The transition from unicellular to multicellular organisms poses the question as to when genes that regulate cell-cell interactions emerged during evolution. The receptor and ligand pairing of plexins and semaphorins regulates cellular interactions in a wide range of developmental and physiological contexts. We surveyed here genomes of unicellular eukaryotes and of non-bilaterian and bilaterian Metazoa and performed phylogenetic analyses to gain insight into the evolution of plexin and semaphorin families. Remarkably, we detected plexins and semaphorins in unicellular choanoflagellates, indicating their evolutionary origin in a common ancestor of Choanoflagellida and Metazoa. The plexin domain structure is conserved throughout all clades; in contrast, semaphorins are structurally diverse. Choanoflagellate semaphorins are transmembrane proteins with multiple fibronectin type III domains following the N-terminal Sema domain (termed Sema-FN). Other previously not yet described semaphorin classes include semaphorins of Ctenophora with tandem immunoglobulin domains (Sema-IG) and secreted semaphorins of Echinoderamata . Our study also identified Met receptor tyrosine kinases (RTKs), which carry a truncated plexin extracellular domain, in several bilaterian clades, indicating evolutionary origin in a common ancestor of Bilateria. In addition, a novel type of Met-like RTK with a complete plexin extracellular domain was detected in Lophotrochozoa and Echinodermata (termed Met-LP RTK). Our findings are consistent with an ancient function of plexins and semaphorins in regulating cytoskeletal dynamics and cell adhesion that predates their role as axon guidance molecules. Plexins were first described as cell surface molecules in the nervous system of Xenopus tadpoles12 and as molecules with sequence similarity to the extracellular domain of Met receptor tyrosine kinases (RTKs)13.Semaphorins were identified as repulsive axon guidance molecules in grasshopper and in chick15. Subsequent studies revealed that Drosophila melanogaster also has a class 5 semaphorin, thus class 5 semaphorins are present in both vertebrates and invertebrates16. The viral semaphorins appear to be all derived from class 7 semaphorins17. The plexins of vertebrates were grouped by sequence similarity into 4 classes (class A-D)18.Semaphorins of bilaterian Metazoa have been grouped by sequence similarity into two invertebrate classes (class 1 and 2) and five vertebrate classes (class 3–7), plus one class for viral semaphorins (class V)19. The Sema domain is exclusive of plexins, semaphorins, and Met RTKs19. Plexins and semaphorins bind to each other through their respective Sema domains22. Met RTKs, on the other hand, bind to secreted growth factors such as hepatocyte growth factor (HGF) that are structurally different from semaphorins23.Both plexins and semaphorins contain a structural hallmark: an N-terminal Sema domain, which is a seven-blade beta-propeller, with each blade formed by four anti-parallel beta-strands24. To date, Neuropilins are considered exclusive of vertebrates25, although in Caenorhabditis elegans, the L1CAM homolog LAD-2 acts as co-receptor for secreted semaphorins26, suggesting that invertebrates may utilize other co-receptors for the binding of secreted semaphorins.For the binding of secreted class 3 semaphorins, neuropilin transmembrane proteins act as required co-receptors in a complex with plexins27. Plexins also contain extracellular IPT domains (immunoglobin-like fold shared by plexins and transcription factors), which are ~95 aa in size, forming a beta-sheet bundle with two central conserved cysteines27. It has been an intriguing finding from crystallographic studies that the plexin extracellular domains form together a ring structure, which consists of a Sema domain followed by three consecutive PSI-IPT domains and three more IPT domains, i.e. Sema-PSI-IPT-PSI-IPT-PSI-IPT-IPT-IPT-IPT-TM29. Exceptions to this stereotypic domain pattern are Plexin-B1, which carries an insert in its second PSI domain, and Plexin-C1, which lacks one PSI and two IPT domains.The Sema domain of plexins is typically followed by a PSI domain , which is ~50–60 aa in size and contains up to 8 conserved cysteines with a central conserved CxWC motif 30. The Rap1/2 G proteins have a pleiotropic network of effectors that control cytoskeletal dynamics and cell adhesion processes31.The intracellular part of plexins contains a Ras-GAP (GTPase activating protein) domain, which inactivates small G proteins of the Rap1/2 families33. In the current study, we surveyed plexins and semaphorins from unicellular eukaryotes to complex metazoan organisms to track their evolutionary origin and to describe their intra- and extracellular domains arrangements. Remarkably, we detected plexins and semaphorins in choanoflagellates, a group of unicellular eukaryotes closely related to Metazoa34. Our phylogenetic analyses suggest that plexins and semaphorins emerged in a common ancestor of choanoflagellates and Metazoa. Whereas plexins have maintained a stable domain arrangement throughout all clades, semaphorins have evolved into diverse classes with different domain arrangements. Interestingly, our analyses of Sema domain-containing proteins also revealed a novel type of Met-like RTK that contains a complete plexin ectodomain structure.Previous genome sequencing studies have recognized that plexins and semaphorins have an early origin in metazoan evolutionWe compiled predicted protein sequences of Sema domain-containing proteins  from genome databases of unicellular eukaryotes and Metazoa by keyword searches  and BLAST searches (see Methods). Protein domains were annotated using conserved domain and secondary structure prediction algorithms (see Methods).C. elegans, D. melanogaster, and H. sapiens18. Plexins of each species were numbered starting with the suffix -1 for the molecule that branches most basally from the phylogenetic tree. Plexins forming monophyletic groups within a species (i.e. that duplicated within a species) were numbered starting with the suffix -A1. For semaphorins, we followed the bilaterian naming conventions, except for the semaphorins that were distinct in domain arrangement from previously described classes and were therefore assigned new class names with numerical index. The protein names of this study should be understood as preliminary effort, and future committee work will be required to revise the nomenclature.Protein name assignment was guided by our phylogenetic analysis based on Sema plus PSI domain sequences, protein domain arrangements, and naming conventions for related proteins in 35. No Sema domain-containing proteins were detected in the genomes of the ichtyosporean Creolimax fragrantissima36 or the filasterean Capsaspora owczarzaki37. In contrast, one plexin and one semaphorin each were found in the genomes of the choanoflagellates Monosiga brevicollis and Salpingoeca rosetta39.To assess if plexins and semaphorins are present in unicellular eukaryotes, we first examined species of the clades Ichtyosporea, Filasterea, and Choanoflagellida, which are phylogenetically closely related to MetazoaFNIII domains. No conserved intracellular domains were detected in Sema-FN1.The choanoflagellate plexins, which we termed Plexin-1 Fig. , are remM. brevicollis and S. rosetta with those of human plexins and generated a conservation plot , which is thought to have a regulatory function40, is less conserved, suggesting that regulation of plexin activity may involve different Rho family members in diverse ways. The conservation pattern of the Sema domain indicated a seven blade propeller pattern. Likewise, protein secondary structure prediction (JPRED) of the choanoflagellate Plexin-1 Sema domain 20 and Nogi et al. (2010)22 have proposed that such residues maintain the structural integrity of the Sema domain in humans. In contrast, residues that are part of known binding surfaces of plexins and semaphorins were not highly conserved, perhaps reflecting the diversity of plexin/semaphorin receptor/ligand pairings , Ctenophora (comb jellies), Placozoa, and Cnidaria, four non-bilaterian lineages that form the sister group of the remaining MetazoaAmphimedon queenslandica43, we detected two full-length plexins with conserved domain structure, termed Plexin-1 and Plexin-A5 , as well as Sema-1A to -1C, which contain a Sema plus PSI domain.In the genome of the sponge -A5 Fig. , and fouMnemiopsis leidyi32 four plexins, termed Plexin-1 to -4  domains  domains following the Sema plus PSI domain.-A2 Fig. . The preNematostella vectensis, a member of the taxon Cnidaria45, we detected a single Plexin-1 and a single transmembrane semaphorin with five detectable TSP1 domains, termed Sema5A  and D. melanogaster (flybase.org).Protostomia and Deuterostomia are the two main clades of bilaterian Metazoa. We first analyzed Protostomia by surveying the genomes of the annelid C. teleta and A. californica, we detected two Plexins, termed Plexin-1 and -2 , which contains an extracellular domain with a complete plexin ectodomain structure and an intracellular tyrosine kinase domain  to represent the clade Vertebrata.To survey Deuterostomia, we analyzed the genomes of the starfish A. planci, we detected a substantial expansion of the plexin, semaphorin, and Met-LP families , termed Plexin-B1 to -B8. In addition, one Met receptor tyrosine kinase was found, along with four Met-LPs. The semaphorins of A. planci fall into four classes: one semaphorin is a member of class 5, termed Sema5A; one a member of class 6, termed Sema6A; four secreted semaphorins that carry after the Sema domain an atypical PSI-like domain (lacking some conserved cysteines) and an Ig domain, termed Sema-SI1 to -SI4 ; and six secreted semaphorins that carry a Sema plus PSI domain, termed Sema-SP1 to -SP6.In the genome of ies Fig. . InteresB. belcheri a compact set of plexins and semaphorins, with two plexins, Plexin-1 and -2, and three semaphorins, Sema5A, 6A, and 6B, and also one Met  and semaphorins in detailed trees that included Bayesian posterior probabilities Fig. . The tresee Figs ,S5.FigurA. queenslandica; Aquee) plexins and Ctenophora  Plexin-1. The next step of the tree showed a polytomic clade (PP = 0.78), containing as clades a Ctenophora sequence (Mleid Plexin-2), an Annelida protein , and a major third polytomic clade (PP = 0.71) that contains all other plexin sequences.Following the topology of the phylogenetic hypothesis inferred for plexins Fig. , we obseC. elegans; Celeg Plexin-1), the second branch (PP = 0.67) contains the remaining non-bilaterian plexins, with proteins of Placozoa , Ctenophora (Mleid Plexin-4), and Cnidaria , and the third branch (PP = 0.53) containing only Protostomia and Deuterostomia sequences.The first branch of this polytomy is a plexin of Nematoda , and a clade that branches into one clade with Smari Plexin-1 and Dmela Plexin-A and another one with all Deuterostomia Plexin-1/Plexin-A proteins . The second major branch (PP = 0.74) has the Mollusca Acali Plexin-2 as a sister species of a clade (PP = 0.62) divided in two: the first one comprising Echinodermata Aplan Plexin-Bs and Aplan Met-LPs (PP = 1.0), and the second comprising Branchiostomidae Bbelc Plexin-2 and human Plexins-Bs, Plexin-C1, and -D1 (PP = 0.76).The first branch (PP = 0.87) of this Protostomia/Deuterstomia-only clade contains all Met sequences, and also Celeg Plexin-2. The second branch (PP = 0.62) splits again into two major branches. One major branch (PP = 0.55) contains a clade with Protostomia Ctele Plexin-2 and Acali Plexin-1, and the Met-LPs of these taxa; and a clade with plexins of Insecta and Chilopoda  splits into two major branches, of which the first clade (PP = 0.99) contains all the Porifera Aquee sequences of this tree. The second clade (PP = 0.92) is divided into two other clades, the first contains only Echinodermata sequences (PP = 1.0) and splits into two other clades, the first (PP = 1.0) containing the Aplan Sema-SI class and the second (PP = 1.0) the Aplan Sema-SP class. The second clade (PP = 0.99) is divided into two clades, the first (PP = 0.97) contains the Protostomia Sema2s while the other comprises the remaining semaphorins (PP = 0.66). This clade is divided into two further clades, the first (PP = 0.96) comprises the Sema5 class, in which all members contain a series of thrombospondin domains. The second clade (PP = 0.98) splits into two clades: the first (PP = 0.86) with semaphorins of Protostomia (Sema1 class) and Deuterostomia (Sema6 class); and the second (PP = 0.99) with Bbelc Sema6B plus the Hsapi Sema3, 4, and 7 sequences.The phylogenetic and structural data led us to a preliminary model about the evolutionary origin of plexins and the different semaphorin classes Fig. . PlexinsA. planci may have occurred through repeated duplication of a chromosomal segment containing a Plexin-B next to a Met-LP gene. Loss of Met genes may have occurred in the lineage leading to Ecdysozoa.Met and Met-LP RTKs are detected in the clades Lophotrochozoa and Echinodermata, and only Met RTKs are detected in the clade Chordata Fig. . No Mets35. It has been proposed that choanoflagellates and Metazoa form together the clade Choanozoa34. As plexins and semaphorins are so far detected in all examined Choanozoa taxa, they may thus be considered a synapomorphy (feature unique to a clade) of Choanozoa.We have presented here a report on the evolution of plexin, semaphorin, and Met RTK proteins. Remarkably, plexins and semaphorins are already present in unicellular choanoflagellates, suggesting that these gene families have emerged in a common ancestor of choanoflagellates and Metazoa, more than 600 million years ago29, and the intracellular part with its Ras-GAP domain30. In contrast to plexins, semaphorins display, -besides their obligatory N-terminal Sema domain-, highly diverse domain arrangements, which could indicate low topological constraint on how semaphorins are presented as ligands to plexin receptors.The domain structure of plexins is largely conserved throughout all clades, suggesting that the receptor function of plexins is closely linked to their structural topology. This applies to both the extracellular parts of plexins, which form a ring structure55. The plexins and semaphorins described in this study may also contain PDZ-binding motifs, but as PDZ-binding motifs are highly diverse in their sequences56, confident determination of PDZ binding will have to await experimental testing.Of note, several plexins and semaphorins of vertebrates carry C-terminal PDZ-binding motifs, which provide a binding site for PDZ-binding domains of proteins such as PDZ-RhoGEF, GIPC1, or PSD-9557. The plexins and semaphorin gene families of Echinodermata and Vertebrata also underwent significant expansions that underlie the diversification of plexin/semaphorin functions in these organisms.Interestingly, the comb jelly (Ctenophora) has the largest number of semaphorins among non-bilaterian Metazoa, and this expansion may be related to the development of a complex nervous system in Ctenophora45. Our findings add to this concept in that plexins and semaphorins are already present in unicellular eukaryotes and may have been later adapted during evolution to regulate more complex intercellular interactions characteristic of Metazoa. Based on the structural similarity of the Sema domain with the beta-propeller domain of α integrins19, it could be speculated that the Sema domain evolved from a common ancestor that is shared with α integrins. Other components of plexins, such as the PSI, IPT, and Ras-GAP domains, are all present in several clades of unicellular eukaryotes (http://pfam.xfam.org/), suggesting that plexins may have formed in a common ancestor of choanoflagellates and Metazoa by domain shuffling of the evolutionary novel Sema domain with other existing domains.Previous studies of the genomes of unicellular Eukaryota and Metazoa have highlighted the early evolutionary appearance of cell adhesion machinery and related signaling pathways, including cadherins and integrins58, it is conceivable that plexins may act as negative regulators against positive cell adhesion forces from molecules such as cadherins and integrins. In this context, it is interesting to note that several choanoflagellate species can form facultative multicellular aggregates, e.g. rosettes34, and plexins may thus be involved in regulating the dynamics of these simple forms of multicellularity.What could be the original function of semaphorins and plexins? As the Ras-GAP activity of plexins seems to be directed towards small G-proteins of the Rap family, which are involved in spatio-temporal control of cell adhesionAn intriguing discovery during our survey of Sema domain-containing proteins are the Met-LP RTKs, which are different from Met RTKs by harboring a structurally complete plexin-like extracellular domain. The absence of Met-LPs from the classic genetic model organisms worm, fly, mouse, and other vertebrates may be the reason that this type of RTK, to our knowledge, has not yet been described. The Met and Met-LP genes may be under different selection constraints regarding ligand binding: Mets have growth factors as ligands that are different from semaphorins; the ligands of Met-LPs are unknown, but it is conceivable that Met-LPs maintained semaphorins as ligands. This interesting possibility awaits further experimental testing.In summary, the evolutionary data indicate that plexins and semaphorins predate the emergence of neurons in Metazoa, and that the axon guidance functions of plexins/semaphorins are therefore later specializations. The basic function of plexins and semaphorins may lie in balancing cell adhesion and cytoskeletal dynamics, thereby coordinating and adjusting cell-cell interactions and physical forces in unicellular and multicellular organisms.www.ncbi.nlm.nih.gov/gene) and Protein (www.ncbi.nlm.nih.gov/protein) databases, Ensembl Genome database (www.ensembl.org), the Uniprot protein database (www.uniprot.org), the Mnemiopsis Genome Project Portal (research.nhgri.nih.gov/mnemiopsis), the JGI genome portals for M. brevicollis (genome.jgi.doe.gov/Monbr1) and C. teleta (genome.jgi.doe.gov/Capca1), and the PFAM protein domain database (pfam.xfam.org). For the ichtyosporean C. fragrantissima and the filasterean C. owczarzaki, we searched by TBLASTN the NCBI genome assemblies at www.ncbi.nlm.nih.gov/assembly/GCA_002024145.1 and www.ncbi.nlm.nih.gov/assembly/GCF_000151315.2, respectively.To identify plexins, semaphorins, and Sema domain-containing Met-like RTKs in unicellular eukaryotes and Metazoa, we surveyed genome databases by keyword  and BLASTP and TBLASTN searches (using a Sema domain peptide sequences as query). Searches were conducted at the NCBI Gene , Expasy Prosite (prosite.expasy.org), and EBI HHMER (www.ebi.ac.uk/Tools/hmmer). Sema domains were further annotated by utilizing JPRED secondary structure prediction59 to identify beginning and end of the 7-blade beta-strand propeller pattern19 .Protein domain features were annotated with domain scan programs of NCBI CCD  and PHOBIUS (phobius.sbc.su.se) algorithms, and transmembrane domains with SOSUI (http://harrier.nagahama-i-bio.ac.jp/sosui) and TMHMM (www.cbs.dtu.dk/services/TMHMM) algorithms. Predicted protein domains are included in the GenPept files of Supplemental data as annotated features.Signal peptides were predicted with SignalP4.1 . Phylogenetic trees were inferred with MrBayes software62, taking in account two partitions (Sema and PSI domains). The program was set to find the best model of substitution during the analysis and found that the WAG model is the one that better describes the evolution of the proteins under study. Since the computational task was long, we used the CIPRES Portal (www.phylo.org)63 for the maximum time allowed (168 hours of eight servers) to complete the task. We obtained the consensus tree after 15 million steps of a Markov chain into two runs of four chains each (with 25% of burning). We examined the parameters convergence using Tracer software (github.com/beast-dev/tracer/releases/tag/v1.7.1) and visualized the tree using the FigTree software (tree.bio.ed.ac.uk/software/figtree). The infile containing the alignment and the script used here are included as supplemental data files.We performed alignments for phylogenetic analysis using the sequences of Sema domains and their adjacent PSI domains, which allowed construction of a tree including semaphorins, plexins, Mets, and Met-LPs. We aligned these sequences separately, using the algorithm M-Coffee of the T-Coffee software platform (tcoffee.crg.cat), which combines different alignment algorithms to produce an optimized alignment34 and mapped the sequences found in each genome into this representation .Supplementary MaterialTable S1File S1File S2File S3


The mobility of magnetic domains forms the link between the basic physical properties of a magnetic material and its global characteristics such as permeability and saturation field. Most commonly, surface domain structure are studied using magneto-optical Kerr microscopy. The limited information depth of approx. 20 nanometers, however, allows only for an indirect interpretation of the internal volume domain structures. Here we show how accumulative high-frame rate dynamic neutron dark-field imaging is able for the first time to visualize the dynamic of the volume magnetic domain structures in grain oriented electrical steel laminations at power frequencies. In particular we studied the volume domain structures with a spatial resolution of ∼100 μm and successfully quantified domain sizes, wall velocities, domain annihilation and its duration and domain wall multiplication in real time recordings at power frequencies of 10, 25 and 50 Hz with ±262.5 A/m and ±525 A/m (peak to peak) applied field. As such, even a small gain in efficiency, by the sheer size of the market, immediately results in a huge energy and cost saving. E.g. for the number quoted above a 0.2% increase in efficiency already compares to an average nuclear power plant’s power production of 1000 MW .Today’s global electricity market sizes to approximately 5 trillion watts. Large power transformers play an important role in this market, being indispensable for transporting electricity along high voltage transmission lines. In 2010, the United States’ the power demand for only large power transformers  alone was 127 309 MW for as reported inGrain-oriented electrical steels (GOES) which are typically used in transformer cores are so-called soft magnetic materials where bulk magnetic domain structure and the mobility of the domains walls determine the efficiency of transformers.2. As transformers are operated at frequencies of power networks the analysis of their surface domains is done with dynamic observation techniques like excitation/scanning synchronization3 and sampling stroboscopy4. Information about domain wall motion, wall spacing, bowing, nucleation and domain refinement of GOES at power frequencies are reported in5 where stroboscopic and even real time experiments are reported all together using light-based Kerr microscopy techniques. The disadvantage of Kerr microscopy is that the insulating coating, which is used in most transformer steel cores, has to be removed for the analysis to expose the surface of the steel. That removal of coating changes the underlying bulk and surface domain structure6. Surface dynamic domain observation can be done by high-voltage scanning electron microscope8 with insulating coating. Recent developments in imaging with magneto-optical indicator films (MOIF) make it possible to perform dynamic investigations at a frequency of 50 Hz while keeping the coating intact9. However, while MOIF enables the visual analysis of domain wall motion it is still limited to surface domain behavior. The experimental accessibility of magnetic domains within the volume of steel is limited to a special “freeze-in” technique, the Libovicky method11, which does not allow obtaining dynamic volume domain information. The combination of dynamic domain investigations with volume information will be presented in this article and will be made now accessible for experimentalists.Domain structures are commonly characterized statically with surface sensitive techniques such as magneto-optical Kerr microscopy9 recently emerged as a valuable complementary technique to the established domain observation methods because it is the only technique available that enables the spatially resolved analysis of bulk magnetic domain walls15 deep in the volume of materials and thus provides unique information. The contrast is based on the small-angle scattering of neutrons at domain walls and the consequent disruption of a predefined interference pattern. The potential has been shown for static radiography12 and tomography16 as well as magnetic domain response studies in static measurements with stepwise changing magnetic field excitation17. Furthermore time averaged measurements in alternating fields were performed and have been published by Betz et al.15 for various power frequencies and fields. The typically long exposure times of several minutes for a single DFI in nGI experiments with standard scintillator based detector systems did not allow to study the dynamic properties of magnetic domain structures in bulk GOES at power frequencies.The unique capability of neutrons to penetrate materials opaque to other non-destructive techniques, e.g. utilizing on light or electrons, makes it possible to study bulk behavior of magnetic domain walls while keeping the insulating coating intact and thus without modifying the domain structure. The dark-field image (DFI) of neutron grating interferometry (nGI)19. The schematical setup is shown in Fig. In this article, we introduce accumulative high-frame rate dynamic neutron dark-field imaging (ADDI) where a standard nGI setup is combined with a fast neutron counting detector providing both time and position for each detected neutron2SiO4) and phosphate. The samples were cut into Epstein strips and annealed at 800 °C for 2 hours in nitrogen atmosphere to undergo stress-relief annealing. As the coating barely attenuates neutrons nGI is ideally suited for the study of samples with intact coating.The high frame rate read-out electronics allow us to measure the time of neutron detection relative to the phase of the applied magnetic field and thus synchronize the detector with sample dynamics and accumulate the time resolved signal for a large number of periods of repetitive processes. This accumulation of periods allowed us to record up to 844 time bins in a 50 Hz cycle resulting in μs time resolution. Thus events are recorded for all phases of the repetitive dynamic process without rejection of majority of events in case of traditional stroboscopic imaging. Specifically tailored data reduction was developed to quantitatively study the dynamic behavior of bulk magnetic domain walls under realistic conditions. We applied the technique to observe domains in GOES with spatial resolution of ~100 μm and successfully obtain domain size, wall velocity, domain annihilation and its duration and domain wall multiplication during magnetization in real time recordings at power frequencies of 10, 25 and 50 Hz with ±262.5 A/m and ±525 A/m (peak to peak) applied field. The GOES sample studied in this paper is in accordance with the standard grade EN 10107: M 100-30p , and has a width of 30 mm, a length of 300 mm and a thickness of 270 μm. It is covered with an isolation layer consisting of forsterite  creates an image that is then used for the data reduction process and makes it possible to tune the statistics in the resulting DFIs. We opt for the summation of 76 images to generate one G0 phase stepping image. For the creation of the time resolved data set we use a running average approach (ii) with a step width of 14 images as a compromise between smoothness of the result and data volume. This means that for the first DFI of the time series we take the sum of time frames 0–75 as defined before. For the second DFI we sum images 13 to 88. This procedure is then continued to cover the whole set of up to 862 time frames. The summation and running average steps create relatively large sets of images that are then analyzed by a batch data treatment (iii) software based on recently developed in-house software21 which enables the application of common nGI data reduction algorithms to large datasets. The flexibility of that type of data treatment allows us to tune for optimal statistics and time resolution and gives us the possibility to analyze the movement of magnetic domain walls with an effective time resolution of 8.8 ms for 10 Hz, 4.4 ms for 25 Hz and 1.8 ms for 50 Hz, as well as spatial resolution with a pixel size of 50 μm.The applied field was recorded with 862 individual time frames for 10 Hz, 684 for 25 Hz and 844 for 50 Hz. In order to increase the statistics in one image of the G0 phase step 76 individual time frames were summed. Each of the 17 steps of the stepped grating is thus composed of 76 time frames and the coverage of the full applied sinusoidal field was realized by a running average approach making the full data treatment a 3 step procedure: (i) Time frame summation according to phase, (ii) Running time average and (iii) Batch data reductionFurther measurements were conducted applying a field of ±525 A/m at frequencies of 10 Hz, 25 Hz and 50 Hz as well as ±262.5 A/m at 50 Hz. As a compromise between statistics and time resolution we opted for time intervals of 8.8 ms for 10 Hz, 11 ms for 25 Hz and 1.8 ms for 50 Hz during post processing. The videos of the behavior of the magnetic domain walls in response to the applied fields can be found in Supplementary Fig. in-situ time resolved measurements displaying the individual domain walls enabled us to characterize bulk domain wall movements by extracting parameters such as number of domains, domain size, domain wall velocity as well as domain wall annihilation duration.The variation of the applied frequency and field strength in Parameter extraction and further analysis is focused on the grain identified in Fig. n, domain size d, domain wall velocity v and annihilation duration Ad. The annihilation duration describes the time period in which some of the magnetic domain walls annihilate to transport magnetic flux at relatively high fields. Annihilation can be observed in the interval indicated by the white, dashed lines in Fig. d indicates the domain wall distance corresponding to the domain width and the angle α is directly correlated with the domain wall velocity v. Ad and d are directly measured physical quantities and v is represented by the slope of the solid line of the triangle in Fig. v we use following equation:2D plots, such as the one in Fig. Md as well as the movement time Mt are defined in number of pixels in the plot and are indicated in Fig. Md represents a length determined by the pixel size of the detector (50 µm), the number of pixels for Mt represents a time and is defined by the time bin duration represented by each pixel in horizontal direction. It varies between measurements with the different applied frequency as described before.The movement distance vn defined byf being the applied frequency.The velocity of the bulk magnetic domain walls is determined by the applied frequency and higher applied frequencies naturally cause the domain walls to move faster. To make domain wall velocities comparable throughout varying applied frequencies we introduce the frequency normalised volume domain wall velocity d for increased frequency at constant field (plot 1–3) and decreased fields for constant frequency (plot 3–4). The white dashed line marks the first zero-field crossing within one period and the time at which d is measured. It is clearly visible that a reduction of the domain wall spacing d is found for increasing frequency. Another feature is the disappearance of some volume domain walls (yellow lines), indicating annihilation. Annihilations can be observed around phases of maximum effective applied field, i.e. phases of π as well as 0 and 2π, for 10 Hz and 25 Hz at ±525 A/m. At a frequency of 50 Hz no annihilation can be observed for both applied fields. For frequency independent comparison of the annihilation duration we introduce the frequency normalised annihilation duration vn defined byVisual comparison of the extracted plots for the varied experimental conditions can be done based on Fig. v, vn and domain size d presented in the table are an average for the domain walls observed in the plots as shown in Fig. d values and averaged them to present the average domain size in Fig. For a quantitative interpretation of the results we extracted the characteristic parameters for each frequency/field combination and summarized them in Fig. d with increasing frequency from 10 to 50 Hz  no change of the domain size from ±525 A/m to ±262.5 A/m, at 50 Hz, can be observed. This is a consequence of the unchanged number of domain walls with decreasing the applied field. Even though the number of domain walls remains unchanged the moving distance Md is reduced due to the lower excitation field and the consequentially lower magnetic flux that needs to be transported. This behavior is also reflected by the decrease in vn  neutron converters and electron multiplier encoded by a Timepix readoutSupplementary MatieralVideo S3aVideo S3bVideo S3cVideo S3d


In the maintenance of large infrastructures such as dams, bridges, railways, underground structures  and others, monitoring of deformations plays a key role in maintaining the safety serviceability conditions and for mitigating any consequences due to ageing factors and possible structural failures. This concern is, today, even more important than in the past due to the relevance of such infrastructures in our society, especially when they are related to communications and transportation.This Special Issue on “Sensors for Deformation Monitoring of Large Civil Infrastructures” is aimed at providing the readers an up-to-date overview of the methodologies, technologies, and data processing techniques in this field. Fifteen articles are presented, including a review paper. Authors of those contributions come from ten countries, a large fraction of them from China.integrated monitoring systems. Signals recorded by different instruments can be conveyed to a control unit that can filter and evaluate all the outputs to feed numerical models able to predict the future behavior of the structure. On the sensor side, several new technologies have been developed and successfully applied in recent years. More and more often, the communication among sensors and with the control unit is based on wireless communications (Wireless Sensor Networks—WSNs).Multiple techniques are described and presented in applications focused on different types of infrastructures. Very often, the corroborative and integrated use of more sensors units and sensor types is adopted. Indeed, the complexity that large infrastructures may feature calls for the use of multiple sensors organized in local geotechnical/structural deformation sensors organized in sensors networks. In Zhao [tunnel deformation monitoring system using ultrasonic transducers is described. By processing the redundant ultrasonic information, the measurement accuracy can be improved under various probe angles, distances, and surrounding temperature variations. Ma [Fiber Bragg Grating-based dynamic tension detection system for overhead transmission line galloping, that may overcome some limitations of traditional monitoring methods adopted to this purpose. WSNs may also be used to detect other processes, as presented in Cheung [Building Information Modelling  techniqing  instruments for deformation monitoring applications. Thanks to the acquisition of dense and redundant 3D point clouds or 2D profiles, this technique may be exploited to obtain information about changes over full surfaces rather than on a small number of control points as in traditional deformation measurement techniques [A recurrent topic in several paper concerns the use of chniques . Yang [1chniques  developechniques  for detechniques  proposedStructural health monitoring (SHM) of long-span bridges is today a hot-topic because of the plethora of such types of structures that have been constructed in recent years, and to the existence of several old ones that are still in full service. As demonstrated by the collapse of “Morandi” bridge in Genua  on 14 August 2018, a great attention should be paid to this problem either by technology developers and by officers in charge of SHM. In addition to the use of a wide range of in-situ monitoring instruments, Meng [ts, Meng  present dam deformation measurement [Advanced Differential Synthetic Aperture Radar Interferometry (A-DInSAR). The major contribution of this paper is the exploitation of such a large number of observed point displacements, which are distributed along the whole structure and are characterized by millimetric accuracy on displacement rates, for the calibration of numerical models. These models are implemented to simulate the structural behavior of a dam under conditions of stress thus improving the ability to maintain safety standards.Dams and water barrages are other important types of infrastructures whose monitoring requires great attention. This special issue also hosts a review of geodetic and remote-sensing techniques for surement , which isurement  the applsurement , the limPersistent Scatterer Interferometry—PSI [subsidence mapping over the entire transportation network of Shanghai (China) including elevated roads, ground highways and underground subways. The precise geolocation and structural characteristics of these infrastructures were combined to effectively guide a more accurate identification of the observed persistent scatterers, showing highly consistency in terms of subsidence velocities and time-series displacements.A-DInSAR observations from satellites are becoming more and more reliable for wide area and long-term deformation monitoring. One of these techniques (namely etry—PSI ) has beeetry—PSI  for subsinertial navigation system (INS) with geodetic instruments to set up a modular Railway Track Geometry Measuring Trolley system. This solution, which may be configured according to different surveying tasks including: precise adjustment of slab tracks, providing tamping measurements, measuring track deformation and irregularities, and determination of the track axis. Gabara and Savicki [Structure-from-Motion (SfM) photogrammetry [dense surface matching [Two papers ,20 deal  Savicki  measurinrammetry  and densmatching , the achphotogrammetric/image-based techniques [unmanned aerial vehicle  carr carrphotcle . We hope that this Special Issue will further promote research on the sensor technology and applications in the field of deformation monitoring of large infrastructures. The authors also hope that the published contributions will foster the process of technology and knowledge transfer from the scientific community to public institutions and private companies who are in charge of maintaining the safety conditions of any types of infrastructures.


Here, we show that the epigenetic reader Kismet (Kis) is required for developmental axon pruning in Drosophila mushroom bodies. Kis binds to cis-regulatory elements of the steroid hormone receptor ecdysone receptor (ecr) gene and is necessary for activating expression of EcR-B1. Kis promotes the active H3K36 di- and tri-methylation and H4K16 acetylation histone marks at the ecr locus. We show that transgenic EcR-B1 can rescue axon pruning and memory defects associated with loss of Kis and that the histone deacetylase inhibitor SAHA also rescues these phenotypes. EcR protein abundance is the cell-autonomous, rate-limiting step required to initiate axon pruning in Drosophila, and our data suggest this step is under the epigenetic control of Kis.Axon pruning is critical for sculpting precise neural circuits. Although axon pruning has been described in the literature for decades, relatively little is known about the molecular and cellular mechanisms that govern axon pruning   •ecr-b1 transcription to initiate developmental axon pruningKismet activates •Kismet promotes H3K36 di- and tri-methylation and H4K16 acetylation•Axon pruning and memory defects of Kismet mutants are rescued by HDAC inhibition Neuroscience; Molecular Neuroscience; Cellular Neuroscience; Model Organism What is well known about this process is that it requires tight regulation of gene expression to execute the necessary signaling pathways in a temporal and tissue-specific manner  (Holometabolous insects provide an attractive model for studying axon pruning as their nervous system undergoes extensive reorganization during metamorphosis . In Droses (MBs) . The MBses (MBs) . Each Kees (MBs) . During es (MBs) .Drosophila, ecdysone is released in large quantities by the prothoracic gland before each of the larval molts and pupation. The ligand is then able to enter the cytoplasm of target cells where it can bind to the Ecdysone Receptor (EcR). The binding of ecdysone to EcR stabilizes its interaction with its dimerization partner Ultraspiracle , a chromatin “reader” that is thought to play a role in chromatin remodeling by binding to methylated histone tails (CHD7 cause CHARGE syndrome (Our laboratory previously identified the chromodomain protein Kismet (Kis) as necessary for proper developmental axon pruning in the  unknown . Kis is ne tails . In humasyndrome , an autoDrosophila MB neurons. We determined that the loss of Kis in the MBs results in pruning defects during metamorphosis, which persist into adulthood and are due to a decrease in expression of ecr-b1. We show that endogenous Kis is enriched at a previously identified region of the genome shown to be important for ecr gene expression and that Kis binds to and is required to promote transcription from at least one cis-regulatory enhancer site in MB neurons. Furthermore, loss of Kis leads to a decrease in the histone marks H3K36 di- and tri-methylation , which have been associated with actively transcribed genes in flies. Additionally, loss of Kis results in a striking loss of H4K16 acetylation (H4K16ac). Adult flies with Kis specifically decreased in the MB neurons display a loss of immediate recall memory, which is rescued by transgenic expression of EcR-B1. Finally, we show that pharmacological intervention via the general histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) can rescue the decrease in ecr-b1 mRNA, axon pruning, and memory defects associated with decreased Kis in MB neurons. Taken together, these data show that Kis-mediated regulation of ecr-b1 is required for proper developmental axon pruning in vivo by mediating the epigenetic marks H3K36me2, H3K36me3, and H4K16ac. These findings suggest that the rate-limiting step required to initiate axon pruning (ecr-b1 expression) is under the epigenetic control of Kis.Here, we investigated the role of Kis in the developmental axon pruning of the kis mutant MB neurons, we utilized the mosaic analysis with a repressible cell marker (MARCM) system to generate homozygous mutant neuroblast clones tagged with a membrane-bound GFP (UAS:mCD8-GFP) using the 201y-Gal4 driver  in be areas D and 1E,be areas C and 1E.be areas . Expresskis mRNA . Additio-L alone , which wcontrols . Similarcontrols . Taken telav-Gal4,UAS:mCD8-GFP driver showed a significant decrease in EcR-B1 immunofluorescence in the MB Kenyon cells compared with those of control MBs at 18–22 h APF  followed by qPCR using chromatin isolated from third instar larval brains to analyze Kis occupancy at the ecr locus. We probed three cis-regulatory element sites between 7 and 33 kb upstream of the ecr-b transcription start site (TSS) (ecr-b1 expression are present in this area (UAS:Kis.RNAi.a can successfully knock down the eGFP-tagged Kis protein and kis-eGFP mRNA (forkhead (fkh) TSS served as a positive control, as it was previously reported to be bound by endogenous Kis (dynamin homolog shibire (shi) as a negative control, since we had previously shown via microarray analysis that loss of Kis did not have any significant effect on shi mRNA expression   A. We chohis area A  B. Upon p binding B. These ecr locus to allow access for the transcriptional machinery. To test this possibility, we immunoprecipitated total histone 3 (H3) protein at the ecr locus in control and Kis knockdown third instar larval brains. We found no significant difference between these conditions at each of the cis-regulatory loci we analyzed  has been suggested to bind to sites distal to TSSs, sites with features suggesting that they are gene enhancer elements. Thus, these data are consistent with at least one other CHD protein binding function  and aged the adults for 5 days after eclosion. At the adult stage, the 201y-Gal4 driver is expressed in a subset of alpha and beta neurons in addition to all the gamma neurons  was shown to genetically interact with the EcR signaling pathway and positively regulate expression of EcR target genes , it might be acting to recruit the HMTs to relevant target genes to transduce effects on transcriptional regulation. To this effect, Kis was previously shown to increase the global association of Ash1 on polytene chromosomes (shi promoter in Kis knockdown animals. Further work needs to be done to tease apart the relationship between Kis, H3K36 methylation, and any other HMTs responsible for this modification in the context of ecr regulation.Few studies have hinted at an epigenetic mechanism of et genes . Howeveromosomes . Althougomosomes , we demoecr-b1 mRNA levels and rescue pruning and memory defects associated with loss of Kis. Consistent with this, SAHA treatment has been previously shown to increase H4K16ac and ultimately increase transcriptional output in cancer cells (ecr-b1 mRNA levels or axon pruning. Therefore, the phenotypic effects we observe with SAHA treatment are specific to animals where Kis function is decreased. Additional work is essential to building a complete understanding of the integration of extrinsic and intrinsic cues that control transcriptional regulation.In the field of epigenetics, much work has been done to understand the cross talk between different histone modifications and their cumulative outcome on transcription. For example, H3K36me2 by the HMT dMes-4 was reported to specifically increase H4K16ac, a modification most well known for its role in de-condensation of chromatin structure and maintenance of active gene expression . We prover cells . Additioecr-b1 and may also be modulating some other factor that can discriminate between dorsal and medial lobes. Recent work has shown that the neuronal architecture of the MB is quite extensive and forms 15 distinct compartments that tile the MB lobes (ecr-b1 expression and pruning in a subset of the neurons that innervate the medial lobes, or, alternately, may be affecting additional factors besides Ecr-B1 that can discriminate pruning between these two lobes. Determining what these factors may be will require additional work.One interesting observation from our work is that Kis appears to affect pruning of the medial lobes of the MB, while largely leaving the dorsal lobes unaffected. This was the case for both our MARCM analysis as well as our RNAi analysis. It is well established that Ecr-B1 is required for axon pruning of both the dorsal and medial lobes . Indeed,MB lobes . Thus, KIn summary, our work has helped to shed light on the epigenetic factors that are involved in the regulated developmental pruning required in Drosophila MB neurons. Determining how axons are properly pruned is fundamental to unraveling the mechanisms that underlie the refinement of neural circuits.One limitation of the current study is the method we use to overexpress the Kismet protein, in that the overexpressed RNA can be targeted by our RNAi strategy. Future studies should consider utilizing an overexpression transgene that cannot be targeted by RNAi. A second limitation to the current study is that fact that, although we have identified a protein that differentially affects pruning in the medial vs. the dorsal lobes of the MB, we cannot fully explain how Kismet was able to accomplish this. Future studies should be cognizant that proteins may regulate the pruning of MB lobes differentially.All methods can be found in the accompanying


Integrin αVβ8, which like αVβ6 functions to activate TGF-βs, is atypical. Its β8 subunit binds to a distinctive cytoskeleton adaptor and does not exhibit large changes in conformation upon binding to ligand. Here, crystal structures, hydrogen-deuterium exchange dynamics, and affinity measurements on mutants are used to compare αVβ8 and αVβ6. Lack of a binding site for one of three βI domain divalent cations and a unique β6-α7 loop conformation in β8 facilitate movements of the α1 and α1’ helices at the ligand binding pocket toward the high affinity state, without coupling to β6-α7 loop reshaping and α7-helix pistoning that drive large changes in βI domain-hybrid domain orientation seen in other integrins. Reciprocal swaps between β6 and β8 βI domains increase affinity of αVβ6 and decrease affinity of αVβ8 and define features that regulate affinity of the βI domain and its coupling to the hybrid domain. The activation mechanism of integrin αVβ8 differs from other integrins. Combining X-ray crystallography, hydrogen deuterium exchange mass spectrometry and mutation, the authors reveal structural features responsible for these differences and provide insights into how typical integrins are regulated. The integrin family grew to its current size in mammals of 24 αβ heterodimers with the cloning of the last β-subunit, β8, in 19913 and the last α-subunit, α11, in 19994. Integrins are force-resistant and provide traction for cell migration and mechanical stability for tissues. However, two integrins, αVβ6 and αVβ8, appear to have evolved primarily to activate transforming growth factor-β1 (TGF-β1) and TGF-β3 and bind with high affinity to a RGDLXX(L/I) motif in the TGF-β prodomain. These integrins activate TGF-β by releasing the growth factor (GF) from the prodomain, which otherwise surrounds the GF and holds it in a latent form in which it cannot bind TGF-β receptors5. αVβ8 is expressed much more highly in the brain than in any other tissues3 and especially highly on glial cells, where it localizes to synapses with other glia and neurons and within synaptosomes6. αVβ8 in the central nervous system is required to activate TGF-β1 complexed with the milieu molecule LRRC33 on microglia cells and for the maintenance of myelin, axons, and neurons in certain regions of the central nervous system and particularly in motor pathways in the brain and spinal cord7.Integrins comprise a family of αβ heterodimers with diverse functions in cell adhesion, migration, and signaling. The integrin family was seeded with its first two members, integrins lymphocyte function-associated antigen 1 (integrin αLβ2) and macrophage antigen 1 (integrin αMβ2) with discoveries in 1982 that they had identical β-subunits and distinct α-subunits8. Retrograde actin flow transmits tensile force through such integrins when they bind to extracellular ligands. This tensile force, together with ligand binding, stabilizes integrins in the extended-open conformation, which, depending on the integrin, has 700- to 4,000-fold higher affinity for ligand than the extended-closed or bent-closed conformations10. Higher affinity results from tightening of the ligand-binding site in the integrin β-subunit βI domain at the β1-α1 loop and α1-helix around the metal ion-dependent adhesion site (MIDAS)  Fig. 11,12.Fig13. Although the conformation of intact αVβ8 is unknown, its ectodomain fragment almost exclusively exhibits an extended conformation16  in the βI domain of other integrins; however, both Asn and Asp can coordinate Ca2+.In contrast, the β8-subunit of integrin αVβ8 has a divergent cytoplasmic domain that binds to the Band 4.1 family Fig. 13. Althon16 Fig. , and thun16 Fig. 14–16. ThHere, we report structural differences and correlating sequence differences between the βI domains of β8 and talin-activated integrin β-subunits that extend well beyond the Asp and Asn ADMIDAS differences, including the β6-α7 loop, and have not previously been discussed or studied. Mutational exchanges between β8 and β6 βI domains and hydrogen-deuterium exchange (HDX) differences between β8 and β6 suggest that that these sequence motif and structural differences have important roles in affinity regulation and may enable affinity regulation without hybrid domain swing-out in atypical β8 Fig. . We furt213RRGDLATIHG223  domains, which link to the N- and C-terminal ends of the hybrid domain distal to its interface with the βI domain. In contrast, all β-subunit domains were better defined in αVβ6 headpiece crystal structures  in integrins. However, replacement of β-MIDAS motif D8 and D9 residues with Asn in β8 results in the absence of any negatively charged sidechains to coordinate Ca2+ and is likely to be sufficient to explain the lack of an ADMIDAS metal ion in αVβ8. Ca2+ binding is also competed by the hydrogen bond of Asn-120 to Gln-302 , as the β-MIDAS motif, where MIDAS is used in a broad sense to include up to two metal ions Fig. . In typi302 Fig. . Gln-302302 Fig. .215GDLATI221 sequence motif in the TGF-β1 peptide binds to αVβ8  at the beginning of the α1-helix with its MIDAS-coordinating residues moves toward the Mg2+. This movement enables coordination of Ser-116, that is, the β-MIDAS S5 residue, to the MIDAS Mg2+ and a hydrogen bond of the Asp-217 sidechain to the SDL1 backbone. The orientations of Arg-215 and Asp-217 sidechains are supported by hydrogen bonds of their backbones to αVβ8. The ligand LATI sequence has an α-helix-like conformation with its hydrophobic Leu-218 and Ile-221 in a pocket formed by the β8 SDL2 loop. In unliganded αVβ8, nine residues in the SDL2 loop, including one of the disulfide-bonded Cys residues, are disordered -Ser motif in the β6-α7 loop Fig. , which eIn place of the Asp/Asn residue at the tip of the β6-α7 loop in typical integrins Fig. , Lys-32715.It appears that the position of the βI domain β6-α7 loop in β8 would accommodate tilting of the α1′-helix and its fusion with the α1-helix, without requiring pistoning of the α7-helix and swing-out of the hybrid domain as seen in typical integrins. The unique features of the β8-subunit may enable intermediate or even complete movement of SDL1 toward the open conformation to be dissociated from pistoning of the α7-helix and swing-out of the hybrid domain Fig. . These fHDX mass spectrometry (MS) on the αVβ6 and αVβ8 headpieces showed similar backbone dynamics of their αV-subunits including slow exchange in the β-propeller domain and interesting differences in dynamics of their β6- and β8-subunits. Multiple peptides covering the βI domain α1 and α1′ helices generally showed more rapid exchange of backbone amide hydrogens in β8 than β6 Fig. , althougThe effect of ligand binding on αVβ6 and αVβ8 was also examined by HDX MS. In the αV β-propeller domain, Tyr-178 and Asp-218 bind Arg-215 of TGF-β1 Fig. . In the 2+ to augment this affinity. Residues in shape-shifting interfaces within the βI domain engaged in distinctive interactions in β8 and β6 were exchanged, including those in the α1 and α1′ helices and the β5-α6 and β6-α7 loops  than wild-type (WT) (2-fold) and αVβ8-mut6 exchanged ADMIDAS residues thought to be important in headpiece opening, these mutants were tested for headpiece opening in presence of ligand and Mn2+. Negative stain EM showed that like αVβ8-WT, and unlike αVβ6-WT, the headpiece of αVβ8-mut3 and αVβ8-mut6 remained closed when bound to pro-TGF-β1 in Mn2+  and a slight shift in the other to state 221. In 1 mM RGD ligand in Mn2+/Ca2+, greater shift to state 3 occurred, and as ligand was increased from 3 to 5 to 10 mM in Mn2+/Ca2+, shifting gradually increased until reaching state 6 and finally, state 8 (open) , shows that affinity for ligand increases along the same continuum. Additionally, only the increase in affinity for ligand can pay the energetic cost required for the structural shifts within the integrin and the crystal lattice.In α4β1 and α5β1 integrins, complete transition from closed to open increases affinity for ligand by 700- and 4000-fold, respectively2+ ion to the backbone carbonyl oxygen of the S5 β-MIDAS residue strongly restrains movement of this key residue and thereby hinders tightening of the ligand-binding pocket including formation of the direct coordination between the S5 Ser sidechain and the MIDAS Mg ion and the hydrogen bonds between the ligand Asp sidechain and the SDL1 backbone. The restraint is provided by the D8 and D9 β-MIDAS residues and the backbone carbonyl oxygen in the β6-α7 loop to which the ADMIDAS Ca2+ ion also coordinates. This hypothesis was verified in plots of S5 and D/N8 β-MIDAS residue Cα atom positions relative to those in an open structure among 45 independent examples of integrin structures SXN motif in the β6-α7 loop Fig. . The firTwo of the mutants with the greatest introduction of β6 residues into the β8-subunit were tested for hybrid domain swing-out. In contrast to results with αVβ6, neither mutant showed the open headpiece when bound to pro-TGF-β1. Our structure of αVβ8 shows that when ligand is soaked in, the βI domain shifts to a state intermediate between closed and open, as observed for typical integrins. Thus far, typical integrins show complete headpiece opening when co-crystallized with ligand, and when observed by EM when bound to ligand. Our results show that substitution with a set of up to 19 putatively atypical β8 residues with typical integrin residues in the βI domain was not sufficient to enable headpiece opening. We were unable to test whether lack of hybrid domain swing-out in β8 was intrinsic to its βI domain. Poor expression of β-subunit chimeras with βI domains swapped between β8 and β6 suggested structural incompatibilities. The βI-hybrid interface in β8 is typical in size and does not have an unusual number of hydrogen bonds , there was no affinity increase compared to WT (αVβ8-mut6). Furthermore, DDT + VTQE + LQKDSGNILQ (αVβ8-mut3) was 3.5-fold lower in affinity than VTQE + LQKDSGNILQ S motif with the Asp or Asn sidechain hydrogen bonding to two adjacent backbone amides to stabilize the tip of the β6-α7 loop. Importantly, the β6-α7 loop in the closed conformation packs against the α1-helix and the backbone carbonyl oxygen of the residue immediately preceding the (D/N)S motif coordinates the ADMIDAS CaIn typical integrins, complete movement of the SDL1/α1-helix to the open state is only allowed when the β6-α7 loop moves away from the α1-helix and toward the hybrid domain, which makes way for tilting of the α1′-helix and its fusion to the α1-helix and is accompanied by pistoning of the α7-helix toward the hybrid domain and swing-out of the hybrid domain, giving the open headpiece conformation Fig. . In β8, Mutations verified an important role for the β6-α7 loop in regulating affinity of both αVβ6 and αVβ8. Replacing the DS motif of β6 with KA from β8 resulted in a 13-fold increase in affinity of αVβ6. Conversely, replacing KA with DS or KAA with DSS decreased αVβ8 affinity by 1.8- and 2.8-fold, respectively. These results demonstrate an important and previously unexpected role of the β6-α7 loop in regulating affinity of both typical integrins and atypical integrin αVβ8. The results suggest that in typical integrins, the DS motif maintains a conformation of the β6-α7 loop that restrains SDL1/α1-helix movement, whereas the lack of this motif and the unengaged conformation of the β6-α7 loop are permissive of SDL1/α1-helix movement toward the open conformation.Our structures of liganded and unliganded αVβ8 suggest that tightening of the ligand-binding pocket in αVβ8 with MIDAS-proximal movement of SDL1 to an open-like conformation is plausible in the absence of hybrid domain swing-out. In the open βI domain conformation of typical integrins, the α1′-helix pivots and invades the space occupied by the β6-α7 loop in its closed conformation. Because the β6-α7 loop in β8 is atypically distal from the α1 and α1′ helices, space is available for α1′-helix pivoting and fusion with the α1-helix. Together with the lack of ADMIDAS metal coordination bonds, the altered β6-α7 loop creates more freedom to accommodate α1-helix movement to its conformation in the open βI domain. Our studies explain why ligand binding does not induce headpiece opening of αVβ8. However, as none of the unusual features of the β8 βI domain would be near the integrin lower legs in the bent integrin conformation, they do not explain the low abundance of the bent conformation for αVβ8.The structure and mutagenesis results reported here suggest that the β8 βI domain may open in the absence of hybrid domain swing-out. The atypical structural features of the β8 βI domain, including the disengagement of the β6-α7 loop from the α1-helix, suggest that complete movement of SDL1 with the α1 and α1′ helices toward the ligand and α1 and α1′ helix merger, resulting in high affinity for ligand, may occur without requiring the α7-helix pistoning and hybrid domain swing-out that is seen in typical integrins Fig. . Thus, αThe unique conformational ensemble of αVβ8 correlates with its unique linkage among integrins not to talin but to Band 4.1. We have revealed in αVβ8 the striking absence of an ADMIDAS, a more mobile SDL1/α1 and α1′ helix, and a β6-α7 loop that is disengaged from the α1-helix. Moreover, our demonstration that a (D/N)S motif in the β6-α7 loop stabilizes typical integrins in their low-affinity state revealed insights into the structural principles that regulate activation not only of atypical integrin αVβ8 but also of typical integrins. Distinct structural features in βI domains may tune them to be activated by adaptors that couple to different cellular cytoskeletal systems.−/− cells exactly as previously described14. Cells were obtained from the authors25, were mycoplasma tested several times per year, and were validated by endoglycosidase H treatment of secreted glycoprotein. Briefly, residues 1–594 of the αV-subunit and 1–456 of the β8-subunit were cloned into modified pcDNA3.1 and ET10 expression vectors, respectively. Mutations M400C in αV and V259C in β8 formed a disulfide to covalently stabilize the heterodimer. Purification with Ni-affinity chromatography, removal of tags, ion exchange, and gel filtration were as described for αVβ617, except the gradient with Sepharose Q was from 50 to 150 mM NaCl and gel filtration was with Superdex 75 in 20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM MgCl2, and 1 mM CaCl2. Fractions were concentrated to 4.5 mg/ml and stored at −80 °C in aliquots. Mutant fragments in the same expression vector were transiently expressed with the αV-subunit using FetcoPro  in suspension Expi293 cells. Supernatants were collected after 6 days and protein was purified as described above. Human pro-TGFβ-1 with a R249A cleavage site mutation was prepared as described20.An αVβ8 headpiece construct with an αV-β8 disulfide was prepared and expressed in HEK293S GntIN-morpholino)ethanesulfonic acid), pH 6.7, 12% polyethylene glycol (PEG) 20,000 at 4 °C. To improve electron density on the β8-subunit, crystals were dehydrated by soaking in solutions that had the starting concentrations of components in the protein and reservoir solutions while raising the concentration of PEG 20,000 to 20% in 2% steps. Additionally, soaking solutions contained 20 mM Mg2+ and 10 mM Ca2+ (unliganded structure) or 1 mM TGF-β1 ligand peptide (G213RRGDLATIHG223), 10 mM Mg2+, and 2 mM Ca2+ (liganded structure). Processing and diffraction limit determination were with XDS26 and CC1/227, respectively. The liganded structure was solved by molecular replacement with αVβ6 (4UM9) using PHASER in Phenix28 and subsequently used to solve the unliganded structure. Structures were refined with PHENIX, built with Coot29, and validated with MolProbity30. Representative electron density is shown in Supplementary Fig. Hanging drop αVβ8 headpiece crystals grew in 0.1 M MES (2-(2+/1 mM Ca2+ or 1 mM Mn2+/0.2 mM Ca2+ with fluorescein isothiocyanate (FITC)-labeled pro-TGF-β3 peptide (FITC-Aminocaproic-GRGDLGRLKK) probe. αVβ8 was serially diluted in 1.4-fold decrements and mixed with 5 nM of probe at 20 °C for 30 min. Fitting fluorescence polarization as a function of integrin concentration at fixed probe concentrations yielded KD values for fluorescent pro-TGF-β3 peptide14.Saturation binding was measured in HBS buffer , supplemented with 1 mM Mg2+/0.2 mM Ca2+ for 30 min and injected in a 24 ml Superdex 75 gel filtration column pre-equilibrated with HBS buffer . The 2:2, 2:1 complexes and unbound αVβ8 and pro-TGF-β1 were well separated14. Peak complex fractions  were loaded on glow-discharged carbon grids and fixed with uranyl formate. About 60 images with 52,000 magnification were collected on FEI Tecnai-12 transmission electron microscope at 120 kV with low-dose model. About 5000 particles were manually picked and subjected to multireference alignment and K-means classification by software SAMUEL31.αVβ8-mut3 or αVβ8-mut6 (15 μg) were mixed with pro-TGF-β1 at molar ratio 1.5:1 in 50 μl of HBS buffer containing 1 mM MnKD values of 12 and 30 nM for αVβ6 and αVβ8 , either alone or mixed with 100 µM TGF-β1 ligand peptide, were diluted 15-fold  with labeling buffer  and then separated in 14 min by a 5–40% acetonitrile:water gradient at 40 µl/min. The separation column was a 1.0 × 100.0 mm2 Acquity UPLC C18 BEH  containing 1.7 µm particles. The back pressure averaged 8800 psi at 0.1 °C33. The error of determining the deuterium levels was ±0.15 Da in this experimental setup. To eliminate peptide carryover, a wash solution of 1.5 M GnHCl, 0.8% formic acid, and 4% acetonitrile was injected after each run.Deuterated and control samples were digested in solution with pepsin  for 5 min on ice, and then injected into an M-class Acquity UPLC with HDX technology (Waters)E mass spectrometer in ion mobility mode. A conventional electrospray source was used and the instrument was scanned over the range 100 to 1900m/z. The instrument configuration was the following: capillary was 3.2 kV, trap collision energy at 6 V, sampling cone at 35 V, source temperature of 80 °C, and desolvation temperature of 175 °C. All comparison experiments were done under identical experimental conditions such that deuterium levels were not corrected for back-exchange and are therefore reported as relative34.Mass spectra were acquired using a Waters Synapt G2-Si HDMSPeptides were identified using PLGS 3.0.1  using six replicates of undeuterated αVβ6 and six replicates of undeuterated αVβ8. Raw data were imported into DynamX 3.0  and filtered as follows: minimum number of products of 3; minimum consecutive products of 2; minimum number of products per amino acid of 0.2; maximum mass error of 10 p.p.m. Those peptides meeting the filtering criteria were further processed automatically by DynamX followed by manual inspection of all processing. Peptides with low signal-to-noise ratios in either bound or free states were removed. The relative amount of deuterium in each peptide was determined by subtracting the centroid mass of the undeuterated form of each peptide from the deuteratered form, at each time point, for each condition. These deuterium uptake values were used to generate uptake graphs and difference maps. Additional experimental details are found in Supplementary Table Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


The vitamin C oxidation products thereby formed are investigated here. DHA and DKG were incubated aerobically at pH 4.7 with peroxide (H2O2), ‘superoxide’ , and illuminated riboflavin . Products were monitored electrophoretically. DHA quenched H2O2 far more effectively than superoxide, but the main products in both cases were 4-O-oxalyl-l-threonate (4-OxT) and smaller amounts of 3-OxT and OxA + threonate. H2O2, but not superoxide, also yielded cyclic-OxT. Dilute Fenton mixture almost completely oxidised a 50-fold excess of DHA, indicating that it generated oxidant(s) greatly exceeding the theoretical •OH yield; it yielded oxalate, threonate, and OxT. 1O2 had no effect on DHA. DKG was oxidatively decarboxylated by H2O2, Fenton mixture, and 1O2, forming a newly characterised product, 2-oxo-l-threo-pentonate . Superoxide yielded negligible OTP. Prolonged H2O2 treatment oxidatively decarboxylated OTP to threonate. Oxidation of DKG by H2O2, Fenton mixture, or 1O2 also gave traces of 4-OxT but no detectable 3-OxT or cyclic-OxT. In conclusion, DHA and DKG yield different oxidation products when attacked by different ROS. DHA is more readily oxidised by H2O2 and superoxide; DKG more readily by 1O2. The diverse products are potential signals, enabling organisms to respond appropriately to diverse stresses. Also, the reaction-product ‘fingerprints’ are analytically useful, indicating which ROS are acting in vivo. Aqueous samples were loaded onto Whatman No. 3 paper and electrophoresed at 2.5–3.5 kV for 30–70 min in a buffer of pH 2  or pH 6.5  .mOG) are calculated relative to orange G. Neutral compounds move a small distance away from the origin owing to electro-endo-osmosis, so mobilities were calculated with a neutral marker (e.g. DHA) as mOG = 0. After long electrophoresis runs, orange G ran off the paper, and mThrR [mobility relative to threarate (mThrR = 1.0) and glucose (mThrR = 0.0)] was used instead of mOG. Ascorbate-related compounds were stained with AgNO3 DHA, which had no affinity for the column, was eluted in H2O., CPL [ proposed to be a mixture of 2-carboxy-l-threo-pentonolactones (2-carboxy-l-xylonolactone plus 2-carboxy-l-lyxonolactone)], an unidentified compound , threone of ROS ,77. CPA e of ROS ): CPA asmpound C . These w2O2 yielded CO2, detected by gas chromatography, suggesting that H might be a C5 compound , which could be formed from DKG (C6H8O7 in the unionised form) thus:2O2 . The theoretical oxidative decarboxylation product of DKG is OTP  was tested by electrophoresis. OAH is a much stronger acid than its parent compound, gluconate, with pKa values ≈ 2.7  . One molk ≈ 3 × 107 M–1 s–1) DHA as a substrate confirmed that this spot was not the approximately co-migrating cOxT, which would have been radioactive [14C]DHA + [O] → ThrO + [14C]OxA) is not expected to be radiolabelled, because it does not retain the C-1 atom of the DHA [14C]DHA by Fenton mixture was [14C]OxA. The predominant reaction products formed from DHA by dilute Fenton mixture were thus OxA and ThrO, far exceeding the formation of the same products by H2O2 or superoxide.Besides OxT, a spot co-migrating with ThrO was also produced . Use of •OH. Other oxidant(s) were evidently also formed, ultimately drawing on atmospheric O2. These oxidising species oxidised DHA predominantly to OxA plus ThrO . As with DHA, the Fenton mixture appeared to act ‘instantaneously’, degrading all the (50-fold molar excess) DKG even if •OH-quenching ethanol was added immediately after the Fenton mixture.The reaction of 1 mM Fenton mixture with 50 mM DKG was also studied . In the 2O2 or superoxide  were produced. There was no major difference between the observed Fenton products  and thosperoxide .1O2 over a longer timescale (24 h), allowing time for detectable ROS formation. This timescale allowed some of the DHA to hydrolyse to DKG during the experiment  in the light but not in the dark [1O2 production from 3O2 by riboflavin in the light is not stoichiometric: each riboflavin molecule can generate many 1O2 molecules, although the yield is not unlimited because riboflavin itself is subject to oxidation by 1O2 [the dark ,70,85, s1O2 generated OTP and 4-OxT  demonstrated that OTP and OxT did not originate from riboflavin itself.DHA plus nd 4-OxT . There w1O2 is incapable of driving an oxidative reaction that can be driven by H2O2 and to a lesser extent by superoxide . Thus, peroxide .1O2) was treated directly with the source of 1O2, most of the DKG disappeared within 24 h , the oxidative decarboxylation product of DKG. OTP is a relatively strong acid (pKa ≈ 2.8), as expected of a 2-oxo-acid, and thus occupies a characteristic position after electrophoresis at pH 2.0.Secondly, we identified the DKG product previously called ‘unknown H’  as 2-oxoThe work thus provides increased clarity of the complex network of AA oxidation pathways, highlighting the numerous different branches.2O2. Here, we tested the effects of different ROS. Studying superoxide's action presents two practical difficulties: (a) superoxide is exceedingly short-lived in aqueous solution , undergoing non-enzymic dismutation, so time-courses are not feasible and (b) the products of superoxide breakdown are a different ROS (H2O2) plus O2, so it may be difficult to determine whether any products observed arise from the action of superoxide (intended) or H2O2 (unintended).Previous work on oxidation of AA metabolites had focussed on H2 is a convenient source of superoxide. KO2 was suspended in hexane and then added to an aqueous substrate solution. When mixed with water, KO2 fizzes briefly (O2 evolution during dismutation) and any superoxide action on DHA or DKG is necessarily completed within <1 s; it is therefore impossible to obtain a time-course. Instead, the only feasible gradation of superoxide action is based on different doses of added KO2, time being kept to a minimum. Another practical issue is that since KO2 + water yields KOH, we required a high buffer concentration.Crystalline KO2O2, generated during superoxide dismutation, we added catalase prior to KO2 to destroy H2O2. Its effectiveness was established by a demonstration that H2O2 that was deliberately added to DHA or DKG in the presence of catalase generated no detectable oxidation products.To minimise the effects of H2+ and H2O2, which generates •OH [2+·EDTA, which has improved solubility and which we find is stable enough to electrophorese as a discrete, negatively charged complex. Theoretically, a mixture containing 1 mol of each component may yield up to 1 mol of •OH:•OH on DHA or DKG by use of Fenton reagent because this mixture evidently continually generates unidentified oxidant(s) using atmospheric O2 as the ultimate electron acceptor. The information recorded for DHA and DKG oxidation by Fenton mixture . With DKG: superoxide is scavenged only very weakly by DKG, which, however, scavenges 1O2 and H2O2 relatively well.With in vivo. Our studies investigated only aqueous media.Similarly, we made the novel observation that the degradation pathways of DHA and DKG vary depending on the type of ROS present. This observation could be exploited to indicate the nature of oxidative stress that an organism is experiencing, based on the signature of the DHA and DKG oxidation products. Some studies in this field have used non-aqueous solvents, e.g. the action of superoxide on DHA to form oxalate + threonate in dimethylformamide , in whic2O2 produces approximately equimolar 3-OxT and 4-OxT plus some cOxT but almost no OxA, whereas superoxide gives predominantly 4-OxT, only traces of 3-OxT and undetectable cOxT but some detectable OxA (with ThrO)  is proposed to occur after the last ROS-dependent step  . The rea2O2 gives much more OTP than does superoxide, whereas these two ROS give similar (low) yields of 4-OxT  steadily yields OTP, whereas superoxide does not. Since DHA does not generate OTP, the yield of OTP can indicate whether or not a particular ROS is scavenged by DKG.Singlet oxygen, the only non-radical ROS tested in this study, shows the most divergent profile of oxidation products . CuriousIn vivo different ROS often occur together, and many ROS react to form other ROS, such as the production of 1O2 [•OH [•OH from H2O2 and superoxide [in vivo. Therefore, analysis of DHA and DKG oxidation ‘fingerprints’ may help to shed light on this important issue. The use of these diagnostic ‘fingerprints’ will require significant further study because of the complexity of ROS reactions in biological systems as well as the complexity of interactions with other molecules. In planta ROS-scavenging activity is likely to be influenced by other metabolites, and it would be useful to analyse the effectiveness of DHA and DKG as scavengers in vivo or in planta.n of 1O2  and •OH 1O2 [•OH  from ozoperoxide . This ca1O2 may represent biologically relevant case of when OTP could be produced in vivo. Although H2O2 has been demonstrated to act as a signalling molecule for a general oxidative stress response, it has been suggested that plant cells may require more site-specific (e.g. from specific organelles) signals [1O2 derived products (different from H2O2 and superoxide products) are more likely to have originated from the chloroplast [1O2 related genes, but not H2O2 related genes [in vivo mainly with the use of radiolabelled tracers, which should, nevertheless, reflect processes that occur with endogenous compounds.It is possible that the various products formed from the reactions of different ROS with different AA metabolites could supply information, making the plant aware of its internal ROS status. Thus, the OxT isomers, cOxT and OTP, might potentially act as signalling molecules, triggering advantageous responses to the presence of specific ROS. Production of OTP from DKG +  signals . The diforoplast . Equallyed genes . The AA ed genes , the oxiin vivo [O-oxalyl-sugars are yet to be explored.Indeed, the formation of DKG itself, which has been detected in vivo ,93, may in vivo , and theOur study highlights the complexity of AA catabolism and demonstrates the numerous branches of AA, DHA, and DKG breakdown. The possibility of their reaction products having further roles within the plant warrants future study. The different AA metabolites investigated here differ in their abilities to scavenge different ROS. The oxidation products of DHA and DKG differ, with only DHA producing cOxT and 3-OxT, and only DKG producing OTP. Different ROS tend to generate different ratios of the oxidation products from these two AA metabolites. The diverse products could potentially act as biological signals enabling the organism to respond appropriately to different stresses. In addition, these products of DHA and DKG breakdown, which are readily resolved by the electrophoretic systems employed here, can potentially serve as analytical fingerprints providing information on the ROS-quenching reactions proceeding in biological systems of interest.


Mixed Lineage Kinase domain-Like (MLKL), a key player in necroptosis, is a multi-domain protein with an N-terminal 4 helical bundle (4HB) and a pseudokinase domain (PsK) connected by brace helices. Phosphorylation of PsK domain of MLKL is a key step towards oligomerization of 4HB domain that causes cell death. Necrosulfonamide (NSA) binds to the 4HB domain of MLKL to inhibit necroptosis. To understand the molecular details of MLKL function and it’s inhibition, we have performed a molecular dynamic study on hMLKL protein in apo, phosphorylated and NSA-bound states for a total 3 μs simulation time. Our simulations show increased inter-domain flexibility, increased rigidification of the activation loop and increased alpha helical content in the brace helix region revealing a form of monomeric hMLKL necessary for oligomerization upon phosphorylation as compared to apo state. NSA binding disrupts this activated form and causes two main effects on hMLKL conformation: (1) locking of the relative orientation of 4HB and PsK domains by the formation of several new interactions and (2) prevention of key 4HB residues to participate in cross-linking for oligomer formation. This new understanding of the effect of hMLKL conformations on phosphorylation and NSA binding suggest new avenues for designing effective allosteric inhibitors of hMLKL. Mixed Lineage Kinase domain-Like (MLKL) protein is the most critical downstream effector of TNF induced necroptotic pathway7. The core necroptotic pathway relies on the hetero oligomerization of receptor interacting protein kinase (RIPK) 1 and 3 to form necrosome8. RIPK3 in turn activates MLKL protein by phosphorylation at Thr357 and Ser3589. It has been hypothesized that the activated MLKL undergoes conformational changes that lead to its oligomerization and permeation through the cell membrane to cause cell death12. Although phosphorylation of PsK domain triggers activation in both human and mouse MLKL, there are significant differences in the activation mechanisms between mouse and human MLKL13. It has also been established that IP-Kinases play a regulatory role in MLKL activation potentially influencing oligomerization and/or membrane recruitment14. While oligomerization of MLKL has been established as a central element in necroptosis, the molecular details of oligomer formation remain elusive18.Necroptosis is a non-apoptotic, caspase free and kinase dependent programmed cell death which leads to plasma membrane rupture and cell lysis5  bind to N-terminal and brace regions to regulate MLKL function. But alternative regulatory mechanisms such as membrane recruitment are also proposed for IP614. In any case, it is clear that phosphorylation of the PsK domain transmits the signal for oligomerization to 4HB domain; the details of this molecular communication are not well-understood.Structurally, MLKL comprises of a N-terminal 4-helix bundle (4HB) domain and a C-terminal pseudokinase (PsK) domain linked together by two brace helices5 Fig. . The pseet al. identified several ATP-pocket binders for hMLKL but they observed that these binders have no direct effect on MLKL and therefore on necroptosis23. The only known effective inhibitor of necroptosis directly binding to MLKL is necrosulfonamide (NSA)6. Exactly how NSA binding to the N-terminal domain blocks necroptosis is also not clearly elucidated. Our goal in this molecular dynamics study is to unravel details of conformational changes that may occur upon NSA binding to the monomeric hMLKL that prevent oligomerization and unleashing of 4HB domain.Studies have been conducted to design MLKL inhibitors for blocking necroptotic pathway. Ma We performed classical molecular dynamic (MD) simulations on a full length monomeric hMLKL protein in three different states - apo, phosphorylated (activated) and Necrosulfonamide (NSA) bound (inhibitor bound) for a total of 1 μs for each state to delineate the molecular details of activation of MLKL by phosphorylation and it’s inhibition by NSA. Our simulations data provide novel insights into the dynamical and structural changes observed in hMLKL upon activation and inhibition. We also rationalize some previously unexplained phenomena associated with NSA binding. This understanding of the role of different structural domains of MLKL can help in designing better hMLKL targeting drugs. It is clear from our results that inhibitors that stabilize the locked conformation of MLKL will be effective in blocking oligomerization and necroptosis.19, NMR structure of human 4HB domain24, but not the full-length hMLKL structure are available. Also, the crystal structure of full-length mouse MLKL structure is available5. We utilized available crystal structures to build a full length chimeric model of monomeric hMLKL structure for performing MD simulations. The full length chimeric model of human monomeric MLKL protein was generated by using published crystal structures of human MLKL kinase domain (PDBID:4MWI), NMR structure of 4HB (PDB:2MSV) and full-length mouse crystal structure (PDB:4BTF) as templates. All the input structures were prepared by using Protein preparation wizard utility in Maestro using Schrödinger Suite molecular modeling package25. All the missing loops and side chains were filled during protein preparation. The mutation in PDB:2MSV was reverted back to wild type construct. Chimeric model was built using advanced homology model panel in Maestro using Prime26. 4HB N-terminal domain was taken from PDB: 2MSV and the PsK domain (residue 193–471) was taken from PDB: 4MWI. The intermediate structure containing the brace helices was taken from mouse PDB: 4BTF. Mouse and human MLKL amino acid sequences were aligned to build a complete model of human MLKL. Steric clashes and other unfavorable interactions observed in the protein reliability report were resolved by running restrained minimization in the protein preparation wizard utility. ATP was added to the model by extracting the ATP coordinates from the structure of ATP bound Kinase in PDB:1ATP27 after it was aligned with the MLKL model. The ATP-bound full length MLKL model was prepared for Molecular Dynamics simulations using protein preparation wizard followed by minimization using Macromodel with default settings28. MLKL is known to bind ATP in the absence of metal ions5, therefore no metal ions were included in the model. Thr357 and Ser358 residues were phosphorylated by using the build panel in Maestro. NSA was added to the phosphorylated model by performing covalent docking mimicking Michaels reaction29. The final three models were fully minimized by using Macromodel with default settings30.Crystal structures of PsK domains of hMLKL31 (version 1.13.7). The structures were solvated with water molecules in a cubic box of side equal to 2 × (m + 5) Å, where m is the maximum distance of any atom in the structure and its center, using the solvate function of HTMD (version 1.13.7). Finally, the systems were built and neutralized using the amber.build function in HTMD32 (version 1.13.7). Na + and Cl− ions were added to neutralize the system and make a salt concentration of 0.1 M. The following parameters were used for building of the systems: ff14SB33 for protein, TIP3P34 for the water model, Na + and Cl− ions from Joung and Cheatham35, ATP from Meagher et al.36, and phosphorylated serine (SEP) and threonine (TPO) from Khoury et al.37.The protonation states of the protein residues at pH 7.0 were estimated using the protein Prepare function in HTMD38, and then the charges for the ACE, NME and cysteine up to atom 39. During this calculation, the charges of the above-mentioned 22 atoms were kept fixed. The final charges were used for the CNS residue. For the Lennard-Jones and bonded parameters, ff14SB was used for all the above mentioned 22 atoms, while GAFF2 parameters were used for the other atoms. The parameters for bonded terms were also derived from GAFF2, except for the C9-S10-C11 angle terms which were taken from the ff14SB parameters for methionine (CT-S-2C angle).For the necrosulfonamide conjugated to Cys86, from the initial structure, we have constructed a residue called CNS and created the parameters for it. For the calculation of the charges, the CNS residue capped with acetyl (ACE) and methylamine (NME) was used as the initial structure  was subjected to the following modifications: the integration step was set to 2 fs, and constraints of 0.1 kcal/mol on the heavy atoms of ATP were added to the default constraints. The default production protocol (in module htmd.protocols.production v6) was used as is.All the systems were simulated using ACEMD1 and V2 was calculated by CPPTRAJ utility of Ambertools41  plots were obtained using VMD. The pairwise interaction energies between residues Lys157 and Cys86 were calculated using CPPTRAJ.Salt bridge interactions are obtained through VMD and all the hydrogen bond interactions are obtained using CPPTRAJ utility of Amber.41 and VMD 1.9.342 software. This section discusses results of the analysis of MD trajectories.We generated a total of 3 μs simulation data for apo, phosphorylated and NSA bound hMLKL systems using classical MD simulations. The data were analyzed by CPPTRAJ utility of AmberTools18Our simulations were able to capture wide inter-domain motions between N-terminal 4HB and PsK domains. The 4HB domain existed in cis state with the PsK domain in the starting conformation in all the three systems but the angle between the two domains fluctuated to a great extent, especially in apo and phosphorylated forms. The angular change between the N-terminal 4HB and PsK domains was measured in terms of angle θ as shown in Fig. Figure Next, we explored the impact of phosphorylation and NSA binding on secondary structure changes in MLKL structure. Similar to cross-correlation matrices, the difference in alpha helical percentages are mainly observed for the brace helices region  (Å) is the simplest method to  assess the structural variability of the protein in different states. We plotted the  Cα RMSD of hMLKL protein for the entire simulation length for apo, phosphorylated and NSA bound states using the average structure from the simulation as the reference point. Figure Root mean square fluctuation (RMSF) (Å) analysis, a measure of the extent of Cα mobility in the protein, is shown in Fig. Cross-correlation matrices help us in investigating the effect of motion of a single residue on a network of residues. The calculated cross-correlation matrices for backbone Cα atoms from apo, phosphorylated and NSA bound MLKL simulations averaged over the entire trajectory are shown in Fig. The cross-correlation matrix of NSA bound MLKL simulation is significantly different from both apo and phosphorylated MLKL as shown in Fig. et al.13. They observed that Lys157 crosslinks with Lys157 from another monomer in the tetrameric form and its mutation led to deficits in liposome permeabilization13. Our simulation data show that NSA binding flips the Lys157 sidechain and locks it in a cation-π interaction with the aromatic ring of NSA and makes it unavailable for crosslinking and tetramer formation.On further analysis of the key interactions of the residues in the brace helices that are affected upon phosphorylation and NSA binding, we observe that Lys157 of the second brace helix in the NSA bound state moved closer to Cys86 compared to apo or phosphorylated states as shown in the radial distribution function (g(r)) plot in Fig. apo and phosphorylated MLKL states. Fig. Flipping of Lys157 causes major perturbations to the interactions in the rest of the brace helices region. For instance, Asp144 of the first brace helix near Lys157, is now freed to stabilize the interactions between N-terminal 4HB and PsK domains of MLKL: the carboxyl group of Asp144 forms a conserved salt bridge with the side-chain of Lys95 from the N-terminal 4HB  and Ser358 (SEP358), the interactions between the residues of the A-loop are stabilized and several other residues in the N and C lobes of the PsK domain form multiple H-bonds and salt bridges. Fig. apo form and is lost in the NSA bound MLKL simulations. Quite strong salt-bridge interactions between phosphoserine-Lysine in a helix-coil have been previously reported because of the negative charge imparted by phosphate group39. Figure Phosphorylation state of a kinase determines its activity and regulation by modulation of its structure and stabilityapo and NSA bound MLKL. In phosphorylated MLKL simulations, we identify a network of residues that is interacting and stabilizing the A-loop dynamics such as salt bridge formation between the gatekeeper residue GLu351 with Lys354 and Glu213  with  Lys354  interact with both 4HB and PsK domain residues directly or indirectly. For example, Asp144 is re-positioned as a hub to establish new direct or indirect interactions between Leu89, Lys95, Glu258 and Arg315 as shown in Fig. apo and phosphorylated forms.Mixed lineage kinase domain-like (MLKL) protein has emerged as a key contributor in necroptosis. MLKL gets activated by RIP3 which phosphorylates threonine and serine on the activation loop in the pseudokinase domain. Upon activation, the 4HB domain unleashes from the PsK domain, gets oligomerized and permeates through the plasma membrane causing cell death. Our goal in this study was to understand two main questions: (1) why phosphorylation of MLKL is important for necroptosis? and (2) how NSA binding on the 4HB domain is preventing necroptosis? Our simulations show increased inter-domain flexibility of phosphorylated MLKL as compared to 23. This new understanding of the role and behavior of different domains of MLKL protein upon phosphorylation and NSA binding can help in designing better hMLKL inhibitors. Our study proposes that inhibitors that stabilize the locked conformation of the 4HB and PsK domains will prevent oligomerization and necroptosis.This study provides new insights into the molecular mechanism of hMLKL protein’s non-catalytic function in necroptosis and rationalizes the previously unexplained phenomena related to successful inhibition of necroptosis by NSA, but not by compounds that bind tightly to the ATP pocket of MLKL. It is clear from our study that inhibitors that replace ATP in the ATP-binding pocket of the PsK domain of MLKL can neither lock the inter-domain conformation nor reorient key 4HB residues to prevent necroptosis as observed by NSA. This explains why the recently reported tight binders of MLKL ATP pocket do not show any anti-necroptotic activitySupplementary InformationSupplementary Dataset 1Supplementary Dataset 2


Dear Editor,The Ras superfamily of GTPases are conserved in eukaryotes from yeast to humans, and play essential roles in the regulation of a variety of key cellular processes, including cell differentiation and proliferation, membrane trafficking, nuclear import and export, cytoskeletal remodeling and mitogenic signaling  protein was expressed in Escherichia coli and its crystal structure has been determined to a resolution of 2.7 Å (Table S1). Two molecules were observed in one crystallographic asymmetric unit. While the 13 residues at the N-terminus are flexible, so each molecule contains 260 residues, spanning A14 to K273. The structure reveals that the G-domain and DnaJ domain are two independent domains, connected by a flexible hinge region  increased the GTPase activity of xRbj Fig. D, which E. coli  shares the lowest similarity with RMSD of 1.41 Å. Previous reports suggest that DnaJ proteins interact with another important chaperone, the DnaK proteins . Further structural studies demonstrated that DnaJ binds to the substrate polypeptide-binding domain (SBD) and nucleotide-binding domain (NBD) of Hsp70, which is a prerequisite for the activation of Hsp70 Below is the link to the electronic supplementary material.