 61 and 73. Helices 61 and 73 are further stabilized by interactions with each other. Helix 25a intercalates between Helices 72 and 73 and also forms an A-minor interaction with Helix 26 . Helix 25a, which was not identified in the Fox and Gutell model, is included in Domain 0. Helix 1 is wholly contained within Domain I.Fox and Gutell’s Helices 47 and 48 are not included in Domain 0 because they are separate from it in three dimensions and are integrated by molecular interactions into Domain III. Thus, Helices 47 and 48 are allocated to Domain III in 2° structureSupplementary Figure S4). Modest adjustments to previous domains are implemented, but as far as possible, differences between 2° structure3D and 2° structurephylo are minimized.The 5S rRNA is placed in proximity to Helix 39 to reflect their locations in 3D space and molecular interactions between the two rRNAs . In all, 43 of 49 base pairs in the secondary structure of Domain 0  are correctly predicted by Mfold . The AU and GU base pairs at one end of Helices 72 and 73 are wrongly predicted. One of these base pairs involves an A that can interact with two possible U’s. Three closing base pairs of the hairpin-loop of Helix 61 are also wrongly predicted. However, this discrepancy results from a non-canonical base pair at this location. In general, the 2° structure of Domain 0 is well-predicted by Mfold, supporting the view that it is a distinct and structurally independent domain.Results from the program Mfold  suggest 3D (0.235) is slightly greater than that of Domains I-VI of 2° structurephylo (0.223). Sphericities of each domain are presented in Supplementary Table S5 (Domains I-VI of 2° structurephylo) and Supplementary Table S6 (Domains 0-VI of 2° structure3D). On average, the domains of 2° structure3D are slightly more compact than those of 2° structurephylo.To evaluate domain models of the 23S rRNA, we estimated the compactness of each domain by computing its sphericity. A sphericity of one is exactly spherical and most compact . The aveAn optimized 2° structure would tend to give the greatest number of 2° interactions and the fewest tertiary interactions. An optimized domain model would tend to give the greatest number of intra-domain interactions and the fewest inter-domain interactions.3D and Structurephylo. Twenty-one tertiary interactions in 2° structurephylo are converted to secondary interactions in 2° structure3D. This difference can be seen in the projections of all base-pairing interactions onto 2° structure3D (phylo (phylo and secondary interactions in 2° structure3D. Eight base-pairing interactions in Helix 1 (residues 1–8 are paired with 2895–2902) and 5 base-pairing interactions in Helix 2 (residues 26–30 are paired with 510–514) are represented as tertiary interactions in 2° structurephylo, but as secondary interactions in 2° structurephylo.The allocation of RNA–RNA interactions between secondary and tertiary interactions differs between 2° structureucture3D  and 2° sD (phylo . The sho2+–RNA interactions) between and within domains for both 2° structure3D and 2° structurephylo. Inter-domain interaction frequencies were scaled by the mean of the number of nucleotides in the two domains. The numerical values of both unscaled (Supplementary Tables S7 and S9) and scaled (Supplementary Tables S8 and S10) interactions for the 2° structurephylo and 2° structure3D, as well as the detailed list of interactions between Domain 0 and Domains I–VI (Supplementary Table S11) are given in the Supplementary Data.We tabulated RNA–RNA interactions  and 2° structure3D (Supplementary Table S10). For both 2° structure3D and 2° structurephylo, the number of scaled intra-domain interactions  is always greater than the number of inter-domain interactions for a given domain . Therefore, the interaction frequencies for the domains including Domain 0 are consistent with independent autonomous structural units. Comparison of the sum of diagonal elements in Structurephylo (Supplementary Table S7) and 2° structure3D (Supplementary Table S9) and 2° reveals that the total number of the unscaled intra-domain RNA–RNA interactions is slightly higher in 2° structure3D (289) than 2° structurephylo (287). This difference suggests the seven structural domains of 2° structure3D are somewhat more independent than six 2° domains of 2° structurephylo, although the difference is small. It can be concluded that the 2° structure3D is certainly not inferior to 2° structurephylo by these criteria.The scaled interaction frequencies are presented for 2° structure3D and 2° structurephylo. The combined results demonstrate a greater accuracy and utility of 2° structure3D over 2° structurephylo.Our goal is to obtain 2° representations that allow facile conceptualization and organization of molecular interactions, 3D architecture, phylogeny and function. We have investigated the utility of 2° structure3D. A region of contiguous base pairs near the center of the rRNA is represented as Helix 26a rather than as the extended single strands in the traditional 2° structurephylo. Helix 26a appears to be critical to ribosomal structure and function, as it is conserved in all three major domains of the tree of life. The importance of Helix 26a is shown by conservation of base pairing in rRNA sequences that represent the most complete available sampling of the phylogenetic tree (Supplementary Table S1) (We have performed an accurate and self-consistent partitioning of the 23S rRNA into helices. rRNA that is double stranded in the 3D structure is double stranded in 2° structureable S1) , and by phylo) regards Helix 26a as two single strands that are carved between multiple domains, treating this helix anomalously from other helices. In the revised domain architecture (2° structure3D), Helix 26a, like other helices, is integrated into a single domain.Consistent treatment of helices as integral and indivisible elements of domains compels a revision of the global domain architecture. The revised domain model includes seven domains, rather than the traditional six. A central domain (Domain 0) forms the core of the 23S rRNA, to which the other six domains are rooted. The previous domain architecture (2° structureSupplementary Tables S5 and S6). Domain 0 is among the most highly networked domain of either model , with a greater scaled frequency of intradomain molecular interactions.Using a consistent structure-based definition of helices, along with (i) calculations of 2° structure-folding propensities, (ii) calculations of sphericity and compactness and (iii) networking analysis of molecular interactions, we have formulated an improved domain model for the 23S rRNA, containing a central core domain (Domain 0). Folding algorithms suggest that Domain 0 is an autonomous folding unit. Domain 0 is compact, showing the greatest sphericity of any domain in either model  from the site of peptidyl transfer is projected onto 2° structurephylo a, the rephylo b, are diucture3D c, the nuucture3D . Similaructure3D  are inco3D suggests that the differences between the LSU and SSU are more modest than previously assumed. In 2° structure3D, the 23S rRNA, like the 16S rRNA, contains a central region to which other domains are rooted, i.e. both rRNAs have a central structural core. In 2° structure3D of the 23S rRNA, like in the 2° structure of 16S rRNA, the structural center is distinct from the functional center (PTC in the 23S rRNA and the decoding center in 16S rRNA) but is in close proximity to it. These observations confirm and extend a previous hypothesis (As noted by Thirumalai, the domain structure of the LSU appears to differ significantly from that of the SSU . The LSUphylo are delimited by a closing helix (3D and not restrain the RNA of a domain to be a continuous strand.Ideally, an RNA domain is an independently stable globular structure composed of a continuous RNA strand, with domain boundaries defined by helicies. In the 2° structure of the 23S rRNA proposed in 1981 by Noller and Gutell , the remng helix a. An addng helix ,48 form E. coli (Domain 0 forms the entry and early portions of the exit tunnel, interacting with Domain V. Domain 0 forms a cradle a for A-sE. coli c and d. Unlike the domains of 16S rRNA, which are predominantly isolated in space and ‘structurally autonomous’ , the domde novo re-determination of the 2° structure of the 23S RNA using 3D structures as the primary data. We propose a revised 2° structure referred to as 2° structure3D. The 2° structure3D lacks an extended central-stranded region in the heart of the 23S rRNA and contains seven 2° domains in contrast to six in the traditional 2° structurephylo. The 2° structure3D contains a central core domain, which we call Domain 0, from which all other domains branch. Domain 0 is highly conserved over phylogeny and provides a structural integrity of 23S rRNA.We have performed a 3D of 23S rRNA of E. coli, as presented here, is readily generalized to other species. Along with the maps discussed in the manuscript, we have also generated 2° structures3D for T. thermophilus, H. marismortui, and S. cerevisiae. Additionally, we have mapped the variety of data onto the 2° structure3D and the conventional 2° structurephylo. On-line editable representations are available in a gallery at http://apollo.chemistry.gatech.edu/RibosomeGallery.The 2° structureSupplementary Data are available at NAR Online: Supplementary Tables 1–11 and Supplementary Figures 1–7.


All globins belong to one of three families: the F (flavohemoglobin) and S (sensor) families that exhibit the canonical 3/3 α-helical fold, and the T (truncated 3/3 fold) globins characterized by a shortened 2/2 α-helical fold. All eukaryote 3/3 hemoglobins are related to the bacterial single domain F globins. It is known that Fungi contain flavohemoglobins and single domain S globins. Our aims are to provide a census of fungal globins and to examine their relationships to bacterial globins.Examination of 165 genomes revealed that globins are present in >90% of Ascomycota and ∼60% of Basidiomycota genomes. The S globins occur in Blastocladiomycota and Chytridiomycota in addition to the phyla that have FHbs. Unexpectedly, group 1 T globins were found in one Blastocladiomycota and one Chytridiomycota genome. Phylogenetic analyses were carried out on the fungal globins, alone and aligned with representative bacterial globins. The Saccharomycetes and Sordariomycetes with two FHbs form two widely divergent clusters separated by the remaining fungal sequences. One of the Saccharomycete groups represents a new subfamily of FHbs, comprising a previously unknown N-terminal and a FHb missing the C-terminal moiety of its reductase domain. The two Saccharomycete groups also form two clusters in the presence of bacterial FHbs; the surrounding bacterial sequences are dominated by Proteobacteria and Bacilli (Firmicutes). The remaining fungal FHbs cluster with Proteobacteria and Actinobacteria. The Sgbs cluster separately from their bacterial counterparts, except for the intercalation of two Planctomycetes and a Proteobacterium between the Fungi incertae sedis and the Blastocladiomycota and Chytridiomycota.Our results are compatible with a model of globin evolution put forward earlier, which proposed that eukaryote F, S and T globins originated via horizontal gene transfer of their bacterial counterparts to the eukaryote ancestor, resulting from the endosymbiotic events responsible for the origin of mitochondria and chloroplasts. Saccharomyces and Candida and in the mold Neurospora was first reported by Keilin Candida norvegensisSaccharomyces cerevisiaeE. coli HMP protein, concurrently discovered by R. Poole's group The presence of hemeproteins capable of reversible reaction with oxygen in the yeasts In this communication, we report on the known and novel putative globins identified in 165 fungal genomes and the results of molecular phylogenetic analyses of the fungal Hbs and their relationship to the bacterial and other eukaryote globins.www.ncbi.nih.gov/Taxonomy/). The Ascomycota with 117 genomes are the most numerous and also exhibit a high percentage of genomes with globins (>90%), except for the Pneumocystidiomycetes, where 3 genomes are devoid of globins. The 33 Basidiomycota genomes have a much lower percentage of genomes with globins (∼59%). Noteworthy is the total absence of globins in Microsporidia and the single Glomeromycota genome. No genomic information is available for the Neocallimastigomycota. It should be pointed out that because about half of the analyzed genomes are not completed, it is not always clear if a given genome lacks globins.The distribution of globins in 165 fungal genomes is summarized in the diagrammatic representation of fungal phylogeny shown in Overall, the fungi have one or two types of globins homologous to members of the bacterial globin F and S families . Except Allomyces macrogynus (Blastocladiomycota) and one as a single domain globin in Batrachochytrium dendrobatidis (Chytridiomycota).An unexpected finding was the presence of two putative globins belonging to the bacterial T family group 1, one as a C-terminal domain in a large 1129 residue protein in E. coli FHb  has three domains, with the ferredoxin reductase-like domain comprising domains cqx2 and cqx3, representing the FAD-binding and NADP-binding domains, respectively. FUGUE searches identify FHbs by providing Z scores >6 for the globin, the cqx2 and the cqx3 domains. The absence of cqx3 is diagnostic of the incomplete FHbs.It is possible to distinguish four subfamilies of F globins in fungi. One, comprises single domain Fgbs present only in the Mucoromycotina (Fungi incertae sedis) . All thrwww.expasy.org/tools) , Candida dubliensis and Schizosaccharomyces pombe (Saccharomycetes) and Filobasidiella neoformans (Basidiomycota) .Another subfamily represents FHbs that have >25 residue N-terminal extensions, of which the first ∼20 residues can be identified as a possible signal peptide via MITOprot and SignalP (g/tools) . These iThe Supplemental Data A. thaliana nonsymbiotic plant Hbs (NsHbs) as outgroup. The Fungi incertae sedis F globins (purple box) cluster separately from the remaining fungal F globins. Closest to them are the Saccharomycete group 1 chimeric FHbs (red) lacking the C-terminal moiety of the reductase domain (Aspergillus clavatus FHbs (blue arrows). The Sordariomycete Fusarium oxysporum has three FHbs (black arrows) that cluster closely together, in contrast to the three Penicillium chrysogenum FHbs (red arrows) representing Eurotiomycetes. The Dothideomycetes (blue box) and the two Leotiomycetes (yellow box) have single FHbs, as do all the Basidiomycota (green boxes). The latter four sequences are split into three separate groups: one and two (green boxes 1 and 2) encompassing a eurotiomycete and a sordariomycete sequence, while the fourth one (green box 3) lies with a dothideomycete FHb.The MAFFT alignment of 62 representative FHb globin domains selected from over 130 sequences was found to have the highest MUMSA score of the five alignments we tried  surrounded by the same 12 Actinomycete sequences as in In contrast to Fungi, where with the exception of the Fungi Incertae sedis, all F globins are chimeric FHbs, bacterial F globins comprise FHbs and single domain Fgbs in silico searches revealed the presence of two previously unknown eukaryote Sgbs, from the rotifer Philodina roseola (ACD54784.1) and the heterolobosan Naegleri gruberi (XP_002677420.1). They represent the only other eukaryote Sgbs known at present and were added to the fungal sequences. In the Bayesian tree based on a MAFFT v.6.850 alignment shown in Tuber melanosporum (star), and most of the Onygenales, a subgroup of the Eurotiomycetes (light brown box A), that appear to be unique in having no FHbs (see Uncinocarpus reeseii and Coccidioides immitis (light brown box B). Note that the Eurotiomycetes (light blue boxes), Sordariomycetes (light purple boxes) and Dothideomycetes, all cluster into clades, 3 (light blue A–C), 4 (light purple boxes A–D) and 2, respectively. The NJ tree based on a TCOFFEE v.9.01 alignment, provided in Supplemental Data In addition to over 70 fungal Sgbs, our FHbs see . The secWithin bacteria, the S globins occur predominantly as a variety of globin-coupled sensors (GCSs), many with more than a single non-globin domain, and two distinct types of single domain globins, the protoglobins (Pgbs) and the Sgbs Next, we selected 40 representative bacterial Sgbs, consisting of 16 Pgbs (including 5 Archaea), and Sgbs with less than 320 residues. The results of a Bayesian analysis based on a TCOFFEE v. 9.01 alignment  and Batrachochytrium dendrobatidis (Chytridiomycota). Physcomitrella patens NsHbs) as outgroup. We also added 10 chlorophyte T1 globins (green), as additional representative microbial eukaryote T1 globins. The Blastocladiomycota T1 sequence (red arrow) clusters with a Lentisphaera, 3 Alphaproteobacteria and 3 Cyanobacteria T1s (blue box A). Next to this cluster lies the Chytridiomycote T1 (black arrow), vicinal to a group comprising 5 Cyanobacteria and 7 Proteobacteria (blue box B). Adjacent is the clade of the 10 Chlorophyte sequences (green box). In the corresponding NJ tree, provided in We also investigated the phylogeny of the newly identified T globins group 1 (T1 globins) of www.ncbi.nlm.nih.gov/Taxonomy) in Dictionary of the Fungi (10th edition) Because the taxonomy of fungi is still in a state of flux, we used the GenBank classification  and Batrachochytrium dendrobatidis (Chytridiomycota) was an unexpected byproduct of our census. In the former, the T globin domain occurs in the C-terminal moiety (826–944) of a 1129 residue chimeric protein. A CDD search Sus scrofa, Homo sapiens and Schizosaccharomyces pombe . Batrachochytrium dendrobatidis, the causative agent of chytridiomycosis in amphibians The finding of group 1 T globins in ad hoc, and different from the fungal globins as outgroup. The main weakness of all the phylogenetic trees we show consists in the low node probabilities. Nevertheless, despite the low posterior probabilities and bootstrap values, too low to provide unequivocal phylogenies, the trees obtained by the Bayesian, RAxML and NJ analyses show mostly broad agreement with each other. In particular, the specific clusters of sequences are generally conserved, even though their tree locations vary, depending on the alignment used and the type of molecular phylogenetic analysis employed.The results of our Bayesian, RAxML and NJ analyses presented in the Earlier, we put forward a model of globin evolution Another aspect of our analyses that requires elaboration, is the selection of representative sequences, fungal on one side and bacterial on the other, necessitated by the large number of available sequences. The selection was based on initial NJ phylogenetic trees based on multiple sequence alignments generated by MAFFT 6.833. In all cases we selected the sequences to maintain representation of the distinct groups, so that e.g. in bacterial FHbs, we included representatives of the major bacterial phyla, such as Actinobacteria, Bacteroidetes, Chlorobi, Deinococcus, Firmicutes (Bacilli) and the five subdivisions of the Proteobacteria in approximate proportion to the actual number of FHbs known.The results of Bayesian analyses based on the alignments of the globin domain only  and of tCandida species, and abnormal FHbs, e.g. in K. waltii . Although the Aspergillus sp. have two FHbs, they appear to be closely related (blue arrows). Te Biesebeke and his collaborators have recently examined the phylogeny of Aspergillus sp. FHbs as well as several related fungi Aspergillus FHbs, one being closer to the Dothideomycetes, and the other to the Basidiomycetes.Both the Dothideomycetes and Eurotiomycetes appear to cluster as independent clades in the trees shown in Malassezia globosa FHb nestles within 11 Actinomycete FHbs, These results points to the possibility of HGT events in fungal phylogeny. Within the last several years, many instances of HGT from prokaryotes to fungi To investigate the relationship of fungal to bacterial FHbs, we carried out Bayesian and NJ analyses based on a TCOFFEE v.9.01 alignment of representative sets of fungal and bacterial FHbs in M. globosa FHb occurs within a clade of Actinomycete FHbs.The complexity of bacterial taxonomy can be reduced by viewing the bacterial taxa at a lower resolution. All the diversity of prokaryotes is encompassed in 37 taxa; these can be grouped into five super taxa: the Archaea, the Actinobacteria, the Clostridia, the Bacilli and the Double Membrane Prokaryotes (DMPs) Aspergillus clavatus (Ascomycota) FHb globin domain shown in Supplementary Data The intermixing of fungal and bacterial FHbs is underscored by the results of PSIBLAST searches using Philodina (“wheel-bearer”) (red arrow) and one amebo-flagellate Naegleria (Heterolobosan) (black arrow) Sgbs. We used the MAFFT alignment even though its MUMSA score was less than the TCOFFEE v.9.01 alignment  and Naegleria (black arrow) Sgbs cluster with the Blastochladiomycota and Chytridiomycota Sgbs and with the Fungi incertae sedis (purple) separated by a Proteobacteria and 2 Planctomycete sequences (blue box A). The similarity of the planctomycete to the fungal Sgbs is underscored by the results of BLASTP searches provided in Supplemental Data Volvox and Chlamydomonas) (green box). The bacterial T1s clustering with the fungal, Euryarchaea and Chlorophyte sequences include all the bacterial T1s that clustered with the 20 Chlorophyte T1s . They both have two FHbs, FHb1 (416/417 residues) and FHb2 (436/439 residues) A. oryzae FHb1 and FHb2 localize to the cytosol and the mitochondria, respectively Aspergillus species FHb1s appears to be positively correlated with hyphal growth A. nidulans, A. terreus, A. fumigatus and A. oryzaeCandida albicans, the most prevalent human fungal pathogen, the response to nitric oxide is complex, accompanied by overexpression of many genes, including only one of the several FHb genes A number of studies over the last decade have demonstrated the FHbs to play a role in the response of pathogenic fungi to nitric oxide, the antipathogenic compound produced by the innate immune system E. coli chemotaxis transducer Tsr, was also able to bind O2 reversibly and elicit an aerotactic response similar to the wild type GCSs Geobacter sulfurreducens (Deltaproteobacteria) GCS, which found bis-histidyl hexacoordination via HisE11 and HisF8 Nothing is known about the structure and function of fungal Sgbs. All we can do is make inferences based on recent results obtained with other members of the bacterial S family. One, is the recent finding by Alam and his collaborators that a Pgb fused to the MCP signaling domain of the This census of putative globin sequences in the Fungi has emphasized the broad presence of globins from the F and S families. We have identified globins in 136 out of 165 available genomes including a novel class of FHbs lacking the C-terminal portion of the reductase moiety as well as 2 previously unidentified T1s. Molecular phylogenetic analyses revealed a complex evolutionary relationship between fungal and bacterial globins. Our results, summarized in http://supfam.mrc-lmb.cam.ac.uk) http://blast.ncbi.nlm.nih.gov//sutils/genom_table.cgi?organism=fungi), and WU-BLAST2 searches . All the sequences were subjected to a FUGUE search http://www-cryst.bioc.cam.ac.uk). Given a query sequence, FUGUE scans a database of structural profiles, calculates the sequence-structure compatibility scores for each entry, using environment-specific substitution tables and structure-dependent gap penalties, and produces a list of potential homologs and alignments. FUGUE assesses the similarity between the query and a given structure via the Z score, the number of standard deviations above the mean score obtained by chance: the default threshold Z = 6.0 corresponds to 99% probability Putative globins and globin domains were identified from the SUPERFAMILY globin gene assignments (http://www.tcoffee.org), PROBCONS http://toolkit.tuebingen.mpg.de/probcons), COBALT www.ncbi.nlm.nih.gov/tools/cobalt/), and MAFFT 6.833 www.ebi.ac.uk/Tools/). The resulting alignments were checked manually for the conservation of the F8 His, and their quality was assessed using MUMSA http://msa.cgb.ki.se).Multiple sequence alignments were obtained using the following algorithms: TCOFFEE v.9.01 All known globin sequences exhibit the Mb-fold It is necessary to point out that since FHbs are chimeric proteins, and since they and the Sgbs have substantial N- and C-terminal extensions, setting the boundaries of the globin domains was an arbitrary decision. After one round of alignment, the sequences were trimmed, assuming the globin domain to start 11 residues prior to the B10 position and to end 15 residues after the H8 position. The trimmed sequences were then used in the final alignments.A total of over 130 F globin sequences, including about 30 of the chimeric incomplete FHbs and over 70 S globin sequences were identified. These were aligned using PROBCONS, MUSCLE v.3.7, MAFFT v.6.850, COBALT and TCOFFEE v.9.01. The quality of the alignments was assessed by MUMSA and the two top scoring alignments were subjected to Bayesian, Maximum Likelihood (ML) and Neighbor-Joining (NJ) phylogenetic analyses.6 generations. Trees were sampled every 100 generations, and the burnin value was set to 2×104. In all analyses convergence of the two parallel runs was observed. Maximum likelihood-based phylogenetic analysis was performed by RAxML version 7.2.3 Bayesian inference trees were obtained employing MrBayes version 3.1.2 Figure S1MAFFT alignments of fungal FHbs.(TIF)Click here for additional data file.Figure S2MAFFT alignments of fungal Sgbs.(TIF)Click here for additional data file.Figure S3Bayesian phylogenetic tree of complete fungal FHbs. Bayesian tree of a MAFFT v.6.850 alignment of 62 complete (globin+reductase domains) fungal FHbs, including all the sequences in (TIF)Click here for additional data file.Figure S4NJ phylogenetic tree of fungal and bacterial FHbs. NJ tree of a TCOFFEE v. 9.01 alignment of 37 representative fungal FHbs and 55 representative bacterial FHbs using two bacterial Pgbs as outgroup. Only bootstrap values >50% are shown. The sequences are identified by the first three letters of the binary species name, the number of residues, and the first three or four letters of the phylum, followed by the first three letters of the family (see mily see . Abbrevi(TIF)Click here for additional data file.Figure S5NJ phylogenetic tree of fungal Sgbs. NJ tree of a TCOFFEE v. 9.01 alignment of 59 fungal, one rotifer and one heterolobosan Sgbs. Only bootstrap values >50% are shown. The sequences are identified by the first three letters of the binary species name, the number of residues, and the first three or four letters of the phylum, followed by the first three letters of the family (see mily see . Abbrevi(TIF)Click here for additional data file.Figure S6Bayesian phylogenetic tree of fungal Sgbs. Bayesian tree based on a TCOFFEE v. 9.01 alignment of 59 fungal, one rotifer and one heterolobosan Sgbs. Support values at branches represent Bayesian posterior probabilities (>0.5). The sequences are identified by the first three letters of the binary species name, the number of residues, and the full phylum and family names (see ames see .(TIF)Click here for additional data file.Figure S7Bayesian phylogenetic tree of bacterial GCSs and Sgbs. Bayesian tree based on a MUSCLE v.6.850 alignment of 60 representative bacterial GCSs including 5 single domain sensor globins (light brown boxes). Support values at branches represent Bayesian posterior probabilities (>0.5). The first three letters of each part of the binary species name is followed by the number of residues and by a letter denoting the nonglobin domain: D – diguanylate cyclase; D* - diguanylate cyclase with additional domains; M – methyl accepting chemotaxis domain; H – histidine kinase domain; S – STAS domain; Pgb – protoglobin; Sgb – single domain sensor globin; U – unidentified domain.(TIF)Click here for additional data file.Figure S8NJ phylogenetic tree of bacterial GCSs and Sgbs. NJ tree of a MUSCLE v.6.850 alignment of 60 representative bacterial GCSs including 5 single domain sensor globins (light brown boxes). Only bootstrap values >50% are shown. The first three letters of each part of the binary is followed by number of residues and by a letter denoting the nonglobin domain: D – diguanylate cyclase; D* - diguanylate cyclase with additional domains; M – methyl accepting chemotaxis domain; H – histidine kinase domain; S – STAS domain; Pgb – protoglobin; Sgb – single domain sensor globin; U – unidentified domain. Abbreviations: ACI – Acidobacteria; Aci – Acidithiobacillales; ACTI – Actinobacteria; Actm – Actinomycete; Alt - Altermonadales; APR – Alphaproteobacteria; ARCH – Archaea; Cre – Crenarchaeota; CYA – Cyanobacteria; Eur – Euryarchaeota; BPR – Betaproteobacteria; GPR – gammaproteobacteria; Dbac – Desulfobacterales; DPR – Deltaproteobacteria; BAC – Bacteroidetes; Bur – Burkholderiales; Caul – Caulobacterales; CHLO – Chloroflexi; Chr – Chromatiales if GPR, Chrooccacales if CYA; CHL – Chlamydia/Verrumicrobia; Clo – Clostridiales; CYA – Cyanobacteria; DEI – Deinococcus; Dmon – Desulfomonadales; Ent – Enterobacteriales; EUR – Euryarchaeotes; FIR – Firmicutes; Gal – Gallionellales; Leg – Legionellales; LENT – Lentisphaerae; Metc – Methylococcales; Met – Methylophilales; Myx – Myxococcales; Nei – Neisseriales; NITR - Nitrospirae; Nos – Nostocales; Oce – Oceanospirillales; Osc – Oscillatoriales; PLA – Planctomycete; Pse – Pseudomonadales; Rhi – Rhizobiales; Rho – Rhodobacterales; Rsp – Rhodospirillales; Ric – Rickettsiales; Rub – Rubrobacterales; THE – Thermus; Thi – Thiotrichales; VER – Verrumicrobia; Vib – Vibrionales; Xan - Xanthomonadales.(TIF)Click here for additional data file.Figure S9NJ phylogenetic tree of fungal and bacterial Sgbs. NJ tree of a TCOFFEE v. 9.01 alignment of 51 fungal, 57 bacterial (including 16 Pgbs), one rotifer and one heterolobosan Sgbs, using two Adgbs (TIF)Click here for additional data file.Figure S10NJ phylogenetic tree of fungal and bacterial T1 globins. NJ tree of a TCOFFEE v. 9.01 alignment of 2 fungal (red and black arrows), 70 bacterial (blue), 4 euryarchaeote (purple) and 10 chlorophyte (green) T1 globins, using 2 Physcomitrella nsHbs as outgroup. Only bootstrap values >50% are shown. The sequences are identified by the first three letters of the binary species name, the number of residues, and the first three or four letters of the phylum, followed by the first three letters of the family (see mily see . Abbrevi(TIF)Click here for additional data file.Table S1Identified and putative globins in fungal genomes.(DOCX)Click here for additional data file.Table S2MUMSA scores for the five multiple alignment algorithms used in this work.(DOCX)Click here for additional data file.Table S3nd iteration using Hits obtained via PSIBLAST 2Aspergillus clavatusnd iteration. (Ascomycota) FHb globin domain (XP_001274889.1), as query, and selecting the first 40 fungal FHbs for the 2(DOCX)Click here for additional data file.Table S4nd iteration using Hits obtained via PSIBLAST 2Ajellomyces dermatidis  Sgb, 214aa (33–214) (XP_002625170.1), as query.(DOCX)Click here for additional data file.Table S5nd iteration using Hits obtained via PSIBLAST 2Coprinopsis cinerea (Basidiomycota) Sgb, 235aa (26–194) (XP_001838134.1), as query.(DOCX)Click here for additional data file.Table S6Hits obtained via BLASTP using the T1 globin domain from the Allomyces macrogynus (Blastocladiomycota) 1129aa chimeric protein (AMAG_16521T0), as query.(DOCX)Click here for additional data file.Table S7Hits obtained via BLASTP using the Batrachochytrium dendrobatidis (Chytridiomycota) T1 globin (BDEG_06358), as query.(DOCX)Click here for additional data file.


Autotransport in Gram-negative bacteria denotes the ability of surface-localized proteins to cross the outer membrane (OM) autonomously. Autotransporters perform this task with the help of a β-barrel transmembrane domain localized in the OM. Different classes of autotransporters have been investigated in detail in recent years; classical monomeric but also trimeric autotransporters comprise many important bacterial virulence factors. So do the two-partner secretion systems, which are a special case as the transported protein resides on a different polypeptide chain than the transporter. Despite the great interest in these proteins, the exact mechanism of the transport process remains elusive. Moreover, different periplasmic and OM factors have been identified that play a role in the translocation, making the term ‘autotransport’ debatable. In this review, we compile the wealth of details known on the mechanism of single autotransporters from different classes and organisms, and put them into a bigger perspective. We also discuss recently discovered or rediscovered classes of autotransporters. These proteins must cross two membrane systems: the inner membrane (IM)—composed of diglyceride phospho- and glycolipids—and the outer membrane (OM)—which is an asymmetric assembly of inner leaflet phospholipids and outer leaflet lipopolysaccharides. Different protein translocation systems exist in the IM that have been studied in much detail; these are the general secretion (Sec) system with its accessory factors, and the twin arginine translocation (Tat) system. Both operate by recognizing specific signal peptides at the N-terminus of transport substrates, which are cleaved during translocation through the IM . The majet al.  in right-handed coiled-coils, or the analogous motif [RxD] in left-handed coiled coils, in this region, sometimes repeatedly. It seems to be a folding core motif , which wThe transition of one handedness to another in the coiled-coil segment might alleviate torsion stress during folding , and theThe current model of type Vc autotransport is displayed in 6.Pseudomonas aeruginosa, named PlpD. Its passenger domain is a lipolytic enzyme and is cleaved autocatalytically after completion of autotransport, not unlike some lipases exported by the classical type Va pathway. But in PlpD, the passenger domain is connected to the translocator β-barrel domain with a POTRA domain (c). Thus, it looks like a gene fusion of the two components of a TPSS [Recently, a novel type of autotransporter was described, and was termed type Vd secretion . It has A domain c. Thus, f a TPSS ,101, albf a TPSS . It is cf a TPSS .Trichodesmium erythraeum [The case of PlpD is interesting in two ways: first, it demonstrates that more classes of unrecognized autotransporters might exist, either because they are not similar enough to the known types and thus not easily found with BLAST in sequenced genomes, or because they are present in groups of organisms underrepresented in the databases (e.g. because they are non-pathogenic). Second, PlpD clearly links type Va and type Vb (two-partner) secretion, and thus puts an end to the debate on whether TPSSs are really autotransporters or not ,103. Eveythraeum .7.E. coli and Invasin of enteropathogenic Yersinia spp. are closely related adhesins with extracellular Ig domains anchored in the bacterial OM—related enough to allow for a chimeric exchange of their transmembrane domain, which still yields functional protein [E. coli to host cells, which leads to pedestal formation. To this end, a second protein (Tir) is secreted into the host cell and is inserted into the host cell plasma membrane from the inside [1-integrins that are located on the surface of the host cell [Intimin of  protein . Intimine inside . The sece inside . Invasinost cell —no otherBoth proteins are well studied for their involvement in pathogenesis. In contrast, numerous models only vaguely describe the membrane anchor of these proteins as a transmembrane β-barrel. They avoid—probably deliberately—a clear discussion on the topology and connectivity between the extracellular domains and the anchor, albeit suggesting that they are autotransporters –111. EveOnly recently, a detailed topology model was published for the Intimin/Invasin family , showingIn contrast to type Va autotransporters, the order of passenger and transport domains are reversed in Intimin and Invasin. Thus, it is the C-terminus, not the N-terminus, that extends from the OM to the host cell. This fact must consequently mean that the mechanism of autotransport is different from type Va autotransport, as a presumed hairpin intermediate would loop through the transport pore pulling a strand in the inverse direction and the exported domain would fold from its N- to its C-terminus  and of quality control proteases (such as DegP) for classical β-barrel proteins [There is an established evolutionary relationship between the transport domains of autotransporters and almost all other OM β-barrel proteins ,116. Thuproteins ,118 and et al. [The difference is the autotransport itself, an event that we strongly believe starts after, or at most synchronously, with OM insertion, and thus represents an addition to the general OM insertion mechanism mediated by the Bam complex. It has been suggested that folded or semi-folded periplasmic intermediates of autotransporters exist. The experiments that lead to this conclusion ,35,119 aet al.  in the pThe same energy considerations hold true for autotransport as such (i.e. the events after membrane insertion). Neither ATP nor a proton gradient is necessary for surface display of the transported passenger domains. Again, the only source of energy for transport available is the free energy of protein folding. Key experiments have recently substantiated this view, showing that folding of type Va autotransporters proceeds sequentially from the C- to the N-terminus, driving the process further ,55. The The major objection raised against the hairpin model of autotransport is the restricted size of the β-barrel pore. The crystal structures of the translocation domains of NalP and other type Va systems and of the type Vc system Hia in principle show that there is enough room for two extended polypeptide strands in the pore (six in the case of type Vc), as discussed in ,51,96. MWe conclude from the literature compiled in this review that there is a general mechanism for autotransport, where the different types of autotransporters follow the general route for β-barrel protein insertion into the OM. An extended signal peptide in many cases ensures slow processing by the Sec machinery, to gain time for proper OM insertion before the passenger domain is released. Moreover, premature folding in the periplasm is inhibited by the known periplasmic chaperone systems, and also by sequence-intrinsic properties of the passenger polypeptides, such as a reduced folding rate, little to no cysteine residues for disulphide formation, high solubility of the unfolded passenger domains and little to no propensity to aggregate when in the unfolded state. Most probably already during membrane insertion a hairpin structure is formed, and the sequential folding of the passenger domain on the cell surface drives the process to completion.


A detailed investigation of its stability, carried out in comparison with the homologous pentameric POZ/BTB domain isolated from KCTD5, highlights a number of interesting features, which are shared by the two domains despite their different organization. Their thermal/chemical denaturation curves are characterized by a single and sharp inflection point, suggesting that the denaturation of the two domains is a cooperative two-state process. Furthermore, both domains present a significant content of secondary structure in their denatured state and a reversible denaturation process. We suggest that the ability of these domains to fold and unfold reversibly, a property that is somewhat unexpected for these oligomeric assemblies, may have important implications for their biological function. Indeed, these properties likely favor the formation of heteromeric associations that may be essential for the intricate regulation of the processes in which these proteins are involved.POZ/BTB domains are widespread modules detected in a variety of different biological contexts. Here, we report a biophysical characterization of the POZ/BTB of KCTD6, a protein that is involved in the turnover of the muscle small ankyrin-1 isoform 5 and, in combination with KCTD11, in the ubiquitination and degradation of HDAC1. The analyses show that the domain is a tetramer made up by subunits with the expected  A large fraction of proteins are oligomeric in their functional state(s). Although protein oligomerization may be incidental, several studies have highlighted the benefits of this process in a variety of different systems. In this framework, the analysis of the structural determinants of protein oligomerization is a field of considerable interest. The characterization of homologous proteins endowed with different quaternary structures is particularly suited to unveil key factors in protein oligomerization , 2.Structural and functional literature studies have highlighted that specific domains, often shared by proteins involved in completely different biological processes, have been specifically designed by evolution to mediate protein-protein interactions and to promote oligomerization. One of the best characterized domains deputed to these roles is the POZ/BTB domain, a motif that is widespread among eukaryotes . From th+ (Kv) channels, the twenty-three KCTD genes present in the human genome encode for proteins implicated in a variety of biological processes not connected with voltage channels. Recent investigations have shown that some members of the family play a key role in protein ubiquitination and degradation as they act as cullin binding adaptors  [Among the proteins embedding POZ/BTB domain(s), the family denoted as KCTD, containing the potassium channel tetramerization domain, has recently received a special attention. Although the name of the family derives from their homology with proteins involved in the formation of voltage-gated K KCTD10) –7. In ot KCTD10) –10. Fina KCTD10) –14. DespBTB) domains and that evolutionary transitions from tetrameric (point symmetry C4) to pentameric (point symmetry C5) associations are very unlikely [BTB), a member of the family that shares significant sequence and functional similarities with the well-characterized KCTD11. KCTD6 is a recently discovered substrate adaptor for cullin-3 that regulates protein levels of the muscle small ankyrin-1 isoform 5 (sAnk1.5) [Structural information have been so far reported only for KCTD5, KCTD11 and KCTD12 , 15–17. sAnk1.5)  and thatsAnk1.5) . We thenBTB (residues 10–110 of KCTD6) was amplified by PCR, using as template the human KCTD6 cDNA (NM_153331) (thermo Scientific) and cloned into the pETM-11 expression vector (Novagen). Escherichia coli BL21(DE3) Star strain (Invitrogen) was transformed with the recombinant vector, grown at 37°C and induced by 0.5 mM isopropyl-β-d-thiogalactoside (IPTG) for 16 h at 22°C. KCTD6BTB domain was purified as already described for KCTD11 with slight modifications [The cell pellets were re-suspended in 50 mM Tris-HCl, 0.1 M NaCl, pH 8.0. The KCTD6BTB plasmid was a gift of Prof. Goldstein (University of Chicago). The recombinant protein was expressed and purified according to the procedure previously reported. The pET28/KCTD5Static light scattering experiments were performed using a MiniDAWN Treos spectrometer (Wyatt Instrument Technology Corp.) equipped with a laser operating at 658 nm and connected online to a size-exclusion chromatography.BTB was analyzed by size-exclusion chromatography connected to a triple-angle light scattering detector equipped with a QELS module (quasielastic light scattering) for mass value. 500 μL sample (1 mg/mL) was loaded on a Superdex 75 10/30 column, equilibrated in 50 mM Tris-HCl and 0.15 M NaCl, pH 8.0 and analyzed as described elsewhere [Purified KCTD6μM. CD spectra were recorded at 20°C using a Jasco J-810 spectropolarimeter equipped with a Peltier thermostatic cell holder. Far-UV measurements (260–190 nm) were carried out using a 0.1 cm path length cell in a 10 mM Tris-HCl, pH 8.0 buffer at a concentration of 5 Chemical denaturation was induced by guanidine hydrochloride (GuHCl) or urea in a range of 0 to 6.0 M and 0 to 8.0 M, respectively, incubating the samples 2 h at room temperature. All the denaturations were investigated by recording the CD signal at 222 nm.BTB, two separate sets of experiments were carried out setting the excitation wavelength at either 280 or 295 nm. For KCTD6BTB, that does not contain Trp residues, only the experiment with excitation at 280 nm was performed. In all cases, the emission was recorded in the range 290–450 nm.Fluorescence experiments were conducted in the same conditions. Fluorescence spectra were collected at 20°C using a Varian Cary Eclipse spectrophotometer and a 1.0 cm path length cell. For KCTD5BTB, spans from residue 10 to residue 110. Pair-wise alignments of KCTD6BTB with POZ/BTB domains of other KCTDs unveil that the closest ones are those of KCTD11 and KCTD21. The sequence identity shared by KCTD11BTB and KCTD21BTB with KCTD6BTB is 61% and 72%, respectively. KCTD6BTB presents sequence identities higher than 50% when compared with KCTD1 (54%), KCTD4 (53%), and KCTD15 (57%).KCTD6 is a protein of 237 residues (UniProt code Q8NC69). Comparative analyses of its sequence with those reported in the sequence databanks clearly indicate that the POZ/BTB domain, located in the N-terminal region of the protein, namely KCTD6http://swissmodel.expasy.org/). The server predicts the occurrence of several β-strands and α-helices in KCTD structure  . The inspection of the region 10–110, corresponding to the BTB domain, unveils the presence of the structural elements (three β-strands and five α-helices) characteristic of this motif. The prediction also suggests that the C-terminal domain of the protein is highly structured, being characterized by the combination of α- and β-structure with coil regions of limited size.To gain insights into the structural features of the protein, we performed a secondary structure prediction session on KCTD6 sequence by using the Swiss Model server  of the purified recombinant product allowed its extensive biophysical characterization. The homogeneity of the purified protein was assessed with a variety of different techniques (SDS PAGE and mass spectrometry). The far-UV CD spectrum of KCTD6BTB is consistent with a properly folded protein  and KCTD21BTB (72%),which are also tetrameric in solution [As POZ/BTB domains of KCTD proteins may be characterized, despite their high sequence identities, by different oligomeric states, we carefully investigated the aggregation state of KCTD6± 350 Da . These eBTB domain were conducted by using far-UV CD spectroscopy by following the CD signal at 222 nm. As the tetrameric KCTD6BTB shares a significant sequence identity (approx. 40%) with the pentameric KCTD5BTB  , the analyses were extended to the latter domain for comparative purposes. The far-UV CD spectrum of the KCTD6BTB sample kept at 100°C is different from that recorded for the native form at 20°C, but is characterized by the presence of a broad minimum at 205 nm and a shoulder at 220 nm  of 62°C, suggestive of a cooperative process in which dissociation and unfolding are coupled. The latter mechanism is supported by the fact that, on increasing the protein concentration, the melting curves present the same shape with an increase in the melting temperature (data not shown), as it should be for a reversible two-state process in which dissociation and unfolding are coupled [Thermal denaturation analyses of the KCTD6t 220 nm . The latspectrum . This de coupled .BTB unveil differences and analogies when compared to KCTD6BTB. The overall shape of the far-UV CD spectrum of KCTD5BTB is similar to that of KCTD6BTB (BTB: (a) the thermally-denatured state of KCTD5BTB is characterized by the presence of a significant content of residual secondary structure and (b) the KCTD5BTB thermal denaturation is also reversible, since the secondary structure is recovered when temperature is lowered to 20°C  the change in molar ellipticity at 222 nm and (b) the change in fluorescence spectrum upon excitation at either 280 nm or 295 nm. The evolution of the CD signal at 222 nm upon the addition of the denaturants is characterized by a sigmoidal shape, with a single inflection point , fluorescence spectra were also collected upon excitation at 295 nm. These spectra show trends similar to those recorded upon excitation at 280 nm. The addition of both denaturants produces a decrease of the intensity and a shift of the wavelength of the maximum from 330 to 360 nm. This finding indicates that the side chains of the five Trp present in KCTD5BTB pentamer, that are buried in the native structure, become solvent exposed in the denatured state. Since Trp45 is located in the initial strand of the sole β-sheet present in KCTD5BTB structure, this finding may suggest that the β-structure is highly perturbed in the denaturation and that the residual structure present in the denatured state corresponds to helical regions. It is also likely that the differences observed in the fluorescence spectra of the two proteins, recorded upon excitation at 280 nm, may be due to the contribution of the Trp45 side chains in the KCTD5BTB domain.The intensity of the fluorescence spectra of the two domains shows clear variations when the concentration of the denaturant approaches the Canalysis . This obad hoc strategies aimed at modulating their activities. Here, we report a characterization of two POZ/BTB domains (KCTD6BTB and KCTD5BTB) isolated from proteins belonging to the family of KCTDs, an emerging class of key factors involved in severe human pathologies. Although KCTD6BTB and KCTD5BTB share a high sequence identity (>40%), these domains are characterized by a different oligomeric organization. Indeed, literature data have shown that KCTD5BTB is pentameric [BTB is tetrameric. The tetrameric association of KCTD6BTB is not surprising taking into account the high sequence identity (>61%) that this domain shares with the tetrameric POZ/BTB domains of KCTD11 and KCTD21. Moreover, the analysis of KCTD6BTB sequence indicates that this domain does not contain the residues that are believed to stabilize the pentameric association of KCTD5BTB [POZ/BTB domains are versatile modules that are often implicated in fundamental biological processes . Previountameric , whereasgure S2) . The findentity >0%, thesegure S2) . It is ientity >6% that thBTB but 93°C for KCTD5BTB. Although the absence of structural data for KCTD6BTB hampers the possibility to relate this marked difference to specific structural determinants, it is likely that the peculiar stability of KCTD5BTB may be related, among other factors, to the presence of an intrasubunit disulfide bridge that connects Cys62 to Cys74 [2 out of 6000 Å2) is buried upon pentamer formation.Even though both domains display a remarkable stability against temperature, a significant difference is emerged: the melting temperature is 62°C for KCTD6to Cys74 . In addiBTB against chemical denaturants (urea and GuHCl) is less remarkable. KCTD6BTB and KCTD5BTB show similar C1/2 values, close to 5.0 M and 2.2 M, when they are denatured by means of urea and GuHCl, respectively. The relatively low value of the ratio C1/2(urea)/C1/2(GuHCl) ≈ 2.2 suggests that electrostatic interactions, that are efficiently impaired by GuHCl, should play a limited role in the stabilization of these oligomeric structures [On the other hand, it is important to note that the stability of KCTD5ructures , 24. TheBTB and KCTD5BTB may have important implications for their biological function. Recent literature data have shown that the POZ/BTB domains are used by KCTDs proteins to form heteromeric associations among different members of the family [The ability of these domains to fold and unfold in a reversible manner is somewhat unexpected for these oligomeric assemblies. Among the few literature examples, it is worth mentioning the tetramerization domain of p53 tumor suppressor protein which shows a reversible and cooperative two-state denaturation against both temperature and GuHCl , 25. Thie family . It has e family  and thate family . Since CIt is intriguing to speculate that the combination observed in KCTDs of well-conserved POZ/BTB, that assures the possibility to form heteromeric associations, coupled with diversified domains may be a more general and widespread feature among oligomeric proteins that allow intricate regulations of biological processes.The secondary structure prediction of KCTD6, an overlay of KCTD5BTB and KCTD6BTB CD spectra, the sequence alignment of KCTD6BTB and KCTD5BTB and a table reporting the comparison between theoretical molecular weights of KCTDs BTB/POZ assemblies and those calculated by light scattering and gel filtration analyses.Click here for additional data file.


PIK3CA) is one of the most frequently mutated genes in human cancer [The gene encoding the p110α catalytic subunit of class IA phosphoinositide 3-kinase  Fig. . The intTo determine the mechanism of activation by oncogenic mutants we first investigated conformational changes that occur in the wild type upon binding to tyrosine-phosphorylated peptides derived from RTKs (pY), and upon binding to phospholipid membranes . We idenWe propose that these events are involved in the transition of a cytosolic, inhibited form of p110α to an open, activated form on the membrane surface. Oncogenic mutants in the ABD (G106V), ABD-RBD (Ras-binding domain) linker (G118D), C2 , and the hot spot mutants in the helical (E545K) and kinase domains (H1047R) upregulated lipid kinase activity by mimicking or enhancing one or more of the conformational events identified in the wild type.Mutations in the ABD and ABD-RBD linker caused both enhanced movement of the ABD domain relative to the kinase domain, and disruption of the C2/iSH2 interface compared to the WT enzyme. This same relationship was seen in mutations in the C2 domain, which also caused disruption of the C2/iSH2 interface along with movement of the ABD relative to the kinase domain. This shows that these two regions are allosterically linked to each other. These mutations can be activated by RTK derived pY, and are hyperactivated compared to the WT enzyme.The presence of the helical domain hotspot mutation located at the nSH2/helical interface fully disrupted this contact and made the mutant insensitive to pY activation, as predicted from previous biochemical and structural data , 6. The The important role of oncogenic mutants of PI3Kα in the development of a variety of human cancers makes the development of novel inhibitory strategies an important goal. The discovery of these unexpected allosteric conformational changes that are enhanced in oncogenic mutants opens up a novel strategy towards designing small molecule inhibitors that function outside of the conserved ATP binding pocket.