The mitochondrial ATP-Mg/Pi carrier imports adenine nucleotides from the cytosol into the mitochondrial matrix and exports phosphate. The carrier is regulated by the concentration of cytosolic calcium, altering the size of the adenine nucleotide pool in the mitochondrial matrix in response to energetic demands. The protein consists of three domains; (i) the N-terminal regulatory domain, which is formed of two pairs of fused calcium-binding EF-hands, (ii) the C-terminal mitochondrial carrier domain, which is involved in transport, and (iii) a linker region with an amphipathic α-helix of unknown function. The mechanism by which calcium binding to the regulatory domain modulates substrate transport in the carrier domain has not been resolved. Here, we present two new crystal structures of the regulatory domain of the human isoform 1. Careful analysis by SEC confirmed that although the regulatory domain crystallised as dimers, full-length ATP-Mg/Pi carrier is monomeric. Therefore, the ATP-Mg/Pi carrier must have a different mechanism of calcium regulation than the architecturally related aspartate/glutamate carrier, which is dimeric. The structure showed that an amphipathic α-helix is bound to the regulatory domain in a hydrophobic cleft of EF-hand 3/4. Detailed bioinformatics analyses of different EF-hand states indicate that upon release of calcium, EF-hands close, meaning that the regulatory domain would release the amphipathic α-helix. We propose a mechanism for ATP-Mg/Pi carriers in which the amphipathic α-helix becomes mobile upon release of calcium and could block the transport of substrates across the mitochondrial inner membrane.  •The mechanism of calcium-regulation in the ATP-Mg/Pi carrier is unresolved.•The human ATP-Mg/Pi carrier is monomeric in the presence and absence of calcium.•In the presence of calcium an amphipathic α-helix binds in a cleft of EF-hand 3/4.•Without calcium the amphipathic α-helix is released from the regulatory domain.•Regulation could occur by the amphipathic α-helix blocking substrate translocation. Mitochondrial carriers are typically located in the mitochondrial inner membrane and fulfil the vital role of shuttling nucleotides, amino acids, inorganic ions, keto acids, fatty acids and cofactors between the mitochondrial matrix and the cytosol Sal1p) SLC25A24, SLC25A25, SLC25A23 and SLC25A54 encoding for the protein short calcium binding mitochondrial carrier (SCaMC) isoform 1 (APC-1), SCaMC-2 (APC-3), SCaMC-3 (APC-2) and SCaMC-1L, respectively SLC25A41, encodes for SCaMC-3L protein In yeast only one APC ortholog exists , also displays a calcium-dependence for activity and has an N-terminal regulatory domain that contains EF-hands. Structures of both the calcium-bound and calcium-free states of the regulatory domain of the human AGC have recently been solved NMR nuclear Overhauser effects have shown that the regulatory domain of HsAPC-1 is more dynamic and less structured in the calcium-free state Here, an independent structural determination of the native human APC-1 regulatory domain was completed, which was combined with an investigation into the oligomeric state of the full-length human APC-1 protein. Furthermore, an extensive comparison of the conformations of EF-hand proteins in the calcium-free, calcium-bound and peptide-bound state was undertaken, which has allowed us to propose a mechanism for the regulation of APC that is consistent with all available experimental data.22.1Q6NUK1) was designed to include an eight-histidine tag followed by a Factor Xa cleavage site (IEGR) at the N-terminus, creating a cleavable purification-tag. The gene was codon-optimized for expression in S. cerevisiae by GenScript and cloned into a pYES3/CT expression plasmid (Invitrogen), with the inducible galactose promoter being replaced by the constitutive promoter for the yeast mitochondrial phosphate carrier, as described previously S. cerevisiae haploid strain W303-1b A construct of HsAPC-1  and 30 column volumes of buffer B  LMNG, 0.1 mg mL− 1 tetra-oleoyl cardiolipin). Factor Xa protease (New England BioLabs) was used to specifically cleave the HsAPC-1 protein from the affinity resin. After Factor Xa cleavage, HsAPC-1 was spun through an empty Proteus midi-spin column (Generon) to remove affinity resin. Protein concentrations were determined using the bicinchoninic acid assay HsAPC-1 was solubilised in lauryl maltose neopentyl glycol (LMNG), and separated from insoluble protein by ultracentrifugation. Solubilised protein was passed through nickel sepharose affinity resin  to selectively bind full-length HsAPC-1, and washed with 50 column volumes of buffer A  LMNG, 0.1 mg mL2.2Lactococcus lactis expression plasmid pNZ8048, and the plasmid transformed into electrocompetent L. lactis NZ9000 using established methods L. lactis lysate was passed over Ni sepharose resin . The resin was washed with 50 column volumes of purification buffer  containing 50 mM imidazole and eluted in purification buffer containing 250 mM imidazole. Second, recovered protein was concentrated, and desalted using a PD10 column . The purification tag was removed from the construct by digestion with Factor Xa protease . Finally protein was loaded onto a Superdex 200 pg 16/60 SEC column  in a running buffer of 20 mM Tris pH 7.5, 20 mM NaCl. Peak fractions were collected and concentrated to approximately 10 mg mL− 1 for crystallography.Primers were design in order to create a construct of HsAPC-1 to include just the regulatory domain and the linker region (residues 14 to 174) of the protein (Forward primer: GAAGGTAGAACCTCCGAAGA. Reverse primer: CCTAGGTCTAGACTCGAGTCATTATTATATATCAATACCTGTGGAATGTTT). The inter-domain loop was not included in this construct. The construct was cloned into the 2.3− 1 tetra-oleoyl cardiolipin. The buffers used to run the molecular weight standards in each case matched the buffers being used in the analysis of each protein.Superdex 200 pg 16/60 SEC columns  were calibrated with molecular weight standards from both the high and low molecular weight standards kits , and unknown protein weights were estimated using the manufacturers recommended protocol. The SEC buffer used for analysis of the HsAPC-1 RD protein was purification buffer with either 5 mM calcium or 5 mM EGTA, with or without 1 mM DTT added as stated. The SEC buffer used for analysis of the full-length HsAPC-1 was 20 mM Tris pH 7.4, 50 mM NaCl, 0.03% (w/v) LMNG and 0.03 mg mLTotal detergent, phospholipid and protein contributions to the total mass could be determined colorimetrically by sugar assay 2.4Crystallisation screening with purified HsAPC-1 RD yielded an initial hit in drop H4 of the JBS Classics screen (Jena Bioscience) with a mother-liquor composed of 30% (w/v) polyethylene glycol 8000, 0.2 M ammonium sulfate. Optimisation around this hit produced square plate-like crystals in 29% (w/v) polyethylene glycol 8000, 0.2 M ammonium sulfate, larger in both width and height than the original hit, approximately 200 × 200 μm in dimension.Crystals of a tetrahedral bi-pyramidal morphology could be grown in 25–30% (w/v) polyethylene glycol 8000, 0.2 M lithium sulfate conditions. These crystals were smaller than the square plate-like crystals, with dimensions of about 40 × 30 × 30 μm, and grew as well defined single crystals.P2 and P212121 crystals, respectively. A long-wavelength dataset of the P212121 crystals used for a calcium-SAD experiment was also collected at Diamond beamline I24.High-resolution diffraction datasets were collected at Diamond beamline I24 and ESRF beamline ID23-2 for the P2 from the square plate-like crystals, and to 2.6 Å in the space-group P212121 from the tetrahedral bi-pyramidal type crystals. Refinement statistics are shown in  2.6Uniprot was used to search for structures of EF-hand proteins with a Pfam 2.7We propose α-helix 4/5 represents a static element connecting the two lobes of the protein together. The pitch of the α-helix 4/5 allowed precise manual alignment 2.8S.P.D.H. expressed, purified and crystallised proteins, performed biochemical assays and carried out the structure determination. All authors were involved in experimental planning, data collection at synchrotrons, data analysis and manuscript preparation.2.9P2 and P212121 forms have been deposited in the Protein Data Bank under accession codes PDB ID: 4ZCU and PDB ID: 4ZCV, respectively.The coordinates and structure factors of the calcium-bound regulatory domain of the ATP-Mg/Pi carrier in both the 33.1L. lactis and purified. HsAPC-1 RD was crystallised using sitting drop vapour diffusion techniques, predominantly in two different conditions. Molecular replacement using the recently solved structure of the HsAPC-1 RD (PDB ID: 4N5X) P212121 or P2 (A construct of the regulatory domain (RD) and the linker region (residues 14 to 174) of HsAPC-1 was expressed in 21 or P2 .As was found previously P2 space group, but not the four chains in the P212121 space-group , and calcium could only be modelled into EF-hand 4 in the three chains of the ce-group B. This iIt was previously suggested that an interaction between the terminal amphipathic α-helix and the hydrophobic pocket of HsAPC-1 RD might be a crystallisation artefact One unusual feature of the interaction between the hydrophobic pocket and its target, the amphipathic α-helix, is that they are both part of the same polypeptide chain. Other EF-hand proteins, such as calmodulin, interact with target α-helices of other proteins. However, the APC regulatory domain is not unique in this type of ‘self-sequestered’ interaction. Other examples are Kv channel interacting protein-1 The interaction between regulatory domain and the amphipathic α-helix resembles that of AGC and the C-terminal α-helix, and given that they are both members of the mitochondrial carrier family of proteins it is tempting to conclude that they might have an analogous mechanism. However, a fundamental difference is that in AGC the carrier domain is inserted between the EF-hands and the target α-helix, whereas in APC the target α-helix is inserted between the EF-hands and the carrier domain.3.24N5X) around α-helices 2 and 3  C. By SDSIt was important to determine whether the observed dimerisation of the isolated RD could be influenced by calcium. Fractions from peak 2 and peak 3 were pooled separately, and analysed by SEC in the presence of calcium or ethylene glycol tetraacetic acid (EGTA), and with or without the reducing agent dithiothreitol (DTT). The elution volumes of the dimer and monomer species were both affected  the dimerisation interface between the two chains of HsAPC-1 does not conform to expected characteristics for a physiologically relevant dimer, (ii) the cysteine involved in covalent dimerisation is not conserved and (iii) no effect of calcium on disulphide bond formation could be observed. Therefore, APC is monomeric and dimers of the HsAPC-1 RD observed in SEC traces and in crystal structures appear to be caused by non-specific disulphide bond formation between Cys15 residues and by crystal packing, respectively. Thus the mechanism of calcium regulation of transport must be different from that of AGC, which is dimeric.3.3The HsAPC-1 RD has been solved in a calcium-bound state. However, to appreciate the mechanism by which the regulatory domain would influence substrate transport, the calcium-free state must also be investigated. Even though attempts have been made here and previously A diverse reference set of EF-hand containing proteins was selected from the PDB with a preference for those containing EF-hands organised in pairs. Both crystal structures and NMR models of calcium-bound and calcium-free states were selected (Supplementary Table 1). In total, over 100 EF-hands were structurally superposed.Calcium-bound EF-hands displayed open or semi-open conformations. The angle of the exiting α-helix relative to the entering α-helix was 70° to 90° and 55° to 70°, respectively . CalciumThe largest variations were seen for the S100-like EF-hands, which have an additional two residues in the calcium-binding loop, and therefore provide a wider variety of possible conformations of the exiting α-helix. Some of the EF-hands within AGC are S100-like, but all four EF-hands of HsAPC-1 are conventional EF-hands, and adopt the fully open conformation as expected for calcium-bound EF-hands (1ZMZ) with an angle of ~ 40°, N-terminal lobe of calmodulin (PDB ID: 1CFD) with an angle of ~ 55° and the N-terminal lobe of calcium-dependent protein kinase (PDB ID: 3HZT) with an angle of ~ 65°.In order to understand the conformational changes that occur when HsAPC-1 goes from the calcium-bound to the calcium-free state, models of the calcium-free state were generated based on deposited structures. Three models of known calcium-free EF-hand pairs  were chosen as reference structures for modelling; centrin (PDB ID: Although the three resulting models had some structural differences from one another, the general motion that they described was consistent A. All caIt is possible that when the amphipathic α-helix is released, it could interact with the carrier domain to inhibit substrate transport, either by blocking access to the binding site or by preventing conformational changes required for transport by the carrier domain D. This m4The evidence presented here indicates that the functional unit of APC, including the isolated regulatory domain, is a monomer. Therefore, APC differs from AGC, which has a regulatory mechanism based on conformational changes within a dimeric regulatory domain. Thus they must be different in the key functional elements involved in regulation. Importantly, any mechanism for calcium regulation of transport must be based on APC functioning as a monomer.So far only structures of the calcium-bound state have been solved  [16], buSupplementary materialAnimation of the calcium-induced changes in HsAPC-1 RD.Morph-model between the calcium-bound HsAPC-1 RD crystal structure and model-2 for the calcium-free HsAPC-1 RD. Structures displayed and coloured as in Fig. 1.The following are the supplementary data related to this article.Transparency document.


Comparing our exact solutions with numerical approximations confirms the veracity of the method. Furthermore, our examples illustrate a delicate interplay between: (i) the rate at which the domain elongates, (ii) the diffusivity associated with the spreading density profile, (iii) the reaction rate, and (iv) the initial condition. Altering the balance between these four features leads to different outcomes in terms of whether an initial profile, located near x = 0, eventually overcomes the domain growth and colonizes the entire length of the domain by reaching the boundary where x = L(t).Many processes during embryonic development involve transport and reaction of molecules, or transport and proliferation of cells, within growing tissues. Mathematical models of such processes usually take the form of a reaction-diffusion partial differential equation (PDE) on a growing domain. Previous analyses of such models have mainly involved solving the PDEs numerically. Here, we present a framework for calculating the exact solution of a linear reaction-diffusion PDE on a growing domain. We derive an exact solution for a general class of one-dimensional linear reaction—diffusion process on 0< Developmental processes are often associated with transport and reaction of molecules, or transport and proliferation of cells, within growing tissues , 2. For One particular biological application where transport and reaction (proliferation) of cells takes place on a growing domain is the development of the enteric nervous system (ENS) –21. ThisOne of the first mathematical models of ENS development, described by Landman et al. , is a PDIn addition to presenting numerical solutions, Landman et al.  also preThe focus of the present work is to consider a linear reaction-diffusion process on a growing domain with a view to obtaining an exact solution of the associated PDE. After transforming the PDE to a fixed domain we obtain a PDE with variable coefficients. The variable coefficient PDE is simplified using an appropriate transformation which enables us to obtain an exact solution using separation of variables. While our strategy for obtaining an exact solution is quite general, we present specific results for linear and exponentially elongating domains. After verifying the accuracy of our exact solutions using numerical approximations, we summarise our results in terms of a concise condition that can be used to distinguish between successful or unsuccessful colonization. We conclude this study by acknowledging the limitations of our analysis, and we outline some further extensions of our approach which could be implemented in future studies.x < L(t), where L(t) is the increasing length of the domain. Domain growth is associated with a velocity field which causes a point at location x to move to x + vτ during a small time period of duration τ [x, we can derive an expression relating L(t) and v, which can be written asWe consider a linear reaction-diffusion process on a one-dimensional domain, 0 < ration τ . By const, so that we have Like others , 4, 22, x-direction with the origin fixed, so that v = 0. Integrating Without loss of generality, we assume that the domain elongates in the positive C, assuming that the population density function evolves according to a linear reaction–diffusion mechanism. The associated conservation statement on the growing domain can be written asx < L(t), where D > 0 is the diffusivity, k is the production rate and v is the velocity associated with the underlying domain growth, given by k > 0 represents a source term which is relevant to ENS development since the precursor cells proliferate [k < 0.We now consider conservation of mass of some density function, liferate –18, 20; x < β, being uniformly colonized at density C0, with the remaining portion of the domain being uncolonized. We suppose that we have zero diffusive flux conditions at both boundaries, x = 0 and x = L(t), and we now seek to find an exact solution, C.To solve ξ < 1. Recalling that L2(t), and hence decreases with time, andthe coefficient of the diffusive transport term is inversely proportional to Cσ(t), represents dilution associated with the expanding domain.the addition of a source term, −The first step in our solution strategy is to transform the spatial variable to a fixed domain, ξ=xL(t) –12, 22, Following Crank  we re-sct, and the transformed variable, T, which means that we can write C can be re-written in terms of the original coordinates, giving C. The Fourier coefficients, an, can be chosen to ensure that the exact solution satisfies the initial condition, given by C is quite general and does not depend on any particular form of the initial condition. We now present the details for a few relevant choices of L(t).ariables , givingx < L. With L(t) = L, we have σ(t) = 0 and t → ∞, we have T → ∞, since D > 0 and L > 0. On the non-growing domain the solution of Before we present results for a growing domain it is instructive to consider the solution of L(t) = L(0)exp(αt), we have σ(t) = α and t → ∞, we have α > 0. This limiting behavior is different to the limiting behavior under non-growing conditions. For an exponentially-elongating domain, the solution of With L(t) = L(0) + bt, we have t → ∞, we have D > 0 and b > 0. For a linearly-elongating domain, the solution of With C generated using the exact solution with plots of C computed numerically. To generate the numerical approximations we discretise ξ < 1, with uniform mesh spacing δξ. The resulting system of coupled ordinary differential equations is integrated through time using a backward Euler approximation with uniform time steps of duration δt. At each time step the resulting system of tridiagonal linear equations is solved using the Thomas algorithm [δξ and δt so that the numerical results are grid-independent.We now present some examples to highlight key features of the model. First we compare plots of lgorithm . All numt = 0,10 and 20, and we see that the exact and numerical solutions are indistinguishable. A summary of the properties of the solutions in the interval 0 ≤ t ≤ 20 is given in a space-time diagram in L(t), and the position of the front, f(t). Here, we define the position of the front to be the spatial location where C = 0.01. This means that we have f(0) = β. Comparing L(t) and f(t) in C profile moves in the positive x-direction as time increases; however, the distance between L(t) and f(t) increases with time such that the C profile does not colonize the domain by t = 20.Results in D. Comparing results in D, as we might anticipate. However, the summary of the time evolution of L(t) and f(t) in D is insufficient for colonization to occur by t = 20. In contrast, the results in D. This time we see that the front reaches L(t), and we have full colonization after t ≈ 16.Results in t ≤ 20. We present a second set of results, in C = 1 for 0 ≤ x < 0.2 and C = 0 for 0.2 ≤ x ≤ 1, whereas the initial condition in C = 1 for 0 ≤ x < 0.75 and C = 0 for 0.75 ≤ x ≤ 1. The situation in t = 20 whereas the situation in t ≈ 14. A third set of results, in k. For k = 0.105, profiles in t = 20. In contrast, when we increase the production to k = 1.705, profiles in t ≈ 20.To further explore the competition between various processes in the model we compare some additional exact and numerical solutions in D, k and the growth rate, b, could affect whether or not the system colonized within a specified time interval.Although all results presented in C, ever reaches the boundary, x = L(t), by some threshold time t★. To explore this we must examine the quantity C(L(t★), t★) by substituting x = L(t★) and t = t★ into C(L(t★), t★) > ɛ, in which case we have successful colonization by time t★. Alternatively, if C(L(t★), t★) < ɛ, we have unsuccessful colonization by time t★. Here ɛ is some user-defined small tolerance. For example, to interpret the results in ɛ = 0.01 to determine the position of the front, and this choice of ɛ could be used to make a distinction between successful and unsuccessful colonization in other applications.Now that we have derived exact solutions describing a linear reaction–diffusion process on a growing domain we can use the new solution to write down a condition which can be used to distinguish between situations which lead to successful colonization from situations which lead to unsuccessful colonization. For our initial condition, given by ɛ. If we choose a slightly larger tolerance, say ɛ = 0.015, our conclusions about the results in ɛ = 0.015, our conclusion about the situations in x = L(t). Instead of reaching x = L(t) by t ≈ 16 with ɛ = 0.01, when we choose ɛ = 0.015, colonization does not occur until t ≈ 60.We now demonstrate how our results are sensitive to the choice of x < L(t), and we present specific results for exponentially-elongating domains, L(t) = L(0)exp(αt), with α > 0, and linearly-elongating domains, L(t) = L(0) + bt, with b > 0. While our approach is relevant for a general class of initial conditions, motivated by Landman et al.’s previous work [C to be localised near one boundary of the domain. Then, using our exact solution, we explore whether the density profile evolves such that it can overcome the domain growth and colonize the entire length of the domain by reaching the other boundary, within some particular time interval.In this work we derive an exact solution for a linear reaction–diffusion PDE on a uniformly growing domain. Our framework is relevant for a general class of uniformly growing domains, 0 < ous work , we consIt is interesting to note, and discuss, several differences between the solution of the linear reaction–diffusion PDE on a non-growing domain, given by nπ)2T), it is tempting to think that we may truncate the infinite series after one or two terms to obtain a useful approximation to the exact solution when T becomes sufficiently large. This kind of approximation is possible in the non-growing case where, as we previously noted, when t → ∞, we have T → ∞. However, different behavior occurs in the growing domain solutions. In particular, for the exponentially-growing domain, as t → ∞ we have t → ∞ we have t. Indeed, we explored whether it is possible to approximate the exact solutions in t than reported here, such as t = 100.Such approximations are well-known to be accurate after a sufficiently long period of time , 27. OneSince we rely on separation of variables and superposition to construct our exact solution, one of the key limitations of our strategy is that the exact solution applies only to a linear reaction–diffusion process. While many reaction–diffusion models are inherently nonlinear, there is a real practical value in the use of linear models, since linear PDE models are often used to approximate the solution of related nonlinear PDE models . For exaC, in principle our solution strategy could also be applied to multispecies reaction–diffusion processes involving several dependent variables, C1, C2, C3, …, that are coupled through a linear reaction network [There are several ways in which the exact solution strategy presented in this work could be extended. Although we have only considered a single species reaction–diffusion processes with one dependent variable,  network , 37. We  network . After t


Arabidopsis protein FPA controls flowering time by regulating the alternative 3′-end processing of the FLOWERING LOCUS (FLC) antisense RNA. FPA belongs to the split ends (SPEN) family of proteins, which contain N-terminal RNA recognition motifs (RRMs) and a SPEN paralog and ortholog C-terminal (SPOC) domain. The SPOC domain is highly conserved among FPA homologs in plants, but the conservation with the domain in other SPEN proteins is much lower. We have determined the crystal structure of Arabidopsis thaliana FPA SPOC domain at 2.7 Å resolution. The overall structure is similar to that of the SPOC domain in human SMRT/HDAC1 Associated Repressor Protein (SHARP), although there are also substantial conformational differences between them. Structural and sequence analyses identify a surface patch that is conserved among plant FPA homologs. Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation or the functions of the FPA SPOC domain cannot be disrupted by the combination of mutations, in contrast to observations with the SHARP SPOC domain.The  Eukaryotic messenger RNAs (mRNAs) are made as precursors through transcription by RNA polymerase II (Pol II), and these primary transcripts undergo extensive processing, including 3′-end cleavage and polyadenylation ,2,3,4. IRecently, the split ends (SPEN) family of proteins was identified as RNA binding proteins that regulate alternative 3′-end cleavage and polyadenylation . They arArabidopsis thaliana and other plants, was found to regulate the 3′-end alternative cleavage and polyadenylation of the antisense RNAs of FLOWERING LOCUS (FLC), a flowering repressor gene . Th. Th26]. in vivo. We mutated two residues, Arg477 and Tyr515, of the surface patch, which are also conserved in the SHARP SPOC domain . The recombinant protein, with an N-terminal hexa-histidine tag, was over-expressed in E. coli BL21 Star (DE3) cells (Novagen), which were induced with 0.4 mM IPTG and allowed to grow at 20°C for 14–18 h. The soluble protein was purified by nickel-charged immobilized-metal affinity chromatography and gel filtration chromatography. The purified protein was concentrated and stored at –80°C in a buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl, 10 mM DTT and 5% (v/v) glycerol. The His-tag was not removed for crystallization.The SPOC domain (residue 433–565) of E. coli B834(DE3) strain using LeMaster media . Signal was detected using SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Scientific).Total protein samples were prepared using extraction buffer containing: 40 mM Tris-HCl, pH 6.8; 0.1 mM EDTA, pH 8.0; 8 M urea; 1.43 M β-mercaptoethanol, 7% v/v Complete Protease Inhibitors (Roche) and 5 mM PMSF. Equal volumes of samples were separated on 8% SDS-PAGE. Proteins were transferred onto Protran nitrocellulose transfer membrane (Whatman) using wet Criterion blotter system (BioRad). The transfer was performed at room temperature for two hours at a stable voltage of 70 V. Membrane was blocked in 3% (w/v) Milk in TBS for 1h at room temperature followed by overnight incubation with anti-FPA antibody   plants fpa-8 and fpa-8 mutants expressing FPA::FPAwt using poly(A)+ purified mRNAs. A probe corresponding to the 5′UTR region of FPA mRNA was used to detect FPA specific mRNAs. RNA size (kb) marker (Ambion). TUBULIN was detected as an internal control. Proximally and distally polyadenylated FPA transcripts are marked with arrows. The ratio of distal:proximal polyadenylated forms is given under each lane. (C). RNA gel blot analysis of FLC transcript in FPA::FPAwt plants. Poly(A)+ RNA was isolated from Col-0, fpa-8 and trangenic lines expressing FPA::FPAwt in an fpa-8 mutant background and detected with a probe recognizing FLC sequence. TUBULIN was detected as a control.(A). FPA protein level in antibody . TUBULIN(PDF)Click here for additional data file.S2 Figfpa-8, 35S::FPA:YFP, FPA::FPA R477A (A) and FPA::FPA Y515A (B) plants were separated on SDS-PAGE. Western blot analysis was performed with FPA antibody. For the loading control TUBULIN antibody was used.Proteins isolated from wild type Col-0, (PDF)Click here for additional data file.S3 Figfpa-8, 35S::FPA:YFP, FPA::FPA R477AY515A plants were separated on SDS-PAGE. Western blot analysis was performed with FPA antibody. For the loading control TUBULIN antibody was used.Proteins isolated from wild type Col-0, (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


The vertebrate-specific proteins astrotactin-1 and 2 (ASTN-1 and ASTN-2) are integral membrane perforin-like proteins known to play critical roles in neurodevelopment, while ASTN-2 has been linked to the planar cell polarity pathway in hair cells. Genetic variations associated with them are linked to a variety of neurodevelopmental disorders and other neurological pathologies, including an advanced onset of Alzheimer's disease. Here we present the structure of the majority endosomal region of ASTN-2, showing it to consist of a unique combination of polypeptide folds: a perforin-like domain, a minimal epidermal growth factor-like module, a unique form of fibronectin type III domain and an annexin-like domain. The perforin-like domain differs from that of other members of the membrane attack complex-perforin (MACPF) protein family in ways that suggest ASTN-2 does not form pores. Structural and biophysical data show that ASTN-2 (but not ASTN-1) binds inositol triphosphates, suggesting a mechanism for membrane recognition or secondary messenger regulation of its activity. The annexin-like domain is closest in fold to repeat three of human annexin V and similarly binds calcium, and yet shares no sequence homology with it. Overall, our structure provides the first atomic-resolution description of a MACPF protein involved in development, while highlighting distinctive features of ASTN-2 responsible for its activity. They reThe only basis for MACPF/CDC activity properly established so far is the oligomerization of many subunits in order to generate a pore-forming complex –19. BindAs a subset of MACPF/CDC proteins, we define PLPs as being proteins which have been identified by sequence homology as sharing common evolutionary ancestry with perforin-1 ,31. Thesin vitro [ASTN-1 was first discovered because antibodies against it block neuron–glial interactions in vitro ; it was in vitro . ASTN-1 in vitro –48, and in vitro  (electro+ endosomes (as well as in non-RhoB+ vesicles) [ASTN-2 is most highly expressed in the cerebellum but also in the hippocampus, cortex and olfactory bulb ,49. The esicles) . As cereesicles) . Intraceesicles) ,40. Thisesicles)   , by copyin vitro in a calcium-independent way with multiple regions of each protein apparently contributing to their interface [b) is complemented by an N-terminal cytosolic domain suspended between two transmembrane α-helices [a). The C-terminus of each protein is known to be detectable on the outside of cells in which they have been expressed and this defines their membrane orientation  [figure 1a; electronic supplementary material, figure S1b). Thus, the endosomal regions of ASTN-2 comprise a smaller N-terminal domain and a larger C-terminal domain whose structure we report here and which we refer to as its endodomain. The ASTN-2 endodomain is exactly equivalent to the cell surface-exposed ectodomain of ASTN-1; and it is the first in a class of integral membrane PLPs (which includes perforin-2) to have its structure determined, though perforin-2 differs from ASTN-2 (and ASTN-1) in having only a single transmembrane helix [ASTN-1 and ASTN-2 have been shown to interact directly with each other nterface . Both AS-helices  (figure ure S1b) . We suggne helix ,58.FiguIn describing here the structure of the endodomain of ASTN-2, we provide a framework for understanding its role in cell migration and tissue development. We describe its unique combination of a canonical MACPF/CDC domain with a minimal epidermal growth factor (EGF)-like repeat (EGF-4), a previously unobserved type of fibronectin type III (Fn(III)) domain in which an additional two β-strands are folded across the core, and an annexin-like domain. We have determined structures of the endodomain at pH 7.5 and pH 5 and of the MACPF/CDC domain alone at pH 4, and find the regions of ASTN-2 resolved to be remarkably insensitive to acidification. We have shown that the ASTN-2 endodomain binds inositol phosphates at a binding pocket located between the EGF-4 and Fn(III) domains and that the annexin-like domain binds calcium, as might be expected. ASTN-2 appears to be unique to vertebrates and its polypeptide sequence is extremely conserved, with 53% identity between jawless fishes and humans  (electr2.2.1.a) using HEK293 cells. Transient DNA transfection with the pHLsec vector previously described [Figure 1b reports the overall structure of the ASTN-2 endodomain at pH 7.5 and comprising the MACPF/CDC, EGF-4, Fn(III) and annexin-like domains. The structure was solved using single isomorphous replacement with anomalous scattering (SIRAS) with a platinum-soaked derivative . The presence of EGF-2 and EGF-3 in the protein that crystallized combined with their invisibility in the crystal structure indicates that they are not important in the folding of the regions of ASTN-2 we do resolve. This is supported by normal modes analysis of the ASTN-2 endodomain which suggests that it constitutes a remarkably tightly folded unit, and by the equivalent interaction of ASTN-2 with inositol phosphates with and without these domains . In normal modes analysis, the first six modes are rigid-body translational and rotational modes in three dimensions, so the first non-trivial mode is mode 7. Normal modes 7–11 indicate only breathing motions within the endodomain, and highlight the combination of the MACPF and Fn(III) as a particularly robust region of the structure . We also solved the structure of the endodomain at pH 5 and of the MACPF/CDC domain alone at pH 4 ; these structures are compared with the neutral pH endodomain structure (figure 1b) below.We expressed and purified an ASTN-2 construct running from its second EGF-like repeat to the C-terminus (underlined in figure 1escribed  enabled 2.2.10-helix (η1) and which packs against the Fn(III) domain interface . The equivalent region in perforin-1 contains 55 residues and two substantial α-helices; and in perfringolysin  approximately 33 residues and three short multi-turn helices . The loop 1 region of the ASTN-2 structure is therefore an outlier with respect to MACPF/CDC proteins as a whole, due to its brevity. The second loop  consists of 54 residues and contains two substantial α-helices (α3 and α4). Loop 2 is solvent exposed in contrast with the packing of loop 1 against the Fn(III) domain. The equivalent region to ASTN-2 loop 2 in perforin-1 contains 59 residues  and in perfringolysin 30 residues (three helices). Thus, while loop 1 of the ASTN-2 MACPF/CDC domain is unusually short, loop 2 is similar in length to that of perforin-1 and quite a bit longer than that of the CDCs. Most critically, however, the two MACPF domain loops are mismatched in length.The N-terminus of the endodomain structure is formed by the canonical MACPF domain consisting of a central, broken, antiparallel four-stranded β-sheet that possesses a distinctive central approximately 90° bend. All MACPF/CDC proteins that form pores have to date been shown to do so using a β-stranded barrel in which each subunit contributes two β-strand hairpins to generate a structure similar to a bacterial outer membrane porin, but formed by multiple subunits rather than a single polypeptide chain ,61. Thisa). This is most striking in loop 1, but even loop 2 is more hydrophobic than the equivalent regions of perforin-1 and perfringolysin, and rather than a hydrophobic–hydrophilic pattern alternating every residue, the alteration seems to be more two-by-two. If loop 2 inserts into a membrane to form a pore this suggests it could do so as a helical structure rather than in the form of a β-hairpin—but whether ASTN-2 really forms membrane pores is unknown.Firstly, the shortened ASTN-2 loop 1, packed against the Fn(III) repeat, is too short to span a bilayer within a β-barrel structure such as the CDCs, perforin and some PLPs are known to form ,61,63,64Secondly, while the exposed, unabbreviated ASTN-2 loop 2 is similar in length to the equivalent region of perforin-1, its large mismatch in length with loop 1 (ASTN-2 loop 1 is more than 30 residues shorter than loop 2) means that they could not pair in β-barrel formation. Yet for each other member of the MACPF/CDC superfamily, the two equivalent loops are similar in length to each other; this mismatch also suggests that ASTN-2 may not be a pore-forming protein, at least not in the way other MACPF/CD protein family members are.10 helical turn (η2) and a final α-helix  which are paired structurally with α1 and α2 to form a head region from which the MACPF domain β-strands extend. This head is linked to the Fn(III) domain by the EGF-4 domain module, which contains β10 and β11 and matches best to the integrin β3-subunit PSI domain EGF-like folds according to the SCOP database (family g.3.11.6) ; the two disulfide bridges in the EGF-4 domain superpose perfectly with those in the integrin β3 subunit PSI domain [b). The EGF-like repeats compared were those from perforin-1, C8α and C8β; the PSI domain of integrin subunit β3; and an EGF repeat from P-selectin as well as EGF-4 from ASTN-2. As shown, it seems that the EGF repeat found in perforin-1 itself and EGF-4 in ASTN-2 share a common line of ancestry, separate from the EGF repeats found in other immune system MACPFs (C8α and C8β). ASTN-2 also seems to be closer to the common point of origin of itself and perforin-1 and thus perhaps to represent a more ancient structural form; indeed the perforin-1 EGF repeat is rather an extreme structure compared with ASTN-2 EGF-4 and the EGF repeats from C8α and C8β.The MACPF domain is completed by a single-turn 3I domain . EGF repThe junction between the EGF-4 and Fn(III) domains contains density which is not part of the ASTN-2 polypeptide and which can plausibly be modelled as an inositol triphosphate bound within a surface cavity. The binding mode of this species and mutational and binding data which support its identification with a phosphorylated inositol are described below.2.3.b,c) (RMSDs 0.23 Å (pH 5) and 1.52 Å (pH 4)). The region of greatest variation at pH 4 is loop 1 between β3 and β4, where 310 helix η1 shifts approximately 2 Å towards the Fn(III) domain on acidification. Yet overall these regions of ASTN-2 seem remarkably insensitive to pH suggesting that, if pH is a triggering factor in ASTN-2 activity as it moves through the endolysosomal system, the sensor is not in these parts of the structure.The movement of ASTN-2 through the endolysosomal system suggests that its activity might be regulated by pH. In an attempt to investigate the effect of reduced pH on the ASTN-2 structure, we obtained crystals of constructs consisting of EGF-3 to the C-terminus (residues 649–1288) at pH 5 and of EGF-3 plus the MACPF domain at pH 4 (residues 649–984), though in neither case was EGF-3 resolved in the resulting structures. The conformations of the lower pH forms of ASTN-2 are very similar to the equivalent regions of the neutral pH structure . The loop between the hairpin strands projects towards EGF-4 and towards the inositol phosphate binding site, suggesting that this point in the structure may be a key point in its conformational regulation.The Fn(III) domain  follows directly on from EGF-4 and has the unexpected feature that it is complemented by a 54-residue β-hairpin from the ASTN-2 C-terminus (β20–β21) that folds across the Fn(III) β-sandwich, clasping the edges of both sheets. As such it seems to lock the C-terminus back to the centre of the endodomain, rigidifying the structure  is occupied by a high-mannose glycan sidechain protected from glucosidase trimming that appears to assist in the stabilization of the MACPF/EGF-4/Fn(III) domains cassette . The principal contacts made by this sugar sidechain with EGF-4 and the Fn(III) surfaces are formed by the two N-acetyl galactosamine moieties and terminal mannoses of the glycan sidechain branches. The role of the glycan sidechain in stabilizing the MACPF/EGF-4/Fn(III) domains cassette is indicated by the effects of a N > Q mutation knocking out its sequon. Such a construct is not secreted during mammalian cell expression, suggesting it is misfolded.The MACPF domain contains two glycosylation sites; one at the top of the domain (N719) was trimmed back by EndoF1  to a single 2.5.b). The residues involved in the interactions are mostly charged  though Phe800 projects towards the domain interface (a) and even of a significant conformational change caused by movement of the MACPF away from the Fn(III) domain which might be enabled if the C-terminal β-hairpin ungrasps the Fn(III) domain. In an attempt to probe the functional significance of the MACPF/Fn(III) interface, we introduced a disulfide bond to lock the two together (Asp791Cys and Thr1134Cys mutations); this resulted in a form of the protein which was secreted poorly from HEK293T cells, although SAXS data suggest that the protein that is secreted is folded like wild-type ASTN-2 .The interface between the MACPF and Fn(III) domains is formed by the truncated loop 1 between strands β3 and β4 of the MACPF domain b. The renterface . Despitec). It is certainly therefore a bona fide Fn(III) domain despite its unique complementation with the β20–β21 hairpin, for which there are no currently described structural parallels.Structural phylogenetic analysis of the Fn(III) domain excluding the long C-terminal β-hairpin (β20–β21) indicates that this domain is closest in structure to Fn(III) repeat 8 from human fibronectin itself, and occupies a single branch with this domain and a Fn(III) domain from tenascin . Annexin itself consists of four tandem repeats of a common fold [b). For example, with the annexin V domains and the ASTN-2 annexin-like domain all oriented equivalently, the most-similar annexin V domain 3 presents a highly positively charged face whereas ASTN-2 presents a mixed face with regions of positive and negative charge.The annexin-like domain is positioned between the Fn(III) domain and the long β-hairpin folded across it (β20–β21) but, functionally, forms the tip of the ASTN-2 endodomain. An annexin-like domain is an unexpected feature of the ASTN-2 structure (α6–α11) which despite undetectable sequence homology with annexin itself superimposes very well on an annexin single repeat (figure 4mon fold  and it ib). This effect is confirmed by mutation of the putative calcium-binding residues within the annexin-like domain, which knock out the stabilizing effect of the presence of calcium (b). Annexin-like domains are known for their capacity to remodel membranes, triggered by calcium binding, and have also been suggested to be involved in the formation of pores in membranes [Like annexin itself, the ASTN-2 annexin-like domain binds calcium, resulting in a stabilization of its structure as evidenced by an increased temperature of melting b. This e calcium b. Annexiembranes —both are2.7.3 and InsP2 in a surface plasmon resonance (SPR)-based experiment (Kd = 30 µM) (a), ASTN-1 does not . A comparison of the sequences of ASTN-2 and ASTN-1 in this region indicated that an arginine apparently interacting with InsP3 in the ASTN-2 crystal structure is replaced by a threonine in ASTN-1. Mutation of ASTN-2 Arg1124 to Thr resulted in a form of the protein which, like ASTN-1, did not bind InsP2 and had much-reduced affinity for InsP3  to demonstrate that ASTN-2 makes a specific choice of InsP3 over mannose-6-phosphate in our hands. We do not, however, know the biological significance of this interaction—which could be either that ASTN-2 binds phosphorylated phosphatidylinositol lipid species, or that it binds to inositol phosphate second messengers, or to another phosphorylated or similarly modified carbon ring system. The location of the binding pocket would permit either or both kinds of phosphorylated inositol species to be bound, though we are not aware of free inositol phosphates being found in either the extracellular or endosomal compartments.As mentioned above, the crystal structure contains density consistent with an inositol triphosphate species being bound in a shallow pocket formed by loops from EGF-4 and the β-hairpin slung across the Fn(III) domain. Initially, we identified difference density of unknown origin in this area which could be modelled as an inositol triphosphate, but faced a need to demonstrate its identity. We were assisted here by the observation that although ASTN-2 interacts with InsP3,4,5)P3 . It is p3.Drosophila embryo [Here we have described the structure of a major portion of ASTN-2, the first structure from an integral membrane MACPF/CDC domain-containing protein. ASTN-2 is closely related to not only ASTN-1 but also the BRINP proteins which, similarly, function in neural cell migration and guidance. Other integral membrane MACPF/CDC proteins include perforin-2, a macrophage-specific form of perforin involved in bacterial killing ,58, and a embryo ,68. The a embryo . We havea embryo .4.4.1.lustal Omega and displayed using Espript 3 [onSurf [c).The full-length cDNA clone of Human ASTN-2  was obtained from Source Bioscience (UK) and the expression constructs reported here are all based on the pHLsec vector . For prospript 3  (electro [onSurf  for plot4.2.The expression constructs were transiently transfected into HEK293T cells using polyethylenimine (PEI) as described before . All the−1, flash-frozen in liquid nitrogen and stored at −80°C until further use.The media containing the secreted recombinant proteins were harvested 5–6 days after transfection, then filtered through 0.22 µm filters and dialysed against phosphate-buffered saline (pH 7.4) overnight before loading onto a pre-equilibrated HisTrap HP column  in a cold room. The column with proteins bound was washed with 10 column volumes of Tris buffer (20 mM Tris–HCl pH 7.5 500 mM NaCl) before being eluted with 10–500 mM imidazole with a gradient concentration in the same Tris buffer. GST-3C was added into the eluates to remove the C-terminal monoVenus-8xhis where necessary. The glycan chains of proteins for crystallization were trimmed using Endo-F1. Subsequently, the eluates were buffer-exchanged into low salt buffer  and loaded onto a pre-equilibrated Q column  and eluted with the same buffer containing a higher concentration of NaCl. This step efficiently removed albumin contamination. Finally, proteins eluted from the Q column were applied to a size-exclusion chromatography column  and homogeneous proteins were pooled together, concentrated to about 10 mg ml4.3.601-1288, 200 nl sitting drops of protein solutions  were mixed with 100 nl of precipitant  and equilibrated against 90 µl of crystallization condition. The small needle-like crystals usually appeared after a week and microseeding with the needle-like crystals yielded reasonably-sized crystals [649-984, the protein was concentrated to 15 mg ml−1 and crystallized in 0.1 M citric buffer, pH 4.0–4.5, 20% PEG 1000. ASTN-2649-1288 was crystallized in 0.1 M MES, pH 5.0–6.0, 5% PEG 6000. Platinum derivative crystals of ASTN2601-1288 were prepared by soaking the crystals with concentrated PtCl4 for 1 h; 25% (vol/vol) glycerol prepared in mother liquor was used as cryoprotectant for both native and platinum-soaked crystals, which were subsequently flash-frozen in liquid nitrogen. Diffraction data were collected at 100 K at Diamond Light Source  on beamlines I02, I03, I04, I04-1 and I24. Although most of the crystals diffracted poorly with a high-resolution limit up to 3.6 Å, a native dataset with higher resolution (3.16 Å) was eventually achieved by screening a number of crystals. An inverse-beam data collection strategy was employed to accurately record the anomalous signal from platinum derivative crystals. Diffraction data were indexed, integrated and scaled using the Xia2 pipeline [Crystallization screening was carried out using commercially available crystallization reagents by vapour diffusion methods in sitting drop format in 96-well plates. For crystallization of ASTN2crystals . For crypipeline .4.4.. The resolution cut-off was set to 6.5 Å and solvent content to 0.6 to obtain initial phases. Density modification with non-crystallographic symmetry (NCS) averaging and phase extension to the full resolution of the native dataset resulted in an interpretable map by RESOLVE [oot [oot and refinement in refmac5 and PHENIX. The crystal structure of the MACPF domain C-terminus at pH 5 and of the MACPF domain alone at pH 4 were solved by molecular replacement using copy A of the structure solved at pH 7.5 in PHASER and refinement was carried out in PHENIX [olprobity software [The structure of ASTN-2 endodomain was solved by single isomorphous replacement with anomalous scattering (SIRAS). A high-redundancy platinum derivative dataset was achieved by combining 21 datasets selected from seven crystals. The Pt sites were initially identified using HKL2MAP  with SHE RESOLVE . A polyaLVE [oot , followeLVE [oot  with extn PHENIX . Surfacen PHENIX . The crysoftware . Data co4.5.2 (C-35B6a), PtdInsP2 (C-45B6a), PtdInsP3 (C-39B6a) and InsP4 (Q-1345) were purchased from Echelon Biosciences. To immobilize the biotinylated inositol phosphate onto the sensor chip, a BIAcore CM5 chip  was first derivatized with streptavidin following the manufacturer's instructions, and inositol phosphates were then injected on to channels 2 and 4 of the biosensor surface, leaving channels 1 and 3 as empty controls. The analyte with twofold serial dilutions was applied at a flow rate of 20 μl min−1 for 180 s followed by 300 s of dissociation time. The biosensor chip was regenerated by 0.1% SDS after each running cycle. To perform the competition assay, the analyte (ASTN-2701-1288) was incubated with a 10-fold molar concentration of InsP4 and mannose-6-phosphate for 45 min, respectively, before being serially diluted in running buffer. The data were fit with the 1 : 1 Langmuir adsorption model (B = BmaxC/(Kd + C), where B is the response of bound analyte and C is the concentration of the analyte in the sample) to calculate the dissociation constant (Kd) using BIAcore BIAanalysis software.The SPR experiments were performed using a Biacore T200 machine  at 20°C in 10 mM HEPES, pH 7.5, 150 mM NaCl, PtdInsP4.6.2 (0.13–64 µM) in 10 mM HEPES, pH 7.5, 150 mM NaCl. Fluorescence measurements were recorded from 298 to 372 K with a 1 K temperature increase each cycle. The melting temperature Tm (the midpoint of the unfolding transition) was calculated from the melting curve.Thermofluor experiments were conducted using a real-time PCR machine . Identic4.7.−1 < q < 0.45 Å−1, where q = 4πsin(θ)/λ and 2θ is the scattering angle. SAXS datasets for WT ASTN-2 were collected at four concentrations: 4.92 mg ml−1, 2.49 mg ml−1, 1.27 mg ml−1 and 1 mg ml−1, and the disulfide bond locking mutant was measured at 1 mg ml−1. Eighteen frames of measurements were recorded and the frames without radiation damage were averaged. The scattering intensity from buffer alone was subtracted from the averaged data to obtain the protein scattering in solution. Data reduction was carried out using the ATSAS package [Rg) was calculated from a Guinier plot using AutoRg [P(r) was calculated in GNOM [−1 < q < 0.15 Å−1). An ab initio model was then calculated from the P(r) function using dammif and dammin [hyre2 [SAXS data were collected on B21 at the Diamond Light Source . The measurements were carried out at 293 K in 10 mM HEPES, pH 7.5, 150 mM NaCl buffer with a momentum transfer range of 0.004 Å package . For wilg AutoRg . Particl in GNOM  using thd dammin . EGF-2 an [hyre2  using tan [hyre2 ). The cr4.8.−1 and using absorbance measurements at 280 nm. Experiments were performed at 20°C, taking sample distribution scans every 6 min. Data were analysed using SEDFIT software using the c method of interpretation to generate sample distributions in s (sedimentation coefficient) without assuming a particular number of species [roFit .Sedimentation velocity experiments were performed using a Beckman Optima XL-I analytical ultracentrifuge equipped with absorbance and interference optics. Double-sector 3 mm centrepieces were used with protein samples at 1 mg ml species . The res4.9.Superimposition of homologous protein structures was performed using SHP  as previ4.10.elNémo [himera [Normal modes analysis was performed using the online server elNémo . The vib [himera .


C) shares overall sequence similarity with the translational GTPase (trGTPase) Hbs1, but whether Ski7 has retained the properties of a trGTPase is unclear. Here, we report the high-resolution structures of Ski7C bound to either intact guanosine triphosphate (GTP) or guanosine diphosphate-Pi. The individual domains of Ski7C adopt the conformation characteristic of active trGTPases. Furthermore, the nucleotide-binding site of Ski7C shares similar features compared with active trGTPases, notably the presence of a characteristic monovalent cation. However, a suboptimal polar residue at the putative catalytic site and an unusual polar residue that interacts with the γ-phosphate of GTP distinguish Ski7 from other trGTPases, suggesting it might function rather as a GTP-binding protein than as a GTP-hydrolyzing enzyme.Ski7 is a cofactor of the cytoplasmic exosome in budding yeast, functioning in both mRNA turnover and non-stop decay (NSD), a surveillance pathway that degrades faulty mRNAs lacking a stop codon. The C-terminal region of Ski7  and adopts the overall structural features of ribosome-associated GTPases. The decay of cytoplasmic mRNAs regulates the output of eukaryotic gene expression in terms of both quantity and quality. In general, mRNA turnover modulates the abundance of normal transcripts in the cell and thereby the quantity of the proteins they encode -bound conformation . Ski7C was incubated with either GTP or GDP for crystallization trials. We obtained crystals of Ski7C in the presence of GDP and magnesium ions using inorganic phosphate as precipitating agent. We solved the structure with a selenomethionine-based single-wavelength anomalous dispersion experiment and refined it at 2.2-Å resolution to Rfree of 24.6%, Rwork of 21.7%, and good stereochemistry (C-GDP-Pi) includes most of the Ski7C polypeptide chain (with the exception of short disordered loop regions) and also includes GDP, a molecule of inorganic phosphate (Pi) and an Mg2+ ion. Manganese was soaked into the crystal prior to data collection to substitute Mg2+ and to unambiguously identify the position of the divalent cation in the electron density using anomalous scattering  wedged in between. The βA and βB strands are not adjacent in the sequence but form a small β sheet that interacts on one side with the barrel of domain II and on the other side with the barrel of domain III (Ski7 domains A. Domain domains B. Domain domains B. The β main III B. As obsmain III .0) packs against domain II and against the linker that connects the two domains. Second, a loop connecting strands β4 and β5 of domain II interacts with the side of the G domain. The trGTPases Hbs1, eRF3, EFTu, and eIF5B share a similar sequel of globular domains . In Ski7C-GDP-Pi, the inorganic phosphate is clearly separated from the β-phosphate of the GDP (with a phosphate-phosphate distance of 4.2 Å)  or engages the small molecules supplied at high concentrations with the crystallization buffer (i.e. GDP and inorganic phosphate) to mimic a GTP-bound state.We compared the nucleotide-binding site in the G domain of Ski7 with that of eIF5B, a trGTPase involved in subunit joining and whose G domain structure has been characterized at high resolution in several apo and nucleotide-bound states  Figure A. G domaructures A and 3B.IF5B-GDP C. The anSki7) packs against one side of the guanine base, at a position usually engaged in hydrophobic stacking interactions  coordinate a monovalent cation (either K+ or Na+) (Ski7 and the main-chain carbonyl of Gly331Ski7 (from switch I)  . The pre or Na+) , althoug or Na+) . In the witch I) A.Ski7 in switch II at the corresponding position of His598eIF5B. This active site histidine residue is conserved in trGTPases, and in the presence of the ribosome and an activating cofactor it re-orients its side chain to stabilize or position the catalytic water  of GMPCP-bound Ski7C (44.0°C) in comparison with GMPPNP-bound protein (47.6°C), indicating a preference for the GTP-bound state  and an additional polar residue near the γ-phosphate of GTP . We envision two possible scenarios of how Ski7C might function. In one scenario, Ski7 could be a trGTPase-like protein in the full sense despite the possession of non-canonical active site residues. In this case, it would be capable of GTP hydrolysis and of switching between active and inactive conformations in the context of the ribosome and of an appropriate cofactor. Such a cofactor would be expected to function analogously to Dom34 and eRF1 but in addition would likely have to stabilize the GDP-bound form of the protein. Alternatively, in a second scenario, Ski7 could have evolved as a GTP-binding protein, i.e. as a pseudo-trGTPase. Here, a Ski7 cofactor would be expected to regulate the interaction with the ribosome with an altogether different mechanism, which does not rely on a conformational switch of the Ski7C domains. Finally, Ski7 might even function without any cofactor. Although the biochemical data available so far would favor the concept of Ski7 as a stable GTP binder, only the identification of the cofactors that might regulate the function of the Ski7C in NSD will allow for clarification of the mechanisms.trGTPases have characteristic features. At the biochemical level, they have low hydrolysis activity in isolation and require the ribosome and a specific cofactor for robust activity. At the structural level, they have a characteristic domain composition, a characteristic conformation in the active GTP-bound state, and a characteristic active site with a monovalent cation in addition to the universal divalent cation present in other GTPases. The work we report here indicates that the C-terminal G domain of C (254–747) was expressed as a fusion protein with an N-terminal thioredoxin polypeptide cleavable with Prescission Protease. The expression was carried out in E. coli BL21 Gold pLyS cells (Stratagene) using Terrific Broth and inducing with 0.1 mM isopropyl β-D-1-thiogalactopyranoside at 18°C for 16 hr. Expression of selenomethionine-derivatized protein was carried out in minimal medium upon addition of amino acids and 50 mg/l selenomethionine prior to induction. Ski7C was purified using a Nickel affinity step  followed by cleavage of the tag with Prescission Protease, purification on heparin resin  and a final size exclusion chromatography step  using 20 mM HEPES (pH 7.5), 100 mM NaCl.Ski7C was crystallized at a concentration of 7 mg/ml with 5 mM nucleotide (GDP or GTP), 1 mM MgCl2, and 5 mM Tris(2-carboxyethyl)phosphine. Crystals grew in 100 mM HEPES (pH 7.0), 700 mM NaH2PO4/K2HPO4, and 3% ethylene glycol. The crystals were cryo-protected in the presence of 25% ethylene glycol and flash-cooled in liquid nitrogen. In the case of the manganese-containing crystals, 10 mM MnCl2 was included in the cryo-protectant. All X-ray diffraction data were collected at 100 K at the Swiss Light Source (SLS) synchrotron in Villigen, Switzerland. The single anomalous diffraction experiment was performed by collecting a dataset at the peak wavelength of the selenium K edge and another dataset was collected from the same crystal at the manganese K edge. The data were processed and scaled with XDS (1 containing one molecule in the asymmetric unit. The data processing statistics are summarized in Ski7with XDS . The cryThe structure was determined and analyzed by E.K. Activity assays were prepared by A.S. Experiments were performed under the direction of E.C and R.G. E.K and E.C wrote the article.


Single crystal silicon (SCS) diaphragms are widely used as pressure sensitive elements in micromachined pressure sensors. However, for harsh environments applications, pure silicon diaphragms are hardly used because of the deterioration of SCS in both electrical and mechanical properties. To survive at the elevated temperature, the silicon structures must work in combination with other advanced materials, such as silicon carbide (SiC) or silicon on insulator (SOI), for improved performance and reduced cost. Hence, in order to extend the operating temperatures of existing SCS microstructures, this work investigates the mechanical behavior of pressurized SCS diaphragms at high temperatures. A model was developed to predict the plastic deformation of SCS diaphragms and was verified by the experiments. The evolution of the deformation was obtained by studying the surface profiles at different anneal stages. The slow continuous deformation was considered as creep for the diaphragms with a radius of 2.5 mm at 600 °C. The occurrence of plastic deformation was successfully predicted by the model and was observed at the operating temperature of 800 °C and 900 °C, respectively. For exaombustor . et al. used the SiC diaphragm to form a capacitive pressure sensor [et al. implemented the SiC piezoresistors on silicon substrate [et al. used the buried oxide layer to isolate the piezoresistors from the substrate and from each other [In order to be used in high temperature applications, silicon has been combined with other advanced materials, such as silicon carbide (SiC) or silicon on insulator (SOI). SiC exhibits good mechanical stability and stable electronic properties at temperatures up to 600 °C . When the sensor . Wu et aubstrate . The perch other . The silet al. previously performed bulge test experiments on micromachined silicon membranes with the temperatures ranging from 900 °C to 1150 °C [The mechanical behavior of the pressurized silicon diaphragm is partly dependent on the material properties of SCS. The yield strength in silicon has been studied by the conventional bending test and tensile test ,12,13,14 1150 °C . HoweverIn order to extend the operating temperatures of existing SCS microstructures, this work investigates the mechanical behavior of pressurized SCS diaphragms at the temperature range of 600 °C–900 °C. Firstly, the design of the test samples and the fabrication process are presented, followed by the details of thermal treatment and profile measurement. Based on the reported critical resolved shear stress of silicon, the occurrence of the plastic deformation is then predicted using the orthotropic properties of silicon by the finite element method. After that, the maximum deflections for the diaphragms with radii from 0.5 mm to 2.5 mm are reported with respect to different annealing temperatures. The slip bands were observed on the surface of the plastically deformed diaphragm by the microscope. Finally, based on the evolution of the measured profiles, the size effect and the temperature effect on the diaphragm behavior are discussed. The testing sample of the SCS diaphragm with a sealed cavity is illustrated in The fabrication of the testing samples has been reported in our previous work . The proDue to the pressure difference between the atmosphere and the sealed cavity, the silicon diaphragms deflected toward the substrate once they were made ready for test. The availability of the wet-etching method and the BESOI wafer allowed good control over the diaphragm thickness, as shown in The probable failure modes associated with the silicon microstructures at elevated temperatures are the plastic deformation or creep. The direct observation of the diaphragm deflection during annealing is experimentally very difficult. However, the brittle failure leads to the permanent change of the diaphragm deformation, and can be detected by studying the evolution of the surface profiles with annealing processes. Based on this concept, a series of experiments were conducted to investigate the size effect and the temperature effect on the deformation of the silicon diaphragms. The microfabricated SCS diaphragm was annealed in a furnace filled with nitrogen gas of atmospheric pressure for three different durations  at each temperature tested in this study. In each annealing process, the furnace temperature rose from 20 °C with a ramp-up rate of 5 °C/min. Then, it was maintained at the annealing temperature for a duration specified in The surface profiles of the test samples were measured with a white light interferometer. All the measurements were taken under the atmospheric pressure at a temperature of 20 °C. At room temperature, silicon is brittle and the Young’s modulus is high. Elastic deformation was measured before annealing Stage A, and the magnitude was proportional to the atmospheric pressure. After annealing, as the samples cooled down to the room temperature, the silicon became brittle again. If the diaphragm deflected plastically during annealing, the measured data afterwards would consist of the plastic deformation and the atmosphere induced elastic deformation. Therefore, the high temperature behavior of the diaphragm can be determined by comparing its surface profile tested before and after the different annealing stages. s and the slip direction ls are listed in The plastic deformation in the single crystal silicon was caused by the dislocation slip, which was initiated by the shearing stress resolved on the slip plane in the slip direction. Wherever the acting resolved shear stress obtained from the operating temperature field exceeded the critical value (the yield strength), the crystallographic slip began. Because of the face-centered-cubic structure of silicon, the dislocations glided along <011> directions on the dense atomic planes {111}. There are a total of 12 primary slip systems for SCS. For each slip system s, the crystallographic orientation of the slip plane nsm, and the macroscopic stress tensor, σ [sm is given by:The resolved shear stress on a slip system s is the scalar product between the orientation tensor, ensor, σ :(1)τs=mThe pressure-induced stress in the test samples can be calculated by the aid of COMSOL Multiphysics 3.5a in 3-dimensional domain. Since the test samples were free to expand during high temperature annealing, there was no thermally induced stress. At the same time, the temperature dependence of the elastic properties of silicon was ignored in the modeling because its effect on the stress field is very small compared to the effect of the applied pressure. The resolved shear stress for all the slip systems could then be obtained by applying the Cauchy stress.Due to the orthotropic properties of SCS, the stress field of one slip system is different from that of the other . It was −3 cm/min [The slip of the dislocation began when the resolved shear stress reached the yield strength at the operating temperature. The onset of the plastic deformation could then be predicted simply by comparing the maximum resolved shear stress with the yield strength at each operating temperature, as shown in 3 cm/min . It can The region where the plastic deformation is likely to take place can also be predicted by the FEA model, as highlighted by the red color in For the pressurized SCS diaphragms annealed at 600 °C, the evolution of their averaged maximum deflections at different annealing stage is recorded in For the diaphragms with the radius of 2.5 mm, they showed very different behavior, as indicated by the two samples’ surface profiles shown in When the annealing temperature was increased to 900 °C, neither plastic deformation nor creep effect was evident when the diaphragm radius was 1 mm or smaller, as indicated in For the diaphragm presented in The experimental results reveal that larger diaphragms have a higher risk of plastic deformation. Once the pressure applied on the test sample is determined, the larger the diaphragm radius, and the higher the stresses are induced. As a result, the slip of the dislocations is more likely to be activated once the critical value is achieved. Meanwhile, high temperature adds to the risk of elastic failure. When it comes to creep, raising temperature increases the diffusion rate of the silicon atoms in the pressurized diaphragms and therefore speeds up the creep progress. For plastic deformation, raising temperature decreases the value of the critical resolved shear stress (CRSS). Therefore, the pressurized diaphragms are more likely to deform plastically in a high-temperature environment. The higher the operating temperature, the larger the plastic deformation is. For the diaphragms with a radius of 2.5 mm, the maximum deformation is about 101 µm after annealing at 600 °C for 137 h, while it is about 112 µm after annealing at 800 °C for 31 h, and is about 135 µm after annealing at 900 °C for 21 h. The creep at 600 °C has a low deformation rate. The experimental results and the FEA prediction of the plastic deformation in the silicon diaphragms are compared and illustrated in Because the plastic deformation is a very complicated process, this work is focused on the mechanical aspect of the phenomenon. A series of experiments was performed to investigate the effect of size and temperature on the deformation of the silicon microstructures. The samples for testing were fabricated by the micromachining technology with good control over diaphragm thickness. By comparing the surface profiles obtained before and after a number of annealing processes, the trend of the diaphragm deformation is obtained. The results are fairly good for engineering applications.The occurrence of the plastic deformation was predicted by comparing the resolved shear stress induced by pressure with the yield strength of SCS, which is a valuable criterion for predicting the elastic failure of SCS microstructures in high temperature applications. The prediction was verified by the experimental results. The accuracy of the FEA modelling can be improved by including the exact material properties and experimental conditions. When the plastic deformation can be avoided in the primary design, the creep deformation must be tested if the device is to operate at a temperature over 600 °C for a long period of time. The experimental results provide good reference data for designing the sensing diaphragm for high-temperature pressure sensors. The profile data imply that either increasing the size or raising the temperature adds to the risk of creep and plastic deformation. The diaphragms with smaller dimensions are more likely to survive in high-temperature environments. However, reducing the radius decreases the sensitivity of the diaphragm to pressure. Therefore, the dimension should be decided so that the device lifetime and sensitivity are well balanced. At the same time, the transduction mechanism needs to be chosen carefully so that the signal change caused by the external pressure can be sensed with a desired resolution.


Here we comprehensively analyze sequences, structures and genomic neighborhoods of one of the most widespread groups of such genes that encode proteins containing a predicted nucleotide-binding domain with a Rossmann-like fold, which we denote CARF (CRISPR-associated Rossmann fold). Several CARF protein structures have been determined but functional characterization of these proteins is lacking. The CARF domain is most frequently combined with a C-terminal winged helix-turn-helix DNA-binding domain and “effector” domains most of which are predicted to possess DNase or RNase activity. Divergent CARF domains are also found in RtcR proteins, sigma-54 dependent regulators of the rtc RNA repair operon. CARF genes frequently co-occur with those coding for proteins containing the WYL domain with the Sm-like SH3 β-barrel fold, which is also predicted to bind ligands. CRISPR-Cas and possibly other defense systems are predicted to be transcriptionally regulated by multiple ligand-binding proteins containing WYL and CARF domains which sense modified nucleotides and nucleotide derivatives generated during virus infection. We hypothesize that CARF domains also transmit the signal from the bound ligand to the fused effector domains which attack either alien or self nucleic acids, resulting, respectively, in immunity complementing the CRISPR-Cas action or in dormancy/programmed cell death.CRISPR-Cas adaptive immunity systems of bacteria and archaea insert fragments of virus or plasmid DNA as spacer sequences into CRISPR repeat loci. Processed transcripts encompassing these spacers guide the cleavage of the cognate foreign DNA or RNA. Most CRISPR-Cas loci, in addition to recognized  Sulfolobus islandicus has been demonstrated  code for RNA-dependent self–non-self recognition mechanisms, which are partially analogous eukaryotic RNA interference (RNAi) systems, and serve as an adaptive immunity system against invasive nucleic acids. The CRISPR-Cas system incorporates fragments of virus or plasmid DNA into the CRISPR repeat cassettes and employs the processed transcripts of these spacers as guide RNAs to cleave the cognate foreign DNA or RNA. Recently, the type-II CRISPR systems have been used as biotechnological reagents of targeted mutagenesis, genome editing or gene-inactivation in eukaryotes  fold  that could be assigned to previously identified CARF families [namely COG1517, PF09455, PF09670, PF09659, PF09651, PF09623, PF09002, Csa3  systems  domain proteins are most abundant. Recently, it has been shown that a WYL domain protein (sll7009) is a negative regulator of the I-D CRISPR-Cas system in In terms of domain architectures, WYL domains are most frequently associated with different predicted DNA-binding N-terminal wHTH domains. However, similar to the CARF domains, WYL domains also show fusions to several enzymatic domains  of the VC1899 family (PF9002) are embedded within a novel Type-VII secretion system gene cluster predicted to function as a DNA-transfer agent and additionally encompassing multiple Ter genes that have been implicated in phage restriction , suggesting a strong link of these proteins to CRISPR-Cas  of which only 9 genomes possess neither Multiple lines of evidence from structural analysis and contextual information from domain architectures and gene neighborhoods suggest that the CARF domains are dedicated ligand-sensors that function primarily in the context of defense against invasive nucleic acids in prokaryotes. Moreover, in the majority of cases Figures  CARF-domThe domain architectures of the CARF proteins show certain parallels to those containing the WYL domain: both domains combine with predicted DNA-binding wHTH domains and/or catalytic effector domains. This similarity of domain architectures implies analogous general functions for the CARF and WYL domains which involve sensing soluble ligands in the context of host-virus conflicts. However, unlike the CARF domain, which commonly combines with C-terminal enzymatic effector domains when encoded within CRISPR-Cas loci, the WYL domains appears to be primarily coupled with wHTH domains in the same contexts. Thus, in CRISPR-Cas systems, the WYL domains are predicted to primarily couple ligand-sensing to transcriptional regulation and less often to direct regulation of effectors that target alien nucleic acids. Some families of CARF proteins, such as Csa3 and NE0113, that lack C-terminal effector domains, and the divergent RtcR-like family domains that are linked to the NtrC-like AAA+ domains are predicted, respectively, to regulate transcription directly or via sigma-54. Taken together, the observations presented here raise two key questions: what are the ligands recognized by the CARF domains and what are the targets of their associated effector domains?in situ and as free NTPs as part of their restriction-avoidance and epigenetic regulatory strategies. Many prokaryotic viruses also encode NAD-utilizing enzymes that modify host proteins, in particular RNA polymerase, with ADP-ribosyl moieties  was used to search for CARF domain-containing proteins and analyzed their genomic context in 2262 completely sequenced prokaryotic genomes. The set of 659 representative genomes was selected for quantitative analysis of co-occurrence of CARF-domain containing proteins and CRISPR-Cas systems as follows: for each genus, a species with the largest genome was selected except for the genera e-value of 0.01, composition based-statistics and low complexity filtering turned off were used to retrieve homologous sequences from the Refseq database. In each iteration, all detected sequences were examined for conserved motifs to detect either potential homologs below the cut-off to be included in the profile or potential false positives to be excluded. For borderline cases, additional profile-profile searches were carried out using the HHpred program with default parameters to evaluate the veracity of those matches  to cluster sequences at different thresholds. Multiple sequence alignments were built using the MUSCLE  to identify related sequences in gene neighborhoods. Each cluster of homologous proteins were then assigned an annotation based on the domain architecture or conserved shared domain. The Pfam database was used as a guide to make preliminary domain identifications followed by detailed analysis  with the Surface→Cavities and Pockets only option. The predicted ligand-binding residues were inferred from the alignment provided in Supplementary File Iterative profile searches with the PSI-BLAST (Altschul et al., E Edgar,  program,E Edgar, . TransmeE Edgar, . For eacThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Aquifex aeolicus EmrA, shows linearly arranged α-helical coiled-coil, lipoyl, and β-barrel domains, but lacks the fourth membrane-proximal domain shown in other pumps to interact with the inner membrane transporter. The adaptor α-hairpin, which binds outer membrane TolC, is exceptionally long at 127 Å, and the β-barrel contains a conserved disordered loop. The structure extends the view of adaptors as flexible, modular components that mediate diverse pump assembly, and suggests that in MFS tripartite pumps a hexamer of adaptors could provide a periplasmic seal.Periplasmic adaptor proteins are key components of bacterial tripartite efflux pumps. The 2.85 Å resolution structure of an MFS (major facilitator superfamily) pump adaptor,  Component interactions of MFS-dependent pumps have been evidenced in vitro, EmrA-EmrB interaction by electron microscopy In the structurally uncharacterised MFS-dependent efflux pumps, such as A. aeolicus, revealing features that appear specific to the MFS efflux pumps.The structure of the closely conserved TolC exit duct has been known for 14 years 22.1Aquifex aeolicus EmrA (aaEmrA), E. coli SoluBL21 cells bearing pET24-aaEmrAΔTM . Cells were broken by two 30 000 psi passages through a cell disruptor. After centrifugation at 150 000×g for 1 h at 4 °C, supernatant was incubated with Profinity IMAC resin (Biorad) and 4 mM imidazole for 1 h at 4 °C. Resin was washed in buffer A  containing 0.1% Triton X-100, then in buffer A alone. Protein was eluted in 25 mM HEPES pH 7.5, 200 mM NaCl and 400 mM imidazole. Imidazole concentration was reduced to 10 mM using an Amicon 10kDA molecular weight cut-off concentrator (Millipore). Final protein concentration was 7 mg ml−1. Selenomethionine-incorporated EmrA was produced using a metabolic inhibition protocol E. coli SoluBL21 (DE3) (Genlantis) cells bearing pET24-EmrAΔTM plasmid were grown at 37 °C to A600 0.5 in M9 minimal media supplemented with 50 μg ml−1 kanamycin, 0.2% glucose, 2 mM MgSO4, 0.1 mM CaCl2 and 0.001% thiamine. At this point 100 mg L−1 threonine, lysine and phenylalanine, 50 mg L−1 leucine, isoleucine and valine and 60 mg L−1 selenomethionine were added and cells grown for a further 45 min. 0.5 mM IPTG was then added for 16 h at 18 °C. Protein was purified as native with the addition of 1 mM TCEP to all buffers. Incorporation of 2 selenomethionine residues was confirmed by mass-spectrometry. The ecEmrA protein lacking the TM domain was produced from plasmid pET24-ecEmrAΔTM and purified as native aaEmrA.To produce native, soluble 2.2aaEmrA was conducted using sitting drop vapour diffusion at 15 °C in CrysChem 24 well plates (Hampton Research). Drops were formed by mixing 2 μl of protein solution with 2 μl of crystallisation reagent  and equilibrated against 500 μl. Crystals grew to maximum size (0.8 mm × 0.2 mm × 0.2 mm) in seven days, and were cryoprotected by stepwise addition of cryoprotectant , before being looped and flash-frozen in liquid nitrogen. Diffraction data were collected at 100 K on beamline I24 . X-ray data sets were indexed and integrated using XDS http://www.rcsb.org/), PDB ID: 4TKO. Root mean square deviations (RMSDs) were calculated over Cα atoms aligned using superpose in the CCP4 suite Crystallisation of 33.1E. coli and 5 homologues, engineered to lack the TM helix (see Aquifex aeolicus EmrA (aaEmrA) from a construct encompassing residues 26 onwards of the 374 residue mature sequence. Native aaEmrA crystallised in space group I4122 with a 78% solvent content and one molecule in the asymmetric unit. Phasing using molecular replacement with adaptor homologues was unsuccessful. Selenomethionine derivatised aaEmrA crystallised as native and phases were calculated using a 3.5 Å resolution single-wavelength anomalous diffraction (SAD) dataset  domain identified to date in all 6 RND and ATP-binding cassette (ABC) adaptor crystal structures A. aeolicus. Some EmrA adaptors have non-conserved C-terminal extensions  but these extra residues are insufficient to form an MP domain, which are usually 84–108 residues long. In fact, the C-terminal residues of ecEmrA are predicted to form a short α-helix The structure contains three linearly arranged domains, an α-helical hairpin, a lipoyl domain and a β-barrel domain . In RND-dependent tripartite pumps, the MP domain is known to make extensive interactions with the periplasmic extension of IM transporters 3.3aaEmrA anti-parallel, two-stranded, α-helical coiled-coil domain is 168 residues and 127 Å long with 11 heptad repeats per helix. The N-terminal helix consists of residues 77–160, and the C-terminal helix residues 165–244 , while coils similar in length to aaEmrA are predicted in pathogenic V. cholerae VceA (∼146 residues/∼115Å long) as well as Thermodesulfobium narugense (∼130 residues/∼95Å long).An alignment of structurally characterised adaptors to date  highlighaaEmrA adaptor α-hairpin with those from MexA, ZneB, AcrA, CusB and MacA results in low RMSDs (1.22–1.70 Å), with the hairpins superposed to the top half of the aaEmrA α-hairpin (residues 116–206)  . The α-h3.4Borrelia BesA lacks an α-hairpin, see aaEmrA β-barrel contains 21 disordered residues, unobserved due to a break in the electron density (residues 322–342). Modelling of this region indicates it can form a 23 Å long loop, approximately the same length as the β-barrel strands (aaEmrA numbering). The loop is one of the most conserved regions in the EmrA structure and is not seen in any of the other EmrA β-barrel loops . If EmrA does facilitate drug transfer through the tripartite pump by binding substrates as suggested The initial observation that adaptor β-barrel topology is also found in domains involved in ligand binding prompted the idea adaptors may have a role in substrate engagement  adaptor . Neither3.6left) right) that are equivalent to the upper TolC α-barrel, small gaps between β-barrel domains can only be sealed by modelling the disordered 21 β-barrel residues. In addition, the TolC-interacting regions of the α-hairpins naturally diverge in the adaptor hexamer, precluding a tip-to-tip TolC-EmrA interaction. Only by docking the TolC open form right). While this length is similar to the cryo-electron microscopy model of the AcrAB-TolC complex formed by non-natural linked fusion proteins left) and the in vitro bipartite MexA-OprM cryo-tomography model (210Å) SoStTripartite efflux pumps span both inner and outer membranes, providing a continuous seal for drugs to bypass the periplasm. Extensive in vivo cross-linking analyses have shown that in AcrAB-TolC a 170 Å long periplasmic seal can be provided by close fit of IM and OM components


UBE3A, a gene encoding a ubiquitin ligase. Current treatments focus on the management of symptoms, as there have not been therapies to treat the underlying molecular cause of the disease. However, this outlook is evolving with advances in molecular therapies, including artificial transcription factors a class of engineered DNA-binding proteins that have the potential to target a specific site in the genome.Angelman syndrome is a monogenic neurologic disorder that affects 1 in 15,000 children, and is characterized by ataxia, intellectual disability, speech impairment, sleep disorders, and seizures. The disorder is caused by loss of central nervous system expression of Here we review the recent progress and prospect of targeted gene expression therapies. Three main issues that must be addressed to advance toward human clinical trials are specificity, toxicity, and delivery.Artificial transcription factors have the potential to address these concerns on a level that meets and in some cases exceeds current small molecule therapies. We examine the possibilities of such approaches in the context of Angelman syndrome, as a template for other single-gene, neurologic disorders. UBE3A is epigenetically imprinted throughout neuronal brain cells, with the maternal allele being preferentially expressed and the paternal allele silenced [UBE3A expression. Approximately 70% of all cases involve a large, 5–7-Mb, de-novo maternal deletion of the chromosome 15q11-q13 region, which includes the critical UBE3A gene [UBE3A (~11%), uniparental disomy (~7%), and imprinting defects (~3%) [Angelman syndrome is a neurodevelopmental disorder that affects 1 in 15,000 children [silenced . In AngeE3A gene . The remts (~3%) . About 1ts (~3%) .UBE3A or that severely reduced expression of the maternal copy of UBE3A were found to be the causative for Angleman syndrome [For gene therapy to be a viable treatment option it is essential that the genetic target is known, and that there is evidence that a postnatal intervention would be beneficial. In 1997 mutations in syndrome . UBE3A isyndrome . Also, tsyndrome . More resyndrome . HoweverThe developmental delay of Angelman syndrome generally becomes noticeable after 6 to 12 months of age. Since the brain has been without UBE3A expression throughout development, an important consideration is whether late-stage  intervention might have clinical value. The first study supporting that it could was based on the observation that αCaM kinase II was inhibited by phosphorylation in a mouse model of Angelman syndrome . SubstitUbe3a produced localized restoration of Ube3a to wild-type levels [Ube3a cDNA could alter the balance of the three known isoforms and result in only a partial functional restoration [Ube3a cDNA, further supporting that the developmental changes may be reversible using late-stage interventions. This initial AAV study also encouraged the exploration of less traditional methods that could be translated to humans.Since the underlying genetic defects are known, gene therapy could, in principle, be applied to replace the missing components. However, replacement of the entire 5–7-Mb region that is deleted in the majority of Angelman syndrome cases is not a viable option due to size limitations imposed by most viral vectors . A pragme levels . The virtoration ,20. The UBE3A that exists in almost all affected individuals. Paternal UBE3A is epigenetically silenced not by promoter DNA methylation but by a long antisense RNA transcript referred to as the UBE3A-ATS , exhibit low toxicity over a long period, and be deliverable by minimally invasive means. Specificity remains one of the most common and problematical issues facing potential molecular treatment. A lack of specificity is common among small molecules that work on a DNA level. For example, chromatin-modifying agents such as 5-azaC and SAHA affect DNA methylation and histone acetylation globally, rather than targeting a specific gene [An ideal therapy would be specific to the targeted gene  [in vivo methodology for evaluating off target binding is chromatin immunoprecipitation analyzed by high throughput sequencing (ChIP-Seq) [One of the largest concerns for ATF-based therapy is off-target effects. It should be noted that concerns about off-target activities of ATFs are somewhat less than for nucleases; ATFs would likely have effects in only the 10-20% of the genome that contains regulatory elements , whereas targets . Off-taras used)  or chromas used) . PerhapshIP-Seq) . SurprishIP-Seq) . An ideaAnother important concern for all interventions is toxicity of the treatment. To date, the only ATF that has been introduced into humans is a zinc finger-based factor that increased VEGF expression, which progressed through Phase I and II clinical trials . A totalA significant obstacle to be overcome for every potential therapy is delivery of the therapeutic to the desired organ. As discussed above, the brain presents a unique challenge due to the blood brain barrier. Methods capable of crossing the blood brain barrier include liposomal delivery, cell penetrating peptides (CPPs), and small molecule drugs that are hydrophobic or capable of binding transcytosis proteins . ATFs haUBE3A. The paternal allele has been re-activated in mice by small molecule drugs that inhibit the transcription of Ube3a-ATS, however these drugs had several off-target effects and delivery limitations. In principle, ATFs may offer several advantages for specificity and delivery. ATFs have advanced in recent years, enabling regulation of specific gene transcripts in the human genome [UBE3A and perhaps a full phenotypic rescue. A similar approach could be applied to other monogenetic neurologic disorders, and potentially even multi-gene disorders.The advances that have taken place in Angelman syndrome research in the last twenty years have made it an ideal candidate for targeted molecular therapy. Recent advances have verified an intact but silenced paternal n genome . The delAAV: Adeno-associated virus; ATF: Artificial transcription factor; ChIP-Seq: Chromatin immunoprecipitation analyzed by high throughput sequencing; CPP: Cell penetrating peptides; CRISPR: Clustered regularly interspaced short palindromic repeat; LTP: Long-term potentiation; TALE: Transcription activator-like effector.The authors declare that they have no competing interests.BJB and DJS conceived, drafted, read, and approved the manuscript.