To exemplify how TRIM proteins could be involved in binding higher order oligomers we discuss three possible models for the TRIM5α/HIV-1 capsid interaction assuming different conformations of B30.2 domains.Many tripartite motif-containing (TRIM) proteins, comprising RING-finger, B-Box, and coiled-coil domains, carry additional B30.2 domains on the C-terminus of the TRIM motif and are considered to be pattern recognition receptors involved in the detection of higher order oligomers . To investigate the spatial architecture of domains in TRIM proteins we determined the crystal structure of the TRIM20Δ413 fragment at 2.4 Å resolution. This structure comprises the central helical scaffold (CHS) and C-terminal B30.2 domains and reveals an anti-parallel arrangement of CHS domains placing the B-box domains 170 Å apart from each other. Small-angle X-ray scattering confirmed that the linker between CHS and B30.2 domains is flexible in solution. The crystal structure suggests an interaction between the B30.2 domain and an extended stretch in the CHS domain, which involves residues that are mutated in the inherited disease Familial Mediterranean Fever. Dimerization of B30.2 domains by means of the CHS domain is crucial for TRIM20 to bind pro-IL-1β  Tripartite motif-containing proteins represent a large family of proteins sharing a conserved domain architecture. The tripartite motif (TRIM) consists of an N-terminal RING domain, one or two B-box domains and a C-terminal coiled-coil domain. The TRIM segment is considered to serve as a scaffold domain that is extended on either side by additional protein-protein interaction modules. The RING- and B-box domains contain zinc finger motifs that confer E3 ubiquitin ligase and self-association activities, respectively. TRIM proteins are involved in diverse cellular functions including regulation of the innate immune system, retroviral restriction, cell proliferation, and differentiation1TRIM20 (Uniprot ID: O15553), which is also named pyrin or marenostrin, is special among other TRIM proteins, because it lacks the RING domain that confers ubiquitin ligase activity. In TRIM20 the RING domain is replaced by a PYD domain, which belongs to the death domain superfamily and is frequently involved in homotypic protein-protein interactions  and it is involved in the modulation of the pro-inflammatory cytokine pro-interleukin-1β (pro-IL-1β)69101113141517TRIM proteins are flexible structural entities that tend to oligomerize upon ligand binding. This behavior makes crystallization of full-length TRIM proteins extremely difficult. Hence, only structures of TRIM protein fragments are known so far. Crystal structures of B30.2 domains from TRIM5α1918Here we present the crystal structure of the TRIM20 coiled-coil/B30.2 fragment at 2.4 Å resolution. This structure shows the overall architecture of TRIM proteins and suggests two distinct spatial orientations between B30.2 and coiled-coil domains. The flexibility between the coiled-coil and B30.2 domains in solution was confirmed by small-angle X-ray scattering (SAXS). We show that the TRIM20 coiled-coil/B30.2 fragment, but not the isolated B30.2- and coiled-coil domains, binds pro-IL-1β, and we discuss different possible binding modes of TRIM5α, a close homologue of TRIM20, to the HIV-1 CA in light of the presented findings.To investigate the oligomerisation state and the spatial domain orientation of TRIM20 we designed a construct that comprises the TRIM20 coiled-coil and B30.2 domains. This design was guided by secondary structure prediction of TRIM proteins, which suggested that the bipartite coiled-coil motif starts immediately after the B-box domain. The alignment of human TRIM5α, −20, −21, −22, and −27 sequences revealed a conserved glutamic acid at position 414 . Therefo1 with 6 chains in the asymmetric unit  are crucial features for the prediction of TRIM proteins. The 2-CC region participates in a 2-helix coiled-coil motif involving residues L427/L430 and residues L513’/L516’ at the N- and C-termini of helix α1, respectively (the prime indicates residues from the second protomer). The 3-CC region participates in a 3-helix coiled-coil motif involving residues L484/V487/V491, I553/I557/L560, and F451’/T455’/L458’ from helices α1, α4, and α1’, respectively  (2 = 2.01). In this model, one of the two B30.2 domains is arranged as in the crystal structure. The second B30.2 domain is rotated perpendicular to the 2-fold axis placing it sideways onto the CHS domain (2 = 1.31) and reveals the conformational space available for the B30.2 domain  experiments. The radius of gyration (RG) for an infinitely diluted sample of TRIM20Δ413 is 4.27 nm, which is consistent with the RG calculated from the structure of the dimer in the 4.29 nm; . However4.29 nm; . Further = 3.62)  and the  = 3.62) . TherefoS domain . Fitting2 domain .The conformation seen in the crystal structure defines one populated state in solution. In additional we see further states in solution where the B30.2 domains are positioned sideways onto the CHS domain. This movement increases the radius where the B30.2 domain could interact with its ligand. In the crystal structure the distance between the B30.2 centers of mass is about 47 Å. Movement of the B30.2 domain sideways increases this distance to 85 Å . Notablyin vitro we performed co-immuno precipitation experiments using purified pro-IL-1β and various constructs of TRIM20. These results show that TRIM20Δ413 directly binds to pro-IL-1β, whereas the isolated CHS- and B30.2 domains do not . Stable complex formation however was only observed after incubating the proteins at 25 °C, whereas no complex formation was observed after incubation at 4 °C . The samclosed state. The hypothesis that this state is flexible enough to be converted into an active conformation is supported by the solution scattering data and the topology of TRIM20Δ413. The best interpretation of the SAXS data is given by an ensemble of TRIM20Δ413 structures with B30.2 domains arranged in various conformations along the CHS domain. The topology enables this movement, because the B30.2 domain is not restricted by a stiff linker.Several crystal structures of isolated TRIM protein domains have been reported and the comparison of these TRIM fragments with the TRIM20Δ413 structure reveals valuable insight into the function of TRIM proteins Fig. 6A. A comparison of the isolated B30.2 domain structures between TRIM20, TRIM21/Fcopen state, where the putative peptide binding sites are accessible even for very large ligands. The modelling of the open state relies on the assumption that the interaction between the proline-rich stretch and the B30.2 domain that is observed in the TRIM20Δ413 structure as an inter-molecular contact is converted into an intra-molecular contact by a rearrangement of the hinge region . It is almost three-times as large as the average surface area of a crystal contact and the biological significance of a protein/protein interface typically increases with its sizeopen state of TRIM20 or the formation of a TRIM20 tetramer.Structural and mutagenesis data support the hypothesis that the interface between the B30.2 domain and the extended proline-rich stretch has biological significance. The buried surface of this interface is extremely large  and pro-IL-1β (31.9 kDa) subunits and indicates that complex formation requires oligomerization of TRIM20Δ413 and/or pro-IL-1β. Additional experiments are required to reveal the exact stoichiometry of the TRIM20/pro-IL-1β complex. Complex formation also requires elevated temperature, because at 4 °C no interaction between TRIM20Δ413 and pro-IL-1β was detected. It is unlikely that the 400 kDa peak in the size exclusion chromatogram indicates temperature induced unfolding and unspecific aggregation, because the isolated components did not show any sign of aggregation at this temperature. The premise of elevated temperature could indicate that larger, temperature-induced structural rearrangements are required for binding.Unfortunately neither the structure of pro-IL-1β nor the exact stoichiometry of this complex is known and attempts to crystallize it were so far unsuccessful. To exemplify how the CHS-domain, which has the same structural topology in many TRIM proteins , is invo3739open- or closed conformation, i. in ii. in open- and closed states (model iii. in 41424344454647closed state (model i.) the assumed peptide binding sites of B30.2 domains are not directly accessible. This model is almost identical to the models suggested by Sanchez et al. (2014) and Goldstone et al. (2014) that were build based on the structures of TRIM25 coiled-coil and TRIM5α B-box/coiled-coil domains, respectively28open state (data not shown) has the same limitation. The docking of symmetric dimers suggests a stoichiometry of TRIM5α to CA of 1:12.We investigated two symmetric dimers with strict 2-fold symmetry . This model relies on the assumption that the inter-molecular interface between the B30.2 domain and the extended stretch is transformed into an intra-molecular interface by a rearrangement of the linker region  PEG 20’000, 8% (w/v) PEG 550 MME using sitting drop vapor diffusion at 20 °C. TRIM20Δ413 (7.5 mg/ml) was mixed in a 1:1 ratio with mother liquor. The obtained crystals were used for micro-seeding in 0.1 M Tris-AcOH, pH 7.4–8.0, 0.26% (w/v) Cystamine, 0.2 M LiSO4, 8% (w/v) PEG 20’000, 8% (w/v) PEG 550 MME using sitting drop vapor diffusion at 20 °C. The protein sample was concentrated to 5 mg/ml and mixed in a 1:1 ratio. Crystals grew within 24 h and were harvested within 18 days. Prior to flash freezing in liquid nitrogen, crystals were soaked in the mother liquor supplemented with 20% (v/v) ethylene glycol. A native data set of a TRIM20Δ413 crystal was recorded at the X06SA beam line of the Swiss Light Source . The protein crystallized in P21 with 6 molecules in the asymmetric unit forming 3 dimers. Data was indexed, integrated and scaled with XDSFirst crystals were found in 0.1 M Tris-AcOH, pH 8–8.5, 0.2–0.4% (w/v) Cystamine, 0.2 M LiSOHow to cite this article: Weinert, C. et al. Crystal structure of TRIM20 C-terminal coiled-coil/B30.2 fragment: implications for the recognition of higher order oligomers. Sci. Rep. 5, 10819; doi: 10.1038/srep10819 (2015).


Neighbouring domains of multidomain proteins with homologous tandem repeats have divergent sequences, probably as a result of evolutionary pressure to avoid misfolding and aggregation, particularly at the high cellular protein concentrations. Here we combine microfluidic-mixing single-molecule kinetics, ensemble experiments and molecular simulations to investigate how misfolding between the immunoglobulin-like domains of titin is prevented. Surprisingly, we find that during refolding of tandem repeats, independent of sequence identity, more than half of all molecules transiently form a wide range of misfolded conformations. Simulations suggest that a large fraction of these misfolds resemble an intramolecular amyloid-like state reported in computational studies. However, for naturally occurring neighbours with low sequence identity, these transient misfolds disappear much more rapidly than for identical neighbours. We thus propose that evolutionary sequence divergence between domains is required to suppress the population of long-lived, potentially harmful misfolded states, whereas large populations of transient misfolded states appear to be tolerated. Single molecule kinetics investigations and molecular simulations are useful tools in elucidating protein assembly mechanisms. Here, the authors use these to show that even naturally occurring tandem repeats undergo transient misfolding and that assembly is much more complex than we previously understood. Proteins comprised of a series of covalently linked domains, often sharing a common fold and similar sequence, are highly abundant, particularly in the eukaryotic proteome2356891011121314151617All-beta, immunoglobulin (Ig)-like domains are involved in many neurodegenerative and non-neuropathic systemic human diseases associated with the formation of deposits or inclusions with amyloid-like characteristics19202122231Here we combine ensemble kinetics and single-molecule microfluidic-mixing methods with molecular simulations to investigate how sequence divergence affects the interplay of native and non-native interactions in avoiding misfolding and aggregation in tandem Ig-like repeats of these domains127Tandem repeats containing two, three or eight domains of I27 were unfolded in the chemical denaturant guanidinium chloride (GdmCl) and then rapidly diluted to monitor ensemble folding kinetics. Compared with isolated I27 domains, an extra folding phase was observed at low denaturant concentrations . This neThe considerable amplitudes of the fast refolding phase hint at a surprisingly large misfolded population. For the two-domain construct I27–I27, the amplitude is 23 (±1)%; for the three- and eight-domain constructs, a simple calculation based solely on the number of covalently linked domains predicts that the proportion of misfolded domains would increase by a factor of 1.3 and 1.8, respectively (see the Methods section for details), relative to the two-domain tandem, in good agreement with the measured amplitude increase of 29 (±1)% and 38 (±1)%, respectively .Misfolded species are also detected in ensemble unfolding experiments. Tandem repeat proteins that have been unfolded, refolded (for 2 min) and then unfolded again, reveal the presence of a population of misfolded proteins that unfold faster than correctly folded molecules  and are E, thus enables us to identify the presence of the different species involved. For these investigations, we used two tandem constructs, I27–I27 and I27–I28, both labelled at position 3 of the first domain (A strand) and at position 83 of the second domain (G strand), with Alexa 488 and Alexa 594 as FRET donor and acceptor, respectively , but also a broad range of additional populations with E between ∼0.2 and ∼0.9. These persist to ∼1 s, when a simpler picture is regained and the two species detected in previous single-molecule experiments are recovered: the natively folded F27–F27, and the stably domain-swapped misfolded state, M27–M27, with mean transfer efficiencies at 0.38±0.02 and 0.90±0.03, respectivelyRemarkably, the transfer efficiency histograms for I27–I27  show tha27–U27; 27–U27/U27–F27; 27–U27 and U27–U28 between 4.6 and 0.5 M GdmCl ∼4 ms after mixing  and extrapolated their transfer efficiency to 0.23 M GdmCl , al M GdmCl . In view27–M27 consist of two strand-swapped domains, each similar in structure to a native I27 folded but the terminal domain still unfolded are stable enough to be metastable refolding intermediates, we engineered and produced the circular permutants corresponding to the central domains of misfolded species as seen in the simulations. Equilibrium denaturation experiments showed that these proteins were strongly destabilized compared with the wild-type structure, but most of them were at least partially folded under native conditions . An analE=0.49±0.03 lies in the previously unassigned region of the FRET efficiency histogram populated at early times during I27–I27 refolding, suggesting that Simulations suggest that, given the positions of the fluorescent dyes on the N- and C-terminal strands of the first and second domain, respectively, all putative intermediates with the central domain (mis)folded are expected to yield transfer efficiencies similar to that of one unfolded I27 monomer labelled at equivalent positions, because folding of the central domain effectively shortens the chain separation of the labels by the length of one I27 domain folding, such that the subsequent folding of the central domain will not cause a FRET efficiency change. Occurrence of this 27–M27 to a degree dependent on its abundance, which is expected to be small because of the high entropic cost associated with the terminal domain (mis)folding first. Given that M27–M27 is per se a small population, we would not be able to detect such an effect and thus did not include this pathway in the model used to fit the data.Note that we cannot exclude a misfolded state with the terminal domain folded and the central domain unfolded . They are characterized by the parallel pairing of the same strand from two domains, and most of them include the self-recognizing region 56–61 previously identifiedComputational investigations of tandem repeats of I27 by Wolynes and co-workers27–U27, whose transfer efficiency we obtained from independent measurements  and F27–F27 (E=0.38±0.02), from independent measurements in the microfluidic mixer On the basis of the results presented above and our extensive knowledge of I27 stability and folding kinetics and mechanism313235ic mixer  or from ic mixer , respecthe chain . MoreoveE≈0.65, which corresponds to the proposed amyloid-like population M2. For comparison, we show in This wealth of prior knowledge places essential constraints on the minimum number of species that need to be included in the model, their kinetic connectivity, some of the rate coefficients and the shapes of the transfer efficiency peaks. The robustness of our analysis  and amyloid-like (M2) misfolds, as well as the direct formation of the native state from unfolded; the native refolding phase is slowed by the need to escape from the misfolded states.E≈0.9 . The presence of such high transfer efficiency species during I27–I28 refolding was most unexpected, and suggests that, contrary to our previous conclusions and expectations, the natural neighbours I27 and I28 can actually form strand-swapped structures, M27–M28, analogous to those seen in I27–I27, albeit far less stable. The ability to form such structures based on native-like interactions in Ig-like multidomain proteins may be connected to the folding mechanism of the individual domains, and may thus affect other protein families38The remainder of the misfolded populations is represented by topologically native-like species (M1) comprising structures with a misfolded central domain and an unfolded terminal domain, transiently accumulated in both I27–I27 and I27–I28 The picture emerging from our results is that avoiding misfolding in multidomain proteins due to the high local concentration of similar protein segments may pose evolutionary challenges analogous to those of avoiding intermolecular aggregationWe have shown that the stable, long-lived misfolded species observed previously1920374345In the predictions from Gō-like simulations, the central domain always comprised residues from the C-terminal portion of domain 1 and the N-terminal portion of domain 2, thus it resembles a circular permutant of I27 (ref Refolding. Unfolded protein in 5 M GdmCl underwent rapid mixing 1:10 (v/v) with PBS (10 mM sodium phosphate (pH 7.4), 137 mM NaCl, 2.7 mM KCl) and the refolding kinetics were recorded using an AppliedPhotophysics SX20 stopped-flow fluorimeter.Unfolding. The kinetics of unfolding were recorded following manual mixing (1:10) on a PerkinElmer fluorimeter at final concentrations of 4–8 M GdmCl.Unfolding of refolded protein. Unfolded protein in 5 M GdmCl was allowed to fold by a 10 × dilution with PBS. The final reaction solution is referred to as ‘previously refolded'. For practical reasons, the time allowed for refolding was 2 min, the refolding half-life of native I27 is ∼70 ms. Excitation was at 280 nm and emission was followed at or above 320 nm. All experiments were conducted in a final dithiothreitol concentration of 5 mM, at 25 °C.−1), assumed equal for all I27 variants, was then subtracted from the stability of the circular permutants measured in the presence of glucose, thus attaining a reliable estimate of these species' stability in native buffer.Given the marginal stability of the circular permutants in native conditions, GdmCl-induced denaturation measurements were conducted in the presence of 1 M glucose. The stabilization due to the addition of glucose was measured for the wild-type protein and this contribution . The average number of neighbouring domains for each domain within a tandem construct of length N is given byThe proportion of misfolded domains was predicted for constructs of 1–8 repeat domains by assuming that the probability of domain pi is the fraction of domains with ni neighbours. Thus, the ratio of 〈n〉 values for constructs of different length, N, would be expected to match the relative abundances of misfolds extracted from the amplitudes obtained in kinetics experiments. This approach neglects the possibility of misfolding between non-neighbouring domains and the inhibition of misfolding by already misfolded domains.where Cysteine residues were introduced by site-directed mutagenesis: E3C in domain 1  and N83C in domain 2 if I27, and K83C for I28 (with the numbering relative to a single domain). DNA sequencing confirmed the mutagenesis. I27–I27 and I27–I28 tandems, with the engineered surface cysteines, were expressed as described previously31Observations of single-molecule fluorescence were made using a custom-built confocal microscope equipped with a continuous wave 488-nm solid-state laser  and an Olympus UplanApo × 60/1.20 W objective. After a dichroic mirror that separates excitation and emission light , fluorescence emission passed through a 100-μm pinhole and was split by a second dichroic mirror  into donor and acceptor fluorescence. Donor fluorescence then passed a filter  before being focused onto a single-photon avalanche diode , while acceptor fluorescence passed a filter (QT 650/100) before being focused onto a single-photon avalanche diode . The arrival time of every photon was recorded with a two-channel time-correlated single-photon counting module .−1 in all cases by varying the pressure applied to the inlet channels according to the viscosity of the solutionsRapid-mixing experiments were performed with a microfluidic-mixing device fabricated by replica moulding in polydimethylsiloxane (PDMS) described in refs E=0.05 were used and normalized to a total area of one. As a result, we have a time series of N histograms (N=23 for I27–I27 and N=25 for I27–I28) measured at times tn  after mixing. Each histogram can be written as a vectorEach time-resolved transfer efficiency histogram was measured between two and five times in different experiments on different days and averaged. Only the parts of the transfer efficiency histogram above hnm with m=1, ..., M is the number of burst events with transfer efficiency values E satisfying Em−Δ/2<E≤Em, where Δ=0.02 is the bin width, and Em is the mean transfer efficiency value of the mth bin. We fit the histograms globally withHere pl(tn) is the relative population of state l at time tn assuming a total of L states  results from our six-species kinetic model (Here ic model  as:p(tn) is a population vector of the formwhere K is the matrix of the interconversion rate coefficients that describes the population change of the six species over time (ver time :p(0)= is the population vector at time zero, starting from the fully unfolded state. In the global fitting procedure, we minimizeand Deviations of the sum of the fitted populations from the measured data were calculated for each bin of every histogram as the residuals:el and wl , which we assume to be constant over time, were fixed to values obtained either from equilibrium experiments (FF and M3), extrapolated from the dependencies on GdmCl , or resulting from fits of individual histograms with the peak parameters of all other populations constrained (M2). In addition to these values, we also knew the native folding rate coefficient kFU/UF→FF=15±5 s−1 (=0.5 kUU→FU) and the rate coefficient for M3 unfolding in 0.23 M GdmCl (kM3→M1∼2 × 10−5 s−1). Instead of fixing the native folding rate coefficient, we minimized χ2 under the constraint that its value must be within the given uncertainty interval. Note that kUU→FU is twice the value of kFU/UF→FF because from the fully unfolded state the probability for a folding event to occur is twice as high. All other rate coefficients were left unconstrained. The extremely slow M3 unfolding indicates that kM3→M1 is much smaller than kM1→M3 and kM1→UU, which, together with the small M3 population, implies a large uncertainty associated with the exact value of kM3→M1 fitted from the data (experiment measurement time is <1 min). The rate coefficients found by the fit, as well as the corresponding set of peak positions and widths, are reported in The peak positons and widths UU→FU and kM3→M1 were varied randomly assuming that the values were normally distributed with a s.d. of 50% of their original values . After each parameter randomization for the fixed positions, widths and rate coefficients, a fit was performed and the outcome recorded, for 104 cycles. Results of this procedure are summarized in χ2 within 50 or 5% of the best χ2 are histogrammed =0.6 nm and contact energy ɛ=ɛPASTA+Δɛ for residues i and j, where ɛPASTA is the relevant entry in the PASTA matrix for parallel β-interactions and Δɛ is an empirical offset . A value of –2.75 kBT for the offset was found to give a maximum transient population of the M2 intermediate most consistent with experiment. Folding simulations were run, starting from fully extended configurations, using Langevin dynamics with a friction of 0.1 ps−1 and a time step of 10 fs. A total of 128 independent runs initiated with different random seeds were performed, each for a duration of 10 μs. A modified version of the GROMACS 4.0.5 molecular simulation package was used for all calculationsThe same coarse-grained model for titin I27–I27 was used here as in previous workSnative={, , …, } was determined based on the native structure using the standard procedure for generating the Gō-type model17SkCD,={(i1+θ(k−i1)L, j1+θ(k−j1)L), (i2+θ(k−i2)L, j2+θ(k−j2)L) …} and SkTD,={(i1+θ(i1−k)L, j1+θ(j1−k)L), (i2+θ(i2−k)L, j2+θ(j2−k)L) …}, where θ(x) is the Heaviside step function and L=93 is the length of a titin domain and linker. The index k is needed to distinguish the different possible native-like misfolds. It corresponds to the point in the sequence where a loop is cut to make the central domain misfold and takes values k∈K={0, 16, 28, 37, 53, 64, 76}. For a native fold, k=0 and SCD,0 and STD,0 are the sets of contacts for the N terminal and C terminal correctly folded domains. In addition, a set of non-native-like contacts SPASTA={ : ɛPASTA<0} was determined. For each set of contacts Sα, a corresponding fraction of contacts could be determined as Qα using:The states shown in rij(x) is the distance between beads (residues) i and j in configuration x, and g=1.2 allows for thermal fluctuations in the distance between residues which are in contact.where The system was initialized in the unfolded state, and then made transitions to another state only if it reached the ‘core region' for that state, defined as:QCD,0<0.4, QTD,0<0.4, QMU<0.4, QUM<0.4 and QPASTA<0.4.‘UU': configurations with QMU>0.8 and QUM<0.5‘MU': configurations with QUM>0.8 and QMU<0.5‘UM': configurations with QMM>0.8‘M3': configurations with QCD,0>0.9 and QTD,0<0.5‘FU': configurations with QTD,0>0.9 and QCD,0<0.5‘UF': configurations with QCD,0+QTD,0)/2>0.9‘FF': configurations with /2}.For the above, we define The lumped states ‘M1' and ‘FU/UF' are defined as: M1=UM ∪ MU and UF/FU=UF ∪ FU.k+1 to k+93 from the full I27–I27 sequence, where k is taken from the set K above. The energy function was otherwise identical to that used in the original Gō-like simulationsF(Q) for the relevant fraction of native-like contacts  were determined for each circular permutant using umbrella sampling on that coordinate, combining the data with weighted histogram analysisGfold was obtained as:Models for the circular permutants of I27 representing the central domains were constructed by simulating a chain consisting of residues R is the molar gas constant, T is the temperature and the position of the dividing surface between folded and unfolded is taken as Q‡=0.4. The free energy surfaces and stabilities are summarized in where How to cite this article: Borgia, A. et al. Transient misfolding dominates multidomain protein folding. Nat. Commun. 6:8861 doi: 10.1038/ncomms9861 (2015).Supplementary Figures 1-9, Supplementary Table 1, Supplementary Discussion and Supplementary References


ATP13A2 lead to Kufor-Rakeb syndrome, a parkinsonism with dementia. ATP13A2 belongs to the P-type transport ATPases, a large family of primary active transporters that exert vital cellular functions. However, the cellular function and transported substrate of ATP13A2 remain unknown. To discuss the role of ATP13A2 in neurodegeneration, we first provide a short description of the architecture and transport mechanism of P-type transport ATPases. Then, we briefly highlight key P-type ATPases involved in neuronal disorders such as the copper transporters ATP7A (Menkes disease), ATP7B (Wilson disease), the Na+/K+-ATPases ATP1A2  and ATP1A3 (rapid-onset dystonia parkinsonism). Finally, we review the recent literature of ATP13A2 and discuss ATP13A2's putative cellular function in the light of what is known concerning the functions of other, better-studied P-type ATPases. We critically review the available data concerning the role of ATP13A2 in heavy metal transport and propose a possible alternative hypothesis that ATP13A2 might be a flippase. As a flippase, ATP13A2 may transport an organic molecule, such as a lipid or a peptide, from one membrane leaflet to the other. A flippase might control local lipid dynamics during vesicle formation and membrane fusion events.Mutations in  Figure Based on the conserved P-type ATPase motifs, 36 human genes are recognized and annotated in databases to encode for P-type ATPases. These include 2 copper-ATPases, 4 NaP-type ATPases use metabolic energy (ATP) to actively pump substrates against an electrochemical gradient. To prevent backflow of transported ligand(s), P-type ATPases use an alternating access mechanism Figure . After sThe transport mechanism of P-type ATPases can be described by the model of Post-Albers Albers, . During +/K+-ATPase, which transports two Na+ ions out of the cell in exchange for three K+ ions per hydrolyzed ATP, and the SERCA Ca2+-ATPases, which transport two Ca2+ ions from the cytoplasm to the lumen of the ER/SR for two to three H+ in the other direction. Other P-type ATPases transport only in one direction using either the forward E1 to E1P step to bind the transported substrate  while others use the E2P step for substrate binding (P4 lipid flippases)  Na+/K+-ATPase for which there is a wealth of structural and kinetic information. SERCA1a was the first P-type ATPase to have its structure solved at high resolution plasmic reticulum (SR/ER) Ca+-ATPase of plants . During each catalytic cycle, the Asp residue is alternately phosphorylated and dephosphorylated by the N-domain and the A-domain, respectively. A Lys in the nucleotide interaction site of the N-domain (KGAPE) interacts with the adenine ring of ATP delivering the γ-phosphate to the active-site residue of the P-domain. This reaction renders a high-energy aspartyl phosphate intermediate. Subsequently, the A-domain subjects the bond to hydrolysis, catalyzed by the Glu residue in the highly conserved signature motif TGE , which now forms the second Ca2+ binding site II. Upon binding of the second Ca2+, the gating residue E309 on M4 will capture the second Ca2+ ion in site II.At the start of the catalytic cycle, cytosolic Ca2+ binding, the rearrangement of the TM helices is transmitted to the P-domain creating a Mg2+-binding site near the critical Asp residue (Asp351 in SERCA1a). Presence of Mg2+ is essential as this cofactor decreases the electrostatic repulsion of the γ-phosphate of ATP by the negatively charged Asp and hence, allows phosphate transfer. In this way, ATPase activity of P-type transporters in the cytosolic domains is tightly coupled to the ion binding in the M-domain, preventing unnecessary ATP hydrolysis. The transition toward the intermediate E1~ P phosphorylated state bends the P-domain and tilts the A-domain that rests on the P-domain. This exerts strain on the linkers between the A-domain and M1, M2, and M3 of the M-domain. As a result, M1-M2 is partially lifted out of the membrane forcing E309 in a fixed position, which closes the Ca2+ entry path (occlusion).Via the induced fit mechanism of Ca2+ by spreading out M1/M2 and M3/M4 away from M5/M6. In addition, this reduces the affinity of the Ca2+-binding sites promoting the luminal release of Ca2+. In exchange, two to three H+ ions bind with high affinity to the E2P state leading to occlusion of the luminal gate and further rotation of the A-domain. The A-domain rotation also brings the TGE loop closer to the phosphorylation site, shielding the aspartyl-phosphate by restricting the access of ADP or water.Following complete γ-phosphate transfer, the ATP-mediated connection between the N- and P-domains is lost. As a consequence, the pump relaxes and the N-domain moves away from the catalytic site and stretches the linker region between the M3 helix and the A-domain. The generated tension triggers rotation of the A-domain and results in transition to the low-energy E2P state. The significant conformational changes associated with the E1P to E2P transition is the rate-limiting step in the catalytical cycle. The rearrangement of the pump opens the luminal exit pathway for Ca2+-ATPase pump is reset to E1 by a series of reversal reactions leading to E2P dephosphorylation and proton countertransport. Entry of a water molecule induces a new rotation of the A-domain, which now precisely positions the Glu of the TGE-loop and the water molecule to catalyze an attack on the aspartyl phosphate. The rotation of the A-domain also repositions M1/M2 and the cation-binding site with the protons becomes occluded. Thereafter, the A-domain disengages from the phosphorylation site resulting in transition of the E2 state to the relaxed E1 conformation associated with release of the counterions.The Ca2+ pump, little is known about the P5-type ATPases to which ATP13A2 belongs. P5-type ATPases are found in all eukaryotic genomes, but are absent in bacterial genomes .In comparison with the well-studied SERCA1a CaThe P5-ATPase membrane topology is unusual , DKTG for auto-phosphorylation (P-domain) and TGE for dephosphorylation (A-domain)   , a milder disease with moderate neurologic symptoms and prominent connective tissue disturbances  which lead to misfolding . The P2A group contains the well-known SERCA and Secretory Pathway/Golgi (SPCA) Ca2+-ATPases, whereas the plasma membrane Ca2+-ATPases (PMCAs) belong to the P2B-ATPases. The P2C-subgroup encompasses the Na+/K+-ATPases and the gastric H+/K+-pumps , which display tissue specific and developmental dependent expression. Only the ATP1A1-3 genes are expressed in the nervous system. The α-subunit forms a hetero-oligomer with a β- and γ-subunit. β is critical for proper targeting and affects the K+ affinity  and α3 (AHC2 and RDP) isoforms. α2 (ATP1A2) is primarily expressed in astrocytes and drives Na+-dependent Glu uptake and removes excess K+ from the extracellular space during neuronal excitation. α3 (ATP1A3) is predominantly expressed in neurons and is involved in post-stimulus recovery , alternating hemiplegia of childhood (AHC), and rapid-onset dystonia parkinsonism (RDP) are autosomal dominant disorders caused by mutations of the NaATP1A2. ATP1A2 mutations lead to FHM2, a severe subtype of migraine with aura and temporary hemiparesis. More than 20 mutations in the α2-subunit are known to cause FHM2 , which is a subtype of migraine with aura originating from the brainstem or involvement of both hemispheres  lead to RDP, which is a rare autosomal dominant movement disorder with variable penetrance, characterized by the abrupt start of dystonia with signs of parkinsonism  remove CaThe human genome encodes 14 P4-type ATPases, which are putative lipid flippases involved in aminophospholipid transport across membrane bilayers. P4-type ATPases are the first class of P-type transporters that do not transport inorganic ions  translocase, which is localized to the plasma membrane and highly expressed in retina and brain, particularly in the cerebellum. The P4 lipid flippase ATP8A2 is involved in localization of PS to the inner leaflet of the plasma membrane  leading to Angelman syndrome (AS). This syndrome is marked by neurobehavioral anomalies that include severe mental retardation, ataxia and epilepsy. AS patients with imprinting mutations or with maternal deletions of 15q11-q13 display little or no ATP10A expression  are a known cause of Kufor-Rakeb syndrome (KRS), an autosomal recessive disorder characterized by juvenile-onset Parkinsonism associated with dementia , is the single P5B-type ATPase in the yeast Saccharomyces cerevisiae and resides in the vacuolar membrane, the yeast equivalent of the mammalian lysosome  74 genes or gene products have been reported as interactors of YPK9. Most interactions are genetic  highlighting genes that when deleted in the absence of YPK9 result in synthetic growth effects or phenotypic suppression or enhancement . The interactors vary both with respect to cellular location and predicted function. Surprisingly, only a few of the interactors are located in the endosome and vacuolar systems (6 out of 74 interactors). The most prevalent interactions participate in vesicular trafficking and protein sorting (12 out of 74 interactors) and transcriptional flux (11 out of 74)   that encode proteins related to mitochondrial function. Negative interactions are defined as genes that upon deletion, in combination with a YPK9 deletion, display aggravated growth effects as compared to the loss of either individual gene. The six genes are related to maintenance of mitochondrial homeostasis , mitochondrial biogenesis  and resistance to oxidative damage (OXR1). Several interactors of YPK9 can thus be related to mitochondrial function, which substantiates a link between YPK9 and protection from oxidative stress.The link between YPK9 have been identified in a genome-wide screen aimed at defining mechanisms by which YPK9 protects cells from Mn2+ toxicity in yeast , cellular transport and vesicular trafficking  or RNA processing (DIM1). Cdc11p and Cdc12p belong to the septin family, which includes highly conserved GTP-binding proteins found in eukaryotes. Septins provide a scaffold to support cell division, polarity and compartmentalization and have been implicated in diverse neurodegenerative disorders in humans, including in PD and α-synuclein mediated toxicity , whereas interactors that increase Mn2+ tolerance  seem to be involved in chromatin organization, histone modification and nuclear transport , while the zebra fish only one (ATP13A2), the late-onset mouse phenotype associated with loss of ATP13A2 would suggest that some of the P5B homologues in mice may be functionally redundant, possibly compensating for each other's loss  . Hypoxic conditions up-regulate transcription of the i.e. the late endosomes and lysosomes, because of a co-localization with LAMP1/2a, Rab7, and Lysotracker. Also the loss of ATP13A2 leads to lysosomal dysfunction and an increase in the size and number of the lysosomes .A role for ATP13A2 in autophagy and mitochondrial clearance has been suggested, but mechanistic details are lacking .The long list of putative physical interactors  family including the proton coupled transporters SLC11A2/DMT1/NRAMP2 and SLC40A1/ferroportin, the putative Zn2+/Mn2+ transporter SLC30A10/ZnT10 and SLC39A14/ZIP14  carrying Fe2+ and trivalent Mn3+   that mediate Zn2+ efflux and 14 members of the SLC39 family of ZRT/IRT-related proteins  that facilitate influx of Zn2+  flippase, ATP13A2 might alter membrane curvature, alter lipid dynamics, organize lipid microdomains or expose/remove important signaling molecules at one or the other membrane leaflet Graham, , which mAlthough the cell biological context in which ATP13A2 is involved is gradually emerging, studying the molecular function and substrate specificity of ATP13A2 using biochemical methods and isolated systems will be required to unravel the substrate specificity and transport properties of ATP13A2. Understanding ATP13A2 at the molecular level will reveal its link to KRS, NCL, dystonia, and PD. This might open new therapeutic possibilities to treat this spectrum of disorders.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the presence of alpha4, the level of PP2Ac ubiquitination is reduced. Using the MID1 RING-Bbox1-Bbox2 (RB1B2) construct containing the E3 ligase domains, we investigate the functional effects of mutations within the Bbox domains that are identified in patients with X-linked Opitz G syndrome (XLOS). The RB1B2 proteins harboring the C142S, C145T, A130V/T mutations within the Bbox1 domain and C195F mutation within the Bbox2 domain maintain auto-polyubiquitination activity. Qualitatively, the RB1B2 proteins containing these mutations are able to catalyze the ubiquitination of PP2Ac. In contrast, the RB1B2 proteins with mutations within the Bbox1 domain are unable to catalyze the polyubiquitination of alpha4. These results suggest that unregulated alpha4 may be the direct consequence of these natural mutations in the Bbox1 domain of MID1, and hence alpha4 could play a greater role to account for the increased amount of PP2A observed in XLOS-derived fibroblasts.MID1 is a microtubule-associated protein that belongs to the TRIM family. MID1 functions as an ubiquitin E3 ligase, and recently was shown to catalyze the polyubiquitination of, alpha4, a protein regulator of protein phosphatase 2A (PP2A). It has been hypothesized that MID1 regulates PP2A, requiring the intermediary interaction with alpha4. Here we report that MID1 catalyzes the  MID1 is a member of the tripartite motif (TRIM) protein family consisting of the N-terminal RING, two B-box, and Coiled-Coil (RBCC) domains . The RINThe Bbox domains, which coordinate two zinc-ions and adopt ββα-RING folds, are required for the efficient ubiquitination of alpha4, an oncogenic protein initially identified as an immunoglobin binding protein (IgBP-1). MID1 RING domain catalyzes only the monoubiquitination of alpha4, but the RING in tandem with Bbox1 or both Bbox domains (RING-Bbox1 and RING-Bbox1-Bbox2) catalyzes the polyubiquitination of alpha4. Neither the individual nor the tandem Bbox domains catalyzes the polyubiquitination of alpha4.Alpha4 down-regulates protein phosphatase 2A (PP2A) in numerous cellular pathways, including the target of rapamycin (TOR) pathway The Bbox1 domain binds alpha4 and this interaction is hypothesized to recruit PP2Ac to MID1 for ubiquitin-mediated degradation In the past decade, there have been numerous reports identifying mutations of MID1 from individuals with ventral midline anomalies that affect the brain, face, heart and genitalia  [27], [2In vivo fluorescence studies using MID fused to a green fluorescent protein tag (GFP-MID1) reveal that a number of XLOS-observed mutations within the C-terminal half of MID1 cause MID1 to form cytoplasmic ‘clumps’ instead of normally being associated with the microtubules In this article, we present data showing that MID1 catalyzes the ubiquitination of PP2Ac in the absence of alpha4. Using the MID1 RB1B2 protein construct, we observed that the RB1B2 proteins with natural mutations within the Bbox1 domain still catalyze the ubiquitination of PP2A but not the polyubiquitination of alpha4. These results suggest that alpha4 may play a greater role to account for the increase of PP2A observed in XLOS-derived fibroblasts.6-tag. Mutations within the DNA for Bbox1  and Bbox2 (C195F) domains were performed using standard PCR mutagenesis protocol using the RB1B2 domain construct. The His6-tagged alpha4 were cloned as previously described 6-tagged using the following primers: 5′CACCATGGACGAGAAGGTGTTCACC3′ and 5′TTACAGGAAGTAGTCTGGGGTACGACG3′. DNA sequencing confirmed the integrity of DNA inserts and mutations before the plasmids were transformed into BL21(DE3) cells.The DNA encoding the MID1 RING-Bbox1-Bbox2  was cloned into an Expresso T7 plasmid  containing a sequence for a C-terminal HisDE3) cells expressing the wild type and mutants MID1 RB1B2 constructs, alpha4, and PP2Ac were grown in LB media at 37°C to an OD600 of ∼0.5 and induced with 0.2 mM isopropyl β-D-1-thiogalactopyranoside for 18 hr at 18°C. The RB1B2 protein and the various mutants were purified as previously described 2+-NTA affinity chromatography approach.The BL21, and 2 µM biotinylated ubiquitin (Ub) in 20 mM Tris–HCl (pH 7.5), and purified recombinant proteins. The ubiquitination assays were incubated at 30°C for 90 minutes and the reaction terminated with 2x SDS-PAGE non-reducing sample loading buffer. The ubiquitination of alpha4 and PP2Ac were performed similarly to the autoubiquitination reaction by adding these proteins to the reaction mixture and using non-biotinylated Ub. The reactions were performed at 30°C for 4 hrs. The reaction products were subsequently evaluated by SDS-PAGE and chemiluminescent western blot (WB) analysis. For the autoubiquitination assay, the antibody used was avidin, which is specific for biotin. For the assays containing the alpha4 and PP2Ac proteins, the antibodies were specific for the C-terminal region of alpha4 (cat #sc-32174) and PP2Ac (cat #sc-376673) and were purchased from Santa Cruz Biotechnology. Control reactions were performed by omitting individual components from the assay. WB digital images were acquired with a Syngene G5:Box chemidoc system.Human Ub-activating enzyme E1, human Ub-conjugating enzymes (UbcH5b), UbcH (E2) enzyme, and biotinylated ubiquitin were purchased from BostonBiochem . The autoubiquitination assays of the MID1 E3 ligase domains were performed in a total volume of 30 µL. The mixture contained 1 unit (generates 1 µmol of phosphate per minute) of thermostable inorganic pyrophosphatase (New England Biolabs), 1 mM DTT, 5 mM ATP-Mginvitro ubiquitination studies using recombinant full-length MID1 and PP2Ac. MID1 was constructed with a maltose-binding protein (MBP) tag at its N-terminus to enhance solubility.To understand how mutations within the Bbox domains of MID1 might affect the regulation of PP2Ac, we began by determining whether MID1 could directly catalyze the ubiquitination of PP2Ac. To this end, we performed As shown in The general consensus in the literature is that alpha4, which interacts with PP2Ac and MID1, is required to recruit PP2Ac to the MID1 We had previously demonstrated that the E3 ligase activity of MID1 was localized to the N-terminal RING-Bbox1-Bbox2 (RB1B2) zinc-binding domains To determine how the RING and Bbox domain contributed to targeting and catalyzing the ubiquitination of PP2Ac, we performed the ubiquitination assay with the following domain constructs: RING (MID1 residues 1–92), RING-Bbox1 , RING-Bbox1-Bbox2 (residues 1–214) and Bbox1-Bbox2 . The LB1B2 protein construct included residues from the linker region between the RING and Bbox1 domains (residues 71–110) but not the RING domain. We previously observed that these four fragments have different levels of E3 ligase activity, as part of the standard autoubiquitination assay and in their ability to catalyze the polyubiquitination of alpha4 In addition to the UbcH5b (UBE2D2) E2 enzyme, our previous studies with MID1 indicated that the RB1B2 protein could interact with multiple E2 enzymes in catalyzing its own polyubiquitination and that of alpha4 To investigate how XLOS-observed mutations within the Bbox domains might affect MID1 E3 ligase functionality, we compared the autoubiquitination activity of the mutant RB1B2 constructs with that of wild type RB1B2 protein. Five mutations within the Bbox1 domain  and one within the Bbox2 domain (C195F) were designed. The A130S mutation, which is not associated with XLOS, was intended to distinguish the role of the hydroxyl from methyl group of the Thr130 mutation. These mutant RB1B2 proteins were expressed and purified similarly as the wild type RB1B2 protein. All mutant protein constructs were soluble and stable at the experimental conditions used.As shown with It is generally acknowledged in the literature that mutations of MID1 are associated with an increase in the amount of PP2Ac, suggesting that MID1 is involved in the regulation of PP2Ac In light of these results, we know that mutations within the Bbox domains are associated with physical abnormalities  and therWe performed the ubiquitination assays with full-length alpha4 using the RB1B2 mutants, and the wild type RB1B2 protein as control. The RB1B2 proteins harboring the C142S and C145T mutations were unable to catalyze the polyubiquitination of alpha4. Only a weak band corresponding to mono-ubiquitinated alpha4 was observed (The RB1B2 construct containing the A130T or A130V mutations was unable to catalyze the polyubiquitination of alpha4 (1PP2A and I2PP2A (see reviews In this study, we provide the first direct evidence that MID1 could catalyze the ubiquitin-modification of PP2Ac and therefore could regulate the function of PP2A . PreviouThe role alpha4 plays in regulating the ubiquitination of PP2Ac by MID1 appears to be complex. On one hand, alpha4 is shown to be a potent regulator of PP2Ac Consistent with our previous studies, the RB1B2 protein construct of MID1 is sufficient for catalyzing the ubiquitination of PP2Ac . It appeIn addition to understanding how natural mutations within the Bbox domains of MID1 affect its E3 ligase activity, these mutations also allows us to gain insights into the mechanism of Bbox domain function. The Cys142 and Cys145 residues are highly conserved  in Bbox1Residue Ala130 is also highly conserved in Bbox1 domains . Even thPerturbation of the Bbox1 domain structure could disrupt the interaction of RB1B2 protein with the alpha4 protein. In a non-biased functional screen, we previously identified a L146Q substitution that abolished the MID1-alpha4 interaction and the subsequent ubiquitination of alpha4 Qualitatively, the mutations within the Bbox1 domain did not affect the ability of the RB1B2 protein to catalyze the di-ubiquitination of PP2Ac. Given that the both RING domain and folded Bbox1 domain are required for catalyzing the diubiquitination of PP2Ac , the diuInterestingly, the RB1B2 protein with the C195F mutation was observed to catalyze the ubiquitination of PP2Ac and alpha4 , 5B. TheIt is intriguing to note that the different mutations within the Bbox1 domain, which should have resulted in same loss of structure and function, do not correlate with the same set of birth defects . The truIn conclusion, MID1 could regulate the function of PP2A by catalyzing the ubiquitination PP2Ac and alpha4. It is possible that many of XLOS-observed mutations, including mutations of the Bbox1 domain, result in an increase of unmodified or monoubiquitinated alpha4, which could subsequently bind and protect PP2Ac from ubiquitin-mediated degradation. Thus, alpha4 may have a significantly greater role in contributing to X-linked Opitz syndrome that includes protecting PP2Ac.


In this paper, we tackle the problem of domain adaptation of statistical machine translation (SMT) by exploiting domain-specific data acquired by domain-focused crawling of text from the World Wide Web. We design and empirically evaluate a procedure for automatic acquisition of monolingual and parallel text and their exploitation for system training, tuning, and testing in a phrase-based SMT framework. We present a strategy for using such resources depending on their availability and quantity supported by results of a large-scale evaluation carried out for the domains of environment and labour legislation, two language pairs (English–French and English–Greek) and in both directions: into and from English. In general, machine translation systems trained and tuned on a general domain perform poorly on specific domains and we show that such systems can be adapted successfully by retuning model parameters using small amounts of parallel in-domain data, and may be further improved by using additional monolingual and parallel training data for adaptation of language and translation models. The average observed improvement in BLEU achieved is substantial at 15.30 points absolute. Recent advances in statistical machine translation (SMT) have improved machine translation (MT) quality to such an extent that it can be successfully used in industrial processes . In practice, however, it is often difficult to obtain sufficient amounts of in-domain data  to train a system with good performance for a specific domain. The main problem is usually vocabulary coverage: domain-specific texts typically contain a substantial amount of special vocabulary, which is not likely to be found in texts from other domains  system to the particular domain of interest  and labour legislation (lab), and two language pairs: English–French (EN–FR) and English–Greek (EN–EL). The crawled data is further exploited for domain adaptation of a general-domain SMT system in several ways: by domain-specific parameter tuning of the main log-linear model and by adaptation of its components. The evaluation experiments carried out in a total of eight evaluation scenarios  confirm substantial and consistent improvements in translation quality for all approaches compared to the baseline.These workflows are then employed to acquire monolingual and parallel data for two domains: environment  translation table entries. Tuning with and for specific domains (while using generic training data) allows the MT system to stitch together translations from smaller fragments which, in this case, leads to improved translation quality. Such tuning requires only small development sets which can be harvested automatically from the web with minimal human intervention; no manual cleaning of the development data is necessary.In addition, additional improvements are realised by using monolingual and/or parallel in-domain training data. Adaptation of language models focuses on improving translation fluency and lexical selection for the particular domain. Adaptation of the translation model then aims at reduction of the out-of-vocabulary (OOV) rate and adding domain-relevant translation variants. All the data sets are available via the European Language Resources Association (ELRA).This paper is an extended and updated version of our previous work published as Pecina et al. , b. CompThe remainder of the paper is organised as follows. After the overview of related work and description of the state-of-the-art in Sect. In this section, we review the current state-of-the-art in the area of web crawling for monolingual as well as parallel data and briefly describe the main concepts of phrase-based SMT (PB-SMT) and its adaptation to specific domains.Web crawling is the automatic process of travelling through the World Wide Web by extracting links of already fetched web pages and adding them to the list of pages to be visited. The selection of the next link to be followed is a key challenge for the evolution of the crawl and is tied to the goal of the process. For example, a crawler that aims to index the web as a whole may not prioritise the links at all, while a focused/topical crawler that aspires to build domain-specific web collections , since they are of only limited or no value for the purposes of studying language use and change  model  if any in-domain data is available, it can be directly used to improve the MT system, e.g., by combining the (limited) in-domain with (more extensive) out-of-domain resources for training; (2) if in-domain data exists but is not readily available, one may attempt to acquire domain-specific data ; (3) finally, if sources of in-domain data cannot be identified, one may attempt to select pseudo in-domain data 〉. If the terms are publicly available online, as is often the case, this approach does not require any domain expertise.Domain-focused web crawling aims to visit (and store) web pages relevant to a specific domain only. A critical issue is the construction of the domain definition see , since eenv; labour law and labour relations, organisation of work and working conditions, personnel management and staff remuneration, employment and labour market for lab. Information about subdomains can prove useful in acquiring more focused collections.For our experiments, we selected English, French, and Greek terms (both single- and multi-word entries) from the “Environment”  and “Employment and Working Conditions”  domains of the EuroVocenv domain is given in Table Each entry was manually assigned a weight indicating the term’s domain relevance, with higher values denoting more relevant terms. Even though a domain expert is required to differentiate relevant terms and assign various weights to them, initial experiments showed that a domain-specific corpus can be constructed  and their weights. The page relevance score nd Golub :\documennd Golub . If \docEven when a page is not classified as relevant, it is still parsed and its links are extracted and added to the list of links to be visited. The fact that we keep links from non-relevant pages allows us to exploit the Tunnelling strategy . Duplicate web pages were detected and removed based on MD5 hashes, and near-duplicates were eliminated by employing the deduplication strategy implemented in the Nutch framework, which involves construction of a text profile based on quantised word frequencies.As shown in column 10 of Table lab domain are much higher than the ones for env for all languages. This is explained by the fact that the web pages related to lab are often legal documents or press releases replicated on many websites.The entclass1pt{minimalab and env, respectively. These datasets are available from the ELRA catalogueThe final processing of the monolingual data was performed on paragraphs marked by Boilerpipe and the language identifier. The statistics from this phase are presented in Table env and lab in English are presented in Table lab data, respectively. This is due to the popularity of these subdomains in comparison with the rest, as well as the fact that the crawler’s goal was to acquire in-domain web pages without a requirement to build corpora balanced equally across subdomains. Another observation is that many documents were classified as parts of two subdomains. For example, 38.09 % of the documents in the English env collection were categorised in both “deterioration of the environment” and “natural environment”. This is explained by the fact that many terms of the domain definition were assigned to more than one subdomain. In addition, many crawled pages contain data relevant to these neighbouring subdomains.Statistics about the distribution of the subdomains of We now describe the procedure for acquisition of parallel data. To this end, we used the focused crawler's bilingual mode of operation (FBC), which is also available as a web service.To guide FBC we used bilingual domain definitions, which consisted of the union of monolingual domain definitions in the targeted languages for the selected domain. In order to construct the list of seed URLs, we manually selected web pages that were collected during the monolingual crawls and originated from in-domain multilingual web sites. We then initialised the crawler with these URLs and forced the crawler to follow only links internal to these sites. By adopting the same crawling strategy mentioned in the previous subsection, FBC follows the most promising links and continues crawling the web site until no more internal links can be extracted.After downloading in-domain pages from the selected web sites, we employed Bitextor to identify pairs of pages that could be considered translations of each other. Specifically, for each candidate pair of pages, we examine the relative difference in file size, the relative difference in length of plain text, the edit distance of web page fingerprints constructed on the basis of HTML tags, and the edit distance of the lists of numbers in the documents. If all measures are below corresponding thresholds, as those are defined in the default configuration of Bitextor, the pair under consideration is considered a pair of parallel pages. The amount of the acquired in-domain bilingual data is reported in columns 3 and 4 of Table After identification of parallel pages, the next steps of the procedure aim at extraction of parallel sentences, i.e., sentence pairs that are likely to be mutual translations. For each document pair free of boilerplate paragraphs, we applied the following steps: identification of sentence boundaries by the Europarl sentence splitter, tokenisation by the Europarl tokeniser Koehn , and senFor each sentence pair identified as parallel, Hunalign provides a confidence score, which reflects the level of parallelism, i.e., the degree to which the sentences are mutual translations. We manually investigated a sample of sentence pairs extracted by Hunalign from the data pool for each domain and language pair (about 50 sentence pairs for each language pair and domain), by relying on the judgement of native speakers, and estimated that sentence pairs with a score above 0.4 are of sufficient translation quality. In the next step, we kept sentence pairs with 1:1 alignment only (one sentence on each side) and removed those with scores below this threshold. Finally, we also removed duplicate sentence pairs.The statistics from the parallel data acquisition procedure are displayed in Table The translation quality of a PB-SMT system built using the parallel sentences obtained by the procedure described above might not be optimal. Tuning the procedure and focusing on high-quality translations is possible, but leads to a trade-off between quality and quantity. For translation model training, high translation quality of the data is less essential than for tuning and testing. Bad phrase pairs can be removed from the SMT translation tables according, for example, to significance testing  and asked native speakers to check and correct them. All 4 evaluators (2 for each language) were researchers with postgraduate education and significant experience in evaluation for NLP tasks. The task consisted of checking that the sentence pairs belonged to the right domain, the sentences within a sentence pair were equivalent in terms of content, and the translation quality was adequate and if not, correcting it.In order to create reliable test and development sets for each language pair and domain, we performed the following low-cost procedure. From the data obtained by the steps described in Sect. Our goal was to obtain at least 3,000 correct sentence pairs  for each domain and language pair. Accordingly, in order to speed up the process, we did not instruct the correctors to amend every sentence pair, but rather allowed them to skip (remove) any sentence pairs that were misaligned. In addition, we asked them to remove those sentence pairs that were obviously from a very different domain (despite being correct translations). The number of manually verified and (if necessary) corrected sentence pairs is presented in the last column in Table According to the human judgements, 53–72 % of sentence pairs were accurate translations, 22–34 % needed only minor corrections, 1–3 % would require major corrections , 2–5 % of sentence pairs were misaligned and would have had to be translated completely (which was not necessary in most cases), and about 4 % of sentence pairs were from a different domain (though correct translations). Detailed statistics collected during the corrections are presented in Table In the next step, we selected 2,000 pairs from the corrected sentences for the test set and left the remaining part for the development set. Those parallel sentences which were not sampled for the correction phase were added to the training sets. The correctors confirmed that the manual corrections were about 5–10 times faster than translating the sentences from scratch, so this can be viewed as a low-cost method for acquiring in-domain test and development sets for MT. Further statistics of all parallel data sets are given in Table We now present our experimental set-up, the baseline  system and its performance. Our primary evaluation measure is BLEU  and domain-specific . We present the results in Table A number of previously published experiments , where similar expressions and phrases often recur. We found that about 5 % of EN–FR development and test sentence pairs also occur in the training data . The duplicates were probably added to later versions of Europarl after the WPT 2005 test sets were released, but this does not affect our domain-adaptation experiments presented in this paper.env domain and 22.47 BLEU points (44.84 % relative) on the lab domain (see columns denoted by gen test data in the training data), the drop in translation quality is evident. It is caused by the divergence of training and test data, which is also illustrated by the increase of the OOV rates (ratios of untranslated words) and perplexity (PPL) of the reference translations of the test sets given language models trained on the target side of the parallel training data (this reflects how well the language model reflects the characteristics of the target language). For both measures, lower scores indicate a better fit.Switching from general-domain to domain-specific test sets yields an average absolute decrease of 21.16 BLEU points (48.22 % relative) on the gen domain to 1.12 % on the env domain and 0.67 % on the lab domain, and the average perplexity increased from 56.6 on the gen domain to 106.4 on the env domain and 98.5 on the lab domain (see Table gen) to specific  domain and makes scoring of hypotheses during decoding difficult. An interesting case is the EN–EL translation direction, where the highest perplexity is surprisingly achieved on the gen domain. This is probably due to the morphological complexity of the target language and nature of the particular test set. After a thorough analysis of the Greek side of this test set, we discovered some inconsistency in tokenisation (introduced by the providers of the data) which contributed to the higher PPL value. This does not, however, influence the findings in this work. In all other cases, perplexity increases for domain-specific data.The OOV rate increases from an average of 0.25 % on the Optimisation of the log-linear combination parameters, which most modern SMT systems are based on, is known to have a big influence on translation performance. A sensible first step towards domain adaptation of a general-domain system is to use in-domain development data. Such data usually comprises a small set of parallel sentences which are repeatedly translated until the model parameters are adjusted to their optimal values.B0), the systems trained on general-domain data and tuned on in-domain data (denoted as P1) improve BLEU by 6.64 points absolute (24.82 % relative) on average . The average absolute improvement over the baseline system B0 is 6.54 BLEU points, which is only 0.1 points less than the score obtained by the P1 systems. In practice, this finding makes the manual correction of development data acquired by our procedure unnecessary since the results obtained using raw parallel data are comparable.A small amount of manual effort was put into the manual correction of the test as well as development data acquired for the specific domains  weight vectors that perform equally well or even better. For this reason, the actual parameter values are not usually investigated. Our experiments, however, show that the parameter values and their changes observed when switching from general-domain to domain-specific tuning are in fact highly consistent, indicating interesting trends  of the general-domain tuned systems (black bars) indicate that the phrase pairs in the systems’ translation tables apply well to the development data that are from the same domain as the training data; a high reward is given to translation hypotheses consisting of phrases with high translation probability . The low negative weights assigned to phrase penalty) imply that the systems prefer hypotheses consisting of fewer but longer phrases. Reordering in the hypotheses is not rewarded and therefore not explicitly preferred  is negative for the systems translating from English and slightly positive for systems translating into English. This reflects the fact that translation from English prefers shorter hypotheses (fewer words), while translation into English prefers longer hypotheses (consisting of more words). This is probably due to the relative morphological complexities of English and the other languages.The high weights assigned to direct phrase translation probability) decrease rapidly, with this weight being close to zero in some scenarios. The translation tables do not provide enough good quality translations for the specific domains, and the best translations of the development sentences consist of phrases with varying translation probabilities. Hypotheses consisting of few (and long) phrases are not rewarded any more (weights of Comparing these findings with the results of the systems tuned on the specific domains (grey bars), we observe that the weights of As can be seen in Fig. From the analysis presented above, we conclude that a PB-SMT system tuned on data from the same domain as the training data strongly prefers to construct translations consisting of long phrases. Such phrases are usually of good translation quality , fluent (formed by consecutive sequences of words), and recurrent (frequent in data from the same domain). Accordingly, they form good translations of the input sentences and are preferred during decoding. This is, of course, a positive trait when the system translates sentences from the same domain. However, if this is not the case and the input sentences contain very few longer phrases from the translation tables, the general-domain tuned system is not able to construct good translations by preferring the longer and (for this domain) inadequate phrases. In this case, shorter phrases could enable better translations to be stitched together.To support this hypothesis, we analysed the phrase length distribution actually seen in the translation of the test sets. The average phrase lengths estimated for various combinations of tuning and test domains and all language pairs are shown in Table Generally, a higher divergence of the test domain from the training domain leads to shorter phrases being used in translation. However, when the systems tuned on general-domain data are applied to specific domains, the average phrase lengths are consistently longer than for domain-specific tuning. The systems are tuned to prefer long phrases but the translation quality is lower. This situation can be interpreted as overtraining; the model overfits the training (and tuning) data and on a different domain fails to form the best possible translations . Nevertheless, preferring translations constructed of shorter phrases (even single words) is not always better. For example, word-by-word translation of non-compositional phrases would generally be erroneous.lab and testing on env, and vice versa), see Table P3. In three scenarios (bold figures), such systems perform as well as the in-domain tuned ones . In the other scenarios, the absolute difference in BLEU is less than 0.4 points. The average gain over the systems tuned on the general domain (B0) is 6.38 points absolute (compared with 6.64 points obtained by P1). This observation is not very intuitive. One would expect that each domain would require specific tuning. However, it seems that the in-domain tuning does not optimize the general-domain trained system to a particular specific domain, but rather to any domain diverging from the general domain in a similar way .As we have already shown, in-domain tuning represents a way to effectively reduce such overfitting. The problem, however, can also be reduced by cross tuning, i.e., tuning on specific domains different from the test domains , the results are very close to those of systems tuned on in-domain data (P1). The average absolute improvement of the systems with default parameters (P4) over the systems tuned on general domain is 4.42 BLEU points (compared with 6.64 points obtained from domain-specific tuning on average).For comparison purposes only, we also report results of non-tuned systems Often, domain-specific parallel data is scarce, or completely unavailable for many vertical sectors and must be prepared by manual translation of monolingual in-domain sentences. We thus investigate how much development data is needed. The only technical requirement is that the parameter optimisation method  must converge in a reasonable number of iterations. For this reason, typical development sets contain about 1,000–2,000 sentence pairs (cf. the size of development sets provided for the WMT translation shared tasks). We vary the amount of sentences in our development sets, tune the systems, test their performance on the test sets and plot learning curves to capture the correlation between translation quality (in terms of BLEU) and gradual increases in the size of the development data.gen/env and gen/lab in Fig. The general shapes of the curves are consistent across all language pairs and thus we provide the curves for the EN–FR translation direction only see Fig. . IncreasIn this section, we explore the potential of adapting the components of the SMT model (language and translation models) by exploiting the crawled domain-specific data in addition to the general-domain data used for training the baseline systems.Improving an SMT system by adding in-domain monolingual training data cannot reduce OOV rates nor introduce new phrase pairs into the translation models. Such data can, however, improve the language models and contribute to better estimates of translation fluency and thus help select better translation hypotheses.In general, there are two ways of using monolingual data for adaptation of the SMT model: the trivial approach is to retrain the existing language model on a simple concatenation of the original general-domain data and the new domain-specific data; a more advanced approach is to build an additional language model based on the domain-specific data only and use it together with the original one. This is possible in two ways  development data, and L3 employs two models combined in the log-linear fashion using weights tuned by MERT on BLEU. The complete results are presented in Table P1), all three methods significantly improve translation quality across all scenarios. In general, the most efficient approach is linear interpolation with an average absolute improvement of 4.88 BLEU points (14.95 % relative). With two exceptions, systems L2 outperform both L1 and L3. In most cases, the improvement is statistically significant. For EN–EL (both directions) in the lab domain, L2 is outperformed by simple concatenation (L1), but this can be explained by the size of the development data used to optimize the interpolation coefficient in L2 (506 sentences), which is probably insufficient. Substantial improvements in BLEU over the system P1 are achieved especially for translations into Greek  despite the smallest size of the monolingual data acquired for this language , T2 (linear interpolation of the two translation models), T3 (two independent translation models in log-linear combination), and compared with the in-domain-tuned systems (P1) in Table T2 and T3) outperform the trivial one (system T0), the difference between the two is marginal. The average increase in BLEU for T2 over T1 is 4.35 points absolute (13.11 % relative) and for T3 over P1 4.56 points absolute (13.87 % relative). In three of the eight scenarios, the difference is not statistically significant, T2 is significantly better in two scenarios, and T3 is better in three scenarios , while in other scenarios the increase in BLEU is between 0.33 and 4.35 points absolute only. This can be explained by the better match between the training and test data, which is evident from the decrease in perplexity of the reference translations given the target language models, as discussed in the following section. This is likely to be caused by the size of the in-domain parallel training data for this language pair and domain which is more than twice as large compared to the EN–FR env data and more than three times larger compared to the EN–EL data, both for the env and lab domains  and the T2 increases BLEU by 4.35 points absolute , but in most scenarios the difference is not statistically significant  improves BLEU by 6.64 points absolute (24.82 % relative). Components of the log-linear combination do not change, so OOV and perplexity remain the same. The average phrase length dropped from 2.63 to 2.04 words, i.e., by 22.5 %. The adapted language model  increased the gain in BLEU to 11.52 points absolute (43.73 % relative). The perplexity of the reference translations given the target language models dropped by 45.4 % on average. The average phrase length decreased to 1.87 words. The language model matches the test data domain better and helps to select better translation hypotheses, which consist of even more  phrases.The main results in terms of BLEU are presented in Table C2) boosts the average relative improvement in BLEU to 15.30 points absolute (58.37 % relative). This step introduces new translation phrase pairs into the translation model and decreases the OOV rate. Compared to the baseline (B0), OOV drops by 30 % on average. In some scenarios (the EN–FR translation in the lab domain), OOV decreases by as much as 50 %, which is a sign of a better match between the test and training data. The target side of the parallel data also improves the language models, with their perplexity falling by an average of 67.5 % relative. The new in-domain material in the translation models also leads to longer phrases being used in the best-scored translation hypotheses. The average phrase length increased compared to the systems with adapted language models only (L2) by almost 20 % to 2.18 words.Finally, adaptation of the translation model , which illustrates the pure effect of such training data. Although with one exception (EN–EL translation of the lab domain), these systems outperform the baseline (B0), the requirement of using general-domain data is evident in all scenarios. The average difference in BLEU of the fully adapted systems (C2) and the systems trained on specific data only (C0) is 7.56 points absolute.For comparison, Table env domain (the trends on the lab domain are the same). Compared to the baseline (B0), the in-domain tuned systems (P1) do not trust the translation model that much and prefer hypotheses consisting of more phrases which are shorter and more reordered. The weight vectors of systems L2 do not change much. A consistent increase, however, is observed for both the language model weight (C2) changed only slightly. A consistent change is observed for the phrase penalty  and two domains , which allowed us to perform a large-scale evaluation using a total of eight test scenarios. The acquired data sets are available from ELRA.Our domain-adaptation experiments focused on the following three components of a PB-SMT model: parameters of the log-linear combination and their optimisation, language model, and translation model. First, we confirmed the observation from previous research that systems trained and tuned on general domain perform poorly on specific domains. This finding is not surprising, but the amount of loss and the fact that it is observed consistently was rather unexpected. The average absolute decrease in BLEU in all the domain-specific evaluation scenarios was 21.82 points (37.86 % relative).We confirm the results of previous research on tuning-only adaptation. Tuning the general domain-trained systems on specific target domain data recovers a significant amount of the loss. Several hundreds of sentence pairs used as development data improved the BLEU score of the baseline tuned on general-domain data by 6.64 points absolute (24.82 % relative) on average. A detailed analysis of the model parameters and phrase length distribution in translations of the test data found that a system trained and tuned on general domain data strongly prefers long and few phrases in the output translations, and thus underperforms on specific domains where such phrases do not occur so frequently. In contrast, the same systems tuned on domain-specific data produce output translations from shorter phrases, allow specific reordering and perform significantly and consistently better on specific domains.Importantly, our findings show that the development data does not have to be manually cleaned and corrected, as parameter tuning on the development set  is quite tolerant to imperfect translations and eventual noise in the development sets. Cross-domain tuning on a different set also offers a good solution when no in-domain development data is available, especially when the domains differ in a similar way. This step has the effect of tweaking the original general-domain system towards shorter phrases and it does not matter much which different development sets are used.The experiments with language model adaptation confirmed previous results. Linear interpolation of the general-domain and domain-specific models increased translation quality by a further 4.88 BLEU points absolute (14.95 % relative) compared to the general-domain systems tuned on in-domain development sets on average and significantly outperformed other techniques (concatenation of training data and log-linear combination of the two models). Adaptation of translation models  increased BLEU scores by 4.56 points absolute (13.87 % relative) compared to the general-domain systems tuned on in-domain development sets. In this case, linear interpolation and log-linear combination produced similar results. In the combined approach, we observed that the effect of using in-domain monolingual and parallel data is largely independent and does not cancel out when these two types of resources are used at the same time. The final BLEU scores increased by 3.78 points absolute (9.66 % relative) with respect to the language-model-adapted systems, by 8.66 points absolute (26.43 % relative) with respect to the in-domain tuned systems, and by 15.30 points absolute (58.37 % relative) with respect to the general-domain baseline, all on average.The pipeline for domain-focused web crawling described in this work proved to be very successful in acquisition of domain specific data—both monolingual and parallel. The experiments then showed a high impact of the acquired resources on domain adaptation of MT. We mainly concentrated on parameter tuning and analysis of its effects. Although we did not especially focuse on adaptation of language models and translation models, the acquired data also significantly improved these components and translation quality in general.