Several homologous domains are shared by eukaryotic immunity and programmed cell-death systems and poorly understood bacterial proteins. Recent studies show these to be components of a network of highly regulated systems connecting apoptotic processes to counter-invader immunity, in prokaryotes with a multicellular habit. However, the provenance of key adaptor domains, namely those of the Death-like and TRADD-N superfamilies, a quintessential feature of metazoan apoptotic systems, remained murky. Here, we use sensitive sequence analysis and comparative genomics methods to identify unambiguous bacterial homologs of the Death-like and TRADD-N superfamilies. We show the former to have arisen as part of a radiation of effector-associated α-helical adaptor domains that likely mediate homotypic interactions bringing together diverse effector and signaling domains in predicted bacterial apoptosis- and counter-invader systems. Similarly, we show that the TRADD-N domain defines a key, widespread signaling bridge that links effector deployment to invader-sensing in multicellular bacterial and metazoan counter-invader systems. TRADD-N domains are expanded in aggregating marine invertebrates and point to distinctive diversifying immune strategies probably directed both at RNA and retroviruses and cellular pathogens that might infect such communities. These TRADD-N and Death-like domains helped identify several new bacterial and metazoan counter-invader systems featuring underappreciated, common functional principles: the use of intracellular invader-sensing lectin-like (NPCBM and FGS), transcription elongation GreA/B-C, glycosyltransferase-4 family, inactive NTPase (serving as nucleic acid receptors), and invader-sensing GTPase switch domains. Finally, these findings point to the possibility of multicellular bacteria-stem metazoan symbiosis in the emergence of the immune/apoptotic systems of the latter. Dictyostelium), and plants as well as certain multicellular prokaryotes such as cyanobacteria and actinobacteria  in bacteria ; (3) as bacteria ; and (4)bacteria .+, or ADP-ribosyltransferases (ARTs) that modify DNA or proteins  proteins . IncreasStudies by us and others revealed that despite the protean manifestations of apoptosis and related phenomena in immunity across diverse eukaryotic lineages, at the molecular level it features certain common protein domains mediating different steps of the process . On the In eukaryotes, these domains are commonly incorporated into lineage-specific protein domain architectures and embedded within signaling webs that are typical of eukaryotic regulatory systems, such as protein kinase cascades  or the ubiquitin/ubiquitin-like (Ub/Ubl)-proteasome system . Until rThese prokaryotic systems also brought to light a hitherto unappreciated commonality with metazoan apoptotic systems, namely the use of small ‘adaptor’ domains in complex formation and effector recruitment . In metaThese findings provided the first hints for the possible provenance of key apoptotic adaptors related to the Death-like and TRADD-N families in bacterial conflict systems. In this study, we expand these findings to show that the classical metazoan-type Death domains are found in bacterial conflict systems that possess a similar array of effectors as the metazoan apoptotic systems. We also identify several previously unrecognized families of the TRADD-N domain in bacteria and metazoans. The new metazoan TRADD-N families often show lineage-specific expansions (LSEs) with a remarkable diversity of domain architectures paralleling their contextual connections in prokaryotic conflict systems. Consequently, we show that the TRADD-N domains define a widespread, conserved functional principle for regulating effector deployment across diverse conflict systems. These observations helped us identify and explain the mechanisms of several novel components of prokaryotic and animal immunity.Despite sharing a basic ternary organization of invader sensing, threshold setting, and effector deployment components, the recently identified conflict systems enriched in multicellular prokaryotes differ in terms of the actual components that play these roles . One majThus, by tracking the EADs we were able to identify other predicted core counter-invader conflict systems beyond the original ternary systems. Their domain compositions pointed to diverse activating mechanisms, such as proteolysis by either trypsin-like or caspase-like peptidases  as query recovered the eukaryotic Death domain in iterative PSI-BLAST searches . A multiple alignment of bDLD3 showed that it shares key conserved residues with the classical metazoan Death domain including tryptophans in helices 2 and 4, and an RxD motif at the beginning of the terminal helix 6 . Predicted effector domains fused to bDLD3 include catalytic domains involved in NAD+/nucleotide-processing   , proteinPNPase]) , proteolPNPase]) , and hydPNPase]) . The verPNPase]) . As withPNPase]) . For exaPNPase]) , in thisMicromonospora, WP_144082196); however, in these it was fused to an unknown module that took the place of the third component VMAP or iSTAND modules of the previously described ternary systems (–6 for iSTAND and p=1.2–2.2 × 10–4 for other STAND NTPases); accordingly, we named it iSTAND2  moiety generated by the TIR domain  counter-invader systems. Thus, the EAD-linked domains help define potential novel effectors and conflict systems that possess them. This postulate helped us identify multiple previously unrecognized sets of conflict systems, two of which utilized lectin fold domains  and werenovel putative carbohydrate binding module; Pfam: PF08305) (Nocardia seriolae) specifying proteins wherein bDLD3 is fused to either NPCBM or PNPase domains (Symploca (NET60851.1), we found bDLD3 in an identical configuration as the above-described fusion with the NPCBM domain, except that the latter domain was replaced by the FGS domain (The first of these are typified by the NPCBM (PF08305) , a membePF08305) . We obse domains , which h domains . In the S domain . This si+-processing domains , peptidases , and protein phosphorylation-related domains   . The cense, FHA)  or a novse, FHA) . The STAse, FHA) , which wse, FHA)  or the Ase, FHA) , which ise, FHA) . We had se, FHA) . As in tse, FHA) , namely se, FHA) . These ose, FHA) . Further–36 and p=3.7 × 10–5, respectively) implies that they are part of the unique immune processes of such organisms, which are regulated by the associated NTPase domains, ternary systems, or other fused signaling domains , and peptidase  domains, and an uncharacterized effector domain also found at the N-terminus of certain fungal heteroincompatibility HetE proteins.In a similar vein, EAD10 led us to a previously unrecognized prokaryotic conflict system that is widespread in bacteria and sporadic in archaea. The archetypal member of this system features an EAD10 domain fused to a constant (but rapidly evolving) C-terminal region comprised of TPR repeats a GreA/B-C domain, and a hitherto undetected version of the PIN domain , in addition to numerous bacterial sequences, recovered several metazoan sequences with significant scores . This search also recovered borderline hits to two starfish proteins . Their relationship to the bacterial proteins was confirmed by reciprocal searches: for instance, a PSI-BLAST search seeded with the above region from the A. planci recovered significant hits to bacteria homologs . Also recovered in these searches were the DNA-binding Death effector domain-2 (DEDD2/FLAME3) proteins of metazoans. Further, a profile-profile search initiated with an alignment generated from these echinoderm sequences and their bacterial homologs unified them with the vertebrate TRADD-N domains (HHpred probability 95.45%), thereby confirming them as TRADD-N domains.Using bacterial TRADD-N homologs as queries in PSI-BLAST searches against the nr50 and the nr90 databases , we recovered novel TRADD-N families across several bacteria and animals. For example, PSI-BLAST searches initiated with a TRADD-N domain of Anneissia japonica, e-value: 10–5, iteration 5). This hit overlapped with a model termed ‘Nucleic_acid_bd’ in the Pfam database (PF13820) defined by animal-specific proteins homologous to NCOA6. Their relationship was confirmed by a profile-profile search with the TRADD-N domain from the cyanobacterium Nostoc  that also recovered the same NCOA6-derived Pfam profile (HHpred probability 98.2%). A multiple sequence alignment helped define the correct boundaries of this conserved domain in the animal NCOA6 proteins and secondary structure prediction along with a sampling of the conserved sequence motifs (see below) showed a complete congruence to the TRADD-N domain structure. Further, profile-profile searches with representatives of each of the newly recovered groups also hit versions of the conventional ACT and structurally related domains  consistent with the previously established shared presence of a RRM-like fold in these domains and TRADD-N . The bacterial TRADD-N domains are found in multidomain proteins, usually occurring in-between distinct N- and C- terminal domains. The N-terminal domains tend to vary greatly even between closely related organisms  . AdditioIn this study, we identified a previously unrecognized version of the TRADD-N domain in the metazoan transcription regulator NCOA6 , a component of SET domain histone methyltransferase complexes such as the ASCOM coactivator, involved in nuclear hormone receptor transactivation . While tCrassostrea) show LSEs of at least 50–66 NCOA6 clade TRADD-N proteins corresponding to 17–20 unique architectural themes  displays a N-terminal region constant region with a cysteine-rich extracellular domain followed by a transmembrane helix, and an intracellular module with a DED and a NCOA6 TRADD-N domain (C. virginica LSE (~10 proteins) consists of proteins with an N-terminal DED-TRADD-N dyad fused to RNA binding KH and RRM domains and multiple macro and C-terminal ART domains resembling PARP-14 that is implicated in antiviral response  domN domain , and (4)N domain , superseresponse . In contl themes . Insteadl themes  or in sol themes .Branchiostoma is another genus with LSEs of the NCOA6 clade TRADD-N domains; different species show 13–129 proteins , brachiopods (33 copies), echinoderms (18–23 copies), hemichordates (22 copies), and cephalochordates (46–71 copies). Notably, these invertebrate proteins show several domain-architectural parallels to the NCOA6 TRADD-N proteins, and like them, the LSEs of DEDD-2 TRADD-N proteins are also characterized by constant regions combining a Death-like superfamily domain  with the TRADD-N domain and variable flanking regions  to be N- or C-terminally positioned domains that are coupled to other effector and signaling domains  Ub-molluscs : these sFinally, it is notable that the TRADD-N domains are shared not just between disparate metazoans with similar ecology but also disparate multicellular bacteria with similar sessile aggregations or colonies  and 4. HSpurred by the observation that the metazoan TRADD-N proteins bring together a wide array of potential effector domains, we carefully analyzed these to detect previously unrecognized effector-deployment systems. We present below two interesting examples that were identified by this analysis.Acropora digitifera. Beyond the GT4 domain, these proteins display high variability of domain architecture with fusions to several other domains suggestive of roles in biological conflicts , suggesting a broad-based strategy to counter immune evasion and inhibition of effectors by the invaders.The GT4 domain is also found in the effector position of another prokaryotic conflict system centered on the AP-GTPase proteins , which aTogether, these observations suggest that the GT4 domain is a potential effector that is used in a variety of bacterial conflict systems both in intracellular counter-invader and inter-organismal conflict. Via lateral gene transfer, it has also been ‘drafted into’ and expanded in the immune systems of certain metazoans. Biochemically characterized metabolic versions of the GT4 family modify a variety of substrates such as glycoproteins, lipids and sugars, and metabolites like mycothiol . This, a–4, iteration 3). Members of this superfamily have two homologous tandem β-α-β-β-β structural units, with the β-sheets of two units -like HNH endonucleases, caspases, ZU5 autopeptidases  and components of the metazoan ASK signalosome, have their ultimate origins in bacterial conflict systems . This woThe findings presented in this work offer a more complete picture of the intertwined evolutionary connections between immune and apoptotic systems of eukaryotes and prokaryotes. Phylogenetically distant multicellular or social forms are threatened by intracellular invaders/pathogens that can rapidly spread from cell to cell across the ensemble. This, coupled with inclusive fitness due to their clonal nature or close relatedness, favors the origin of apoptotic mechanisms of defense that limit the invasions to the initially infected cells. Other components of the metazoan toolkit, such as components of the calcium stores system and adhesion molecules, have also been reported to have their roots in bacteria with multicellular tendencies  . Hence, The tracing of the classical metazoan Death-like domain to the bacteria and the identification of the TRADD-N diversification adds to the functional understanding of these adaptor domains. Specifically, the predictions made regarding the mode of action of the TRADD-N domain could help understand the poorly understood immune systems of invertebrates. The prediction of shared immune mechanism across diverse invertebrates with comparable ecological features could help understand epidemics in marine ecosystems. Finally, we also believe that the predicted nucleic acid sensor and catalytic domains such as GT4 described here might help develop unique biochemical reagents to detect and modify biomolecules.https://ftp.ncbi.nih.gov/blast/documents/blastclust.html) (RRID:SCR_016641) to identify conserved gene neighborhoods based on conservation between different taxa. Additional filters were then used to output valid neighborhoods for further analysis: (1) nucleotide distance constraints , (2) conservation of gene directionality within the neighborhood, and (3) presence in more than one phylum.The NCBI Taxonomy database was used to obtain the names and ranks of taxa. For contextual analysis of prokaryotic gene neighborhoods, the GenBank genome files corresponding to unique GenBank genome assemblies (GCA ids) were used as the starting material. Specific neighborhoods were extracted using a Perl script that reports upstream and downstream genes of the anchor gene. Proteins encoded by these genes were then clustered using BLASTCLUST (SCR_001010) (SCR_005305) (PSI-BLAST (RRID:_001010)  and JACK_005305)  were useSCR_008184)  the non-redundant (nr) protein database of the National Center for Biotechnology Information (NCBI) frozen at October 1, 2020; or (2) the same database clustered down to 90, 70, or 50% similarity using the MMseqs program (RRID:_008184) ; or (3) SCR_010276) (SCR_011810) (SCR_016504) (Profile-profile searches were run using HHpred (RRID:_010276)  against _010276) , and (3)_011810) , HMM3 or_011810)  programs_016504) .As significant patterns in sequence divergence help identify proteins in conflict systems, we used statistically significant differences in mean column-wise Shannon entropy of alignments as a measure tested using the t-test . Positiowhere P is the fraction of residues of amino acid type i, and M is the number of amino acid types.SCR_015501) and the IQ-TREE (SCR_017254) programs. For the former, the JTT model was used with 10 rate categories of sites and the -slow option for an exhaustive tree search. For the latter, an exhaustive model search determined the WAG model with four gamma-distributed rate categories and empirical state frequency determined from the alignment as the most appropriate for tree construction.Phylogenetic trees were constructed using the FastTree  (RRID:SC IQ-TREE  (RRID:SCSCR_003047) (http://www.pymol.org) (RRID:SCR_000305) and MOL* programs (http://molstar.org). All other data processing (knitr and dplyr libraries), network analysis (igraph libraries), and graph visualization were done in the R language. Delimited datasets used in these analyses are available in the source data files and at ftp://ftp.ncbi.nih.gov/pub/aravind/Death_TRADDN/. PDS were computed thus   run agaited thus : organiswhere 2 test for the bias in location of the domains in architectures was performed using its implementation in the R language in form of the chisq.test command.The hypergeometric test was used to test for association of systems described herein with multicellularity in prokaryotes thus : each pr In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Review Commons.][Editors' note: this paper was reviewed by Acceptance summary:This paper presents a complex protein and genome sequence-analytic study that connects apoptotic biomolecular systems and counter-invader immunity systems. The authors identify true bacterial homologs of the Death-like 8 and TRADD-N superfamilies and show that these domains have emerged as part of a radiation of effector-associated α-helical adaptor domains. They also discover new counter-invader systems using intracellular invader-sensing lectin-like domains (NPCBM and FGS), glycosyltransferase-4 family sugarmodification domains, inactive NTPases serving as nucleic-acid-receptors and invader-sensing GTPase switches. Reviewer #1The authors present a very complex protein and genome sequence-analytic study that connects apoptotic biomolecular systems and counter-invader immunity systems: (1) They identify true bacterial homologs of the Death-like 8 and TRADD-N superfamilies. (2) These domains have emerged as part of a radiation of effector-associated α-helical adaptor domains that likely, via homotypic interactions, bring together diverse effector and signaling domains.(3) The TRADD-N domain links effector deployment to invader-sensing in multicellular bacterial and metazoan counter-invader systems. (4) They discover new counter-invader systems using intracellular invader-sensing lectin-like domains (NPCBM and FGS), glycosyltransferase-4 family sugarmodification domains, inactive NTPases serving as nucleic-acid-receptors and invader-sensing GTPase switches.Significance:The discoveries described in this manuscript are fundamental for the understanding or evolution and the network of apoptotoc/immunity biomolecular systems. This is a very good work.Place in the literature:This will be a seminal paper.Audience:All life scientists with general interest.My expertise:Bioinformatics, sequence analysis, gene function discovery, natural products.We thank the reviewer for the positive comments. We address the specific comments and our responses to them below.1. Several times, the authors mention "sensitive sequence analysis" without actually telling what they have in mind. Clearly, the approach involves including hits even if sequence statistical measures are not fully supportive/borderline. This is not an error if other indicators  support the conclusion. It would be great if the authors transparently showed when their sequence analysis needed to become extra-"sensitive".By sensitivity of sequence analysis we mean the capacity for improved detection of homology both in terms of range (i.e. detection of more homologs) and depth (i.e. detection of more distant homologs). The former is an issue because of the continuum of sequence divergence between homologs; hence, a single starting point might not be sufficient to detect all homologs in iterative sequence searchers. The latter arises from absence of “bridging” sequences in the current database between two distant homolog groups. Both are specifically important when studying proteins in biological conflict systems due to their rapid sequence divergence. Sensitivity can be increased by using curated, successively constructed multiple alignments that encompass an increasing diversity of sequences to construct the profile for RPS-BLAST, HMM and profile-profile HHPRED searches. We have now added more details to the Materials and methods explaining this .2. As a reader, I would appreciate a compression package of all the alignments, domain definitions, etc. in a computer-readable form  as supplementary material. This would greatly help to make the work reproducible.We now provide in the supplementary material a compressed archive containing all alignments generated in this study as text alns. We will also be also submitting these alignments to the Pfam database for wide dissemination across the community upon publication.Minor Comments:The manuscript is written clearly, all conclusions are backed by data. Yet, it would be helpful to have Results and Discussion being clearly distinguished.Given that certain involved inferences are closely related to the actual observations, we have kept those beside the paragraphs reporting the latter. However, in response to this suggestion we now have a separate Results section that includes all the primary findings and a Discussion section (page 22) that only includes the more general context and implications of the findings in the former section along with possible evolutionary hypotheses.Reviewer #2 ):The paper by Kaur et al. describes use of bioinformatics to look for relationships between genes encoding cell death proteins in bacteria and metazoans. While the focus is on adaptor proteins related to TRADD-N domains, many other domains are also covered.With the caveat that I am not a bioinformatician or computational biologist, I found this paper to be outstanding. The clear and informative figures provide a rapid way for interested readers to see the composition, origin, and relationships of their favorite proteins.The discussion is intelligent and revealing of evolutionary relationships and plausible explanations of how cell suicide could have been adaptive. They convincingly argue that prokaryotes use these proteins for defense: to detect foreign molecules, transmit signals, and implement responses.The abundance, complexity, and ubiquity of these proteins amongst single-celled organisms suggests defense against other, invading, organisms was an early and very important adaptation during evolution, and perhaps more surprisingly, use of these proteins for the suicide of cells, in order to protect their relatives, was also an early and widely adopted strategy.(Significance (Required)):I am not qualified to verify the bioinformatic analysis.I could not come up with any suggestions for how this manuscript could be improved.We thank the reviewer for the positive comments on the impact, significance and import of the study.


We observe conductive domain walls forming in zig-zagging structures, that are composed of head-to-head and tail-to-tail domain wall segments alternating on the nanoscale. Remarkably, both types of segments possess high conductivity, unimaginable in oxide ferroelectrics. These effectively 2D domain walls, dominating the 3D conductance, can be mobilized by magnetic fields, triggering abrupt conductance changes as large as eight orders of magnitude. These unique properties demonstrate that non-oxide ferroelectrics can be the source of novel phenomena beyond the realm of oxide electronics.Atomically sharp domain walls in ferroelectrics are considered as an ideal platform to realize easy-to-reconfigure nanoelectronic building blocks, created, manipulated and erased by external fields. However, conductive domain walls have been exclusively observed in oxides, where domain wall mobility and conductivity is largely influenced by stoichiometry and defects. Here, we report on giant conductivity of domain walls in the non-oxide ferroelectric GaV 4S8, extending the source of phenomena beyond the realm of oxide electronics.Conductive domain walls have been usually found in ferroelectric oxides. Here, the authors report on giant conductivity of domain walls and their magnetically avalanche-like expulsion event in non-oxide ferroelectric GaV Domain walls (DWs) are interfaces between adjacent domains, which can host spatially confined states with novel functionalities forbidden in bulk4. Notable examples of such properties emerging at DWs include spatially confined magnetic states in TbMnO35 and GaV4Se86 as well as local electronic properties in nonpolar ferroelastics, such as ferroelectricity in ATiO3 (A = Sr and Ca)8 and flexopiezoelectricity in WO39.In order to minimize the energy of a ferroic material, energetically equivalent domain states can form with different orientations of the ferroic order parameter. The number of domain states is dictated by the symmetry reduction upon the para-to-ferroic transition and the population of the domains can be controlled by the conjugate field14. Such conducting DWs have been observed in several materials, like BaTiO315, LiNbO317, h-RMnO3 (R = Er and Ho)19, and BiFeO322.An intriguing aspect of DWs in ferroelectric insulators is their electrical conductivity, often originating from the discontinuity of the normal component of the polarization across DWs 25 which render the walls immobile and thus curtail their usefulness and flexibility. Additionally, oxide materials are prone to defects, often hampering the utility of their conducting DWs.All ferroelectrics in which conductive DWs have so far been reported are oxides, where DW conductivity usually requires a specific strain configuration of the crystal, an improper character of the ferroelectricity or other unusual properties4S8, investigated by macroscopic transport studies as well as by piezoresponse force microscopy (PFM) and conductive atomic force microscopy (c-AFM). This compound belongs to the lacunar spinel family, where the V4 molecular clusters have been reported to accommodate different number of 3d electrons32. The heterovalent substitution of Ga3+ by Ge4+ or by Zn2+ has been demonstrated to respectively increase or decrease the number of 3d electrons occupying the V4 molecular units31. The stability of the two line compounds, GaV4S8 and GeV4S8, both categorized as narrow-gap molecular Mott insulators34, also points to the high flexibility in the valency of the V4 clusters. This multi-valency may provide an intrinsic mechanism to screen charged DWs via mobile carriers, either conduction electrons or holes, without requiring defects and off-stoichiometry. This intrinsic screening process would naturally make charged DWs also conductive. Hence, this material possesses several ingredients that are crucial to realize ideal DW functionalities and are not found to coexist in any oxide material. For example, we found that such charged DWs govern the overall conductivity of this material even though they constitute only a minor fraction of the 3D bulk volume. Furthermore, a “digital” alternation of conducting head-to-head (HH) and tail-to-tail (TT) segments occurs on the nanoscale within the conductive DWs. This may imply the coexistence of both electron- and hole-conduction channels in close proximity, unprecedented in oxide materials. Finally, this compound is a proper ferroelectric in which conductive DWs emerge in the unstrained crystal spontaneously, such that they retain their flexibility, facilitating their electric and magnetic control. We demonstrate that the in situ magnetic erase of DWs, which takes place through an avalanche-like DW expulsion process, changes the conductance of the material by up to eight orders of magnitude.In this study, we report on highly conductive DWs in a non-oxide ferroelectric, GaV4S8 undergoes a ferroelectric transition from a cubic (R3m) phase at TJT ≃ 45 K, due to a cooperative Jahn–Teller effect lifting the degeneracy of the V4 cluster orbitals28. Since the high-temperature crystal structure already lacks the inversion symmetry, the cubic-to-rhombohedral deformation necessarily leads to the onset of electric polarization. The ferroelectric nature of the rhombohedral phase has been demonstrated by pyroelectric current measurements36, PFM38, lattice dynamics studies39 and by dielectric spectroscopy40, as well as supported theoretically42. The breaking of the 1–P4) with the polarization vectors along the four 〈111〉-type cubic body diagonals, as shown in Fig. ∘ with each other, thus, no ±P domains can form in a single crystal43. Lamellar domain patterns, formed of two alternating ferroelectric domains and separated by {100}-type mechanically and electrically compatible 109∘ DWs, were observed by PFM38.GaV4S8 undergoes a magnetic ordering transition at TC = 13 K to a cycloidal (Cyc) state and subsequently to a ferromagnetic (FM) state below 6 K45. By magnetic fields in the sub-Tesla range, the Cyc state is turned to a Néel-type skyrmion lattice (SkL) and then to the FM state. The critical fields strongly depend on the orientation of the field with respect to the polar axis, being also the magnetic easy axis43. In addition to the polarization induced by the rhombohedral distortion, all the three magnetic phases exhibit sizable magnetoelectric polarizations36.Within the polar phase, GaV4S8 is exploited to control the population of domains by either electric (Ep) or magnetic (Hp) poling fields and to probe their volume fractions via either electric or magnetic properties. As illustrated in Fig. Ep applied along the [111] axis favors the P1 domain with polarization along the field direction, resulting in the P1 mono-domain state. However, when applying −Ep, the material acts as a “poling-diode” due to the lack of inversion domains: Upon the suppression of the P1 domain state, a P2–P3–P4 multi-domain state is created. Figure Ep∥[111] (for details of the poling protocol see the Methods part). The unpoled state is dominated by the P1 domain, as reflected by the large positive value of the polarization (~1.75 μC/cm2). This domain imbalance, likely arising from internal strains within the crystal, is further supported by the larger magnitudes of two low-field peaks at ~30 and ~70 mT, as compared to the two high-field anomalies at ~120 and ~140 mT. The former two are associated with the subsequent Cyc → SkL → FM transitions within the P1 domain with magnetic easy axis parallel to the magnetic field, while the latter two indicate the same transitions taking place simultaneously in the other three domains with easy axes oblique to the field43. Poling with +Ep enhances the polarization, which saturates at Ps ≈ 2.3 μC/cm2 for Ep > +4.4 kV/cm, resulting in the P1 mono-domain state. In contrast, −Ep decreases the measured polarization, indicating an enhanced volume fraction of the P2, P3, and P4 domains. When the P1 domain is fully suppressed, the polarization should reach the value of −Ps/3 ≈ −0.8 μC/cm2. Thus, the smallest experimentally obtained value of +0.7 μC/cm2, recorded after poling with −Ep = 7.4 kV/cm, still points to a multi-domain state formed of all the four domain states. In parallel to the electric-field-driven promotion (+Ep) or suppression (−Ep) of the P1 domain, the two low-field peaks in the susceptibility are enhanced or suppressed, respectively, contrary to the two high-field anomalies.Figure 46, the domain population can be efficiently controlled also by magnetic field. Poling with Hp along the [111] axis is expected to promote the P1 mono-domain state, irrespective of the sign of Hp, since the anisotropy energy is an even function of Hp. Indeed, Fig. Hp and its saturation for 1 mono-domain state is achieved. The domain population, as probed via the magnetic susceptibility, shows the same trend in Fig. Due to the coincidence of the magnetic easy axis with the polar axisEp or Hp, follows a typical semiconducting behavior both above and below the structural transition with only a tiny anomaly at TJT  plane, have the same out-of-plane piezoelectric signal. In the rhombohedral phase of GaV4S8, nine types of mechanically compatible DW can emerge: Three {100}-type uncharged and six {110}-type charged DWs  and darker domains. To fulfill mechanical compatibility and charge neutrality, the dark ones must be P2 domains, separated from P1 domains by (010)-type head-to-tail (HT) DWs. The P2 domain also forms a larger mono-domain island embedded in the P1–P2 lamellar pattern. On the right side of the PFM image, a P3–P4 lamellar pattern is seen with the same (010)-type HT DWs. Here, the two domains share the same color, as expected, and only the DWs in between appear darker. Charge neutrality is confirmed for both lamellar patterns by the c-AFM image.To identify the type of conductive DWs and to reveal the microscopic mechanism of DW conductivity, we carried out simultaneous PFM and c-AFM studies. Figure 2 mono-domain island from the P3–P4 lamellar pattern, while the upper part separates the P1–P2 and the P3–P4 lamellar patterns, as also displayed schematically in Fig. 2–P3 junctions, alternating with insulating P2–P4 segments. The P2–P3 junctions, being  The c-AFM data in Fig. The upper part of the complex DW structure represents the typical 2D conductive DW sheets observed in this material. It is composed of zig-zaging P1–P2 lamella (right side) and a P3–P4 lamella (left side). Correspondingly, the zig-zag DW is composed of HH and TT segments. While both types are conductive, the conductivity of the TT DWs was found to be higher, as seen in the c-AFM image. This conductivity difference is slightly increased towards larger bias voltages, as demonstrated in the nonlinear I–V curves for both polarities. To eliminate possible artefacts in the I–V characteristic due to voltage-driven DW displacements, the data points in the I–V curve were extracted from c-AFM images recorded at different voltages, using line scans perpendicular to TT and HH DWs. While the difference between the conductivity of HH and TT DW segments was reproducible for zig-zag DWs, we could not quantify the difference between their intrinsic conductivity for the following reasons. At first, it is not clear to what extent the internal conductivity of the DWs governs the measured conductivity and how much it is affected by the tip-surface contact. Moreover, due to the close proximity of HH and TT DWs, the current path from the tip to the large-area back electrode is ill defined. Thus, the measured conductivity may represent a weighted average of the internal conductivities of TT and HH segments, rather than their individual values.In order to gain further insights about the charge transport in the zig-zag DWs, we studied the I–V characteristic of HH and TT segments. The PFM and c-AFM images in Fig. 4S8? In materials with charged DWs, the conductivity is often governed by screening charges, i.e., by mobile carriers with charge opposite to the bound charge of the DW, which are accumulated from the bulk48. If the same mechanism applies for the zig-zag DWs in GaV4S8, the conductive nature of TT and HH segments implies that both positive and negative screening charges can be accumulated from the bulk. In this scenario, the conductivity difference observed between HH and TT DWs indicates that mobile electrons and holes have different mobility. While the multivalent character of V4 clusters49 may provide an intrinsic pathway both for mobile electrons and holes, as mentioned in the introduction, the unambiguous assignment of the microscopic conductivity mechanism requires further studies. It is worth mentioning here that the strain gradient, suggested to be a driving mechanism for unconventional DW functionalities52, at the zig-zag DWs may influence the local conductivity. Nevertheless, the simultaneous conductivity of TT and HH DWs is a unique feature of this compound, as opposed to oxide ferroelectrics, where typically one type of charge carriers, either electrons or holes, are provided by defects and/or off-stoichiometry. Thus, such extrinsic mechanisms lead to an enhanced conductivity at one type of charged DW and reduced conductivity at the oppositely charged one, compared to the bulk48. In fact, the high DW mobility (demonstrated below) and the non-reproducible formation of domain patterns from cooling to cooling imply that the concentration of defects is not high in our single crystals and the physics is likely not governed by defects. The role of defects in DW pinning has been documented well in various systems56. In contrast, the mobility of the DWs in GaV4S8 is well exemplified by their magnetically driven displacement and erase (discussed below).How do TT and HH DWs conduct in GaV59. Figure μ0H ≈ 8 − 12 T, which is shown in Fig. 357. In contrast, no conductivity enhancement was observed for either uncharged (010)-type DWs or the interior of the domains. In addition to its negative MR, the conductive DW is gradually displaced by the field and exits the scanned region above 11 T, as seen in Fig. After characterizing the conductive DWs, we study the effect of magnetic field on their charge transport, which may be strong for multiferroic DWs35. Since the field-driven polarization must be different for the different domain states, in principle, the charge density induced by the discontinuity of the polarization component normal to the wall could be tuned magnetically, which could charge originally neutral (010)-type DWs. For fields along the [111] axis, this effect should be the strongest for DWs involving the P1 domain. However, we found that P1–P2 DWs remains insulating, excluding this scenario. Thus, we believe a likely explanation of the giant negative MR is the enhancement of carrier mobility due to the suppression of spin scattering by magnetic field. Since a field-polarized FM state, with magnetization co-aligned with the magnetic field and not with the easy axes of the domains, can be achieved in GaV4S8 by fields ~1 T, the magnetic state at conductive DWs should be distinct from that in the bulk, to leave room for spin-dependent scattering in fields >1 T. Indeed, such magnetic states confined to DWs have been reported in GaV4S837 and GaV4Se86.Does the magnetic field affect the density or the mobility of the carriers at DWs? Due to the multiferroic nature of the material, the polarization can be altered by magnetic fieldμ0H ≥ 8 T, trigger irreversible changes in this material. This phenomenon is best seen at 10 K in the field-dependent resistivity and polarization curves respectively displayed in Fig. 4, which is close to the ratio observed in macroscopic measurements at the same temperature in Fig. Besides the giant negative MR of conductive DWs, magnetic fields above a threshold value, typically 1 mono-domain in μ0H = 12 T, which persists after switching off the field -cut crystal was contacted by silver paste in a top-bottom capacitor geometry. The poling fields, either electric or magnetic, were applied along [111] direction at 50 K and the sample was cooled to 4 K, where the poling fields were switched off and the sample was kept shorted for 15 min. Pyroelectric current was measured during heating with a heating-rate of 7 K/min. Magnetic field was swept at a rate of 1 T/min when recording the magneto-current in the magnetic field-dependent polarization studies. The polarization was obtained by integrating the pyroelectric current or magneto-current over time.All experiments were performed on insulating single crystals of GaVThe dc resistivity was measured by the four-probe method, where a Keithley electrometer (6517B) was used to apply a source voltage of ±8 V between terminal 1 and 2, located at opposite (111) faces of the sample, as well as to measure current through these terminals. The voltage drop between terminals 3 and 4 was recorded by a Keysight electrometer (B2987A). Terminal 3 and 4 was adjacent to and co-planar with terminal 1 and 2, respectively. Each co-planar contact pair nearly covered the corresponding surface with a small gap between them. During the poling procedures, the terminals, 1 and 3 as well as terminals 2 and 4, were shorted and the poling fields were applied from 50 K down to 4 K, where the poling fields were switched off before starting the measurements during heating the sample. For temperature- and field-dependent measurements, resistivity was recorded after stabilizing the temperature and the magnetic field in each step in order to avoid pyroelectric and magnetoelectric contributions, respectively.The dc magnetization measurements were carried out with a Physical Property Measurement System (14 T—PPMS) of Quantum Design, while the ac susceptibility was measured with a Magnetic Property Measurement System (5 T MPMS—SQUID) of Quantum Design. For electric-field poling, a home-designed probe was developed for the MPMS system and the same poling protocol was followed as for the polarization measurements. The slight difference in the critical fields for the magnetic transitions in Fig. Cryogenic AFM measurements were performed in an attoLiquid2000 setup in an atmosphere of 20 mbar He exchange gas using conductive Pt/Ir-coated ANSCM-Pt tips. The unique cryogenic setup allows for scanning probe studies in magnetic fields up to 12 T, but only supports the detection of the out-of-plane piezoelectric coefficient. Measurements were performed on an as-grown (111)-face of the crystal, with a polished parallel (111)-face of the sample attached to a back electrode. For PFM measurements, an external Stanford Instruments SR830 lock-in amplifier was used to apply an excitation voltage of 5 V to the tip at a fixed frequency of 17.87 kHz, while the back electrode was grounded. The signal was optimized for maximum domain contrast by setting the phase shift such that all signal appears in the X-channel, when scanning the interior of the domains. PFM measurements while changing magnetic fields were performed using an Intermodulation Products Multifrequency Lock-in Amplifier in resonance tracking mode. The c-AFM measurements were performed by applying dc bias voltages of up to 60 V to the bottom electrode and detecting the current flowing through the tip with a FEMTO I/V converter. The I–V curve is obtained from c-AFM images recorded at 15 K at various voltages of up to ±120 V. The value of current at a fixed voltage for HH and TT DWs is obtained by taking the mean current value of cross-sections of fixed length across multiple DWs.Supplementary Information


Individual linearly polarized laser pulses of 800 nm wavelength and 100 fs duration above a certain threshold fluence reverse the sample magnetization, independent of the magnetization direction, the so-called toggle switching. Local deviations from this deterministic behavior close to magnetic domain walls are studied in detail. Reasons for nondeterministic toggle switching are related to extrinsic effects, caused by pulse-to-pulse variations of the exciting laser system, and to intrinsic effects related to the magnetic domain structure of the sample. The latter are, on the one hand, caused by magnetic domain wall elasticity, which leads to a reduction of the domain-wall length at features with sharp tips. These features appear after the optical switching at positions where the line of constant threshold fluence in the Gaussian footprint of the laser pulse comes close to an already existing domain wall. On the other hand, we identify the presence of laser-induced domain-wall motion in the toggle-switching event as a further cause for local deviations from purely deterministic toggle switching.We present a microscopic magnetic domain imaging study of single-shot all-optical magnetic toggle switching of a ferrimagnetic Gd The reversal of magnetization at the fastest possible time scales and microscopic length scales is one of the most important issues regarding the use of magnetic materials in future data-storage or information-processing devices. One route towards that goal is the control of magnetization by ultrafast laser pulses without resorting to magnetic fields, the so-called all-optical switching (AOS) –15. AOS Important for applications of all-optical toggle switching is the reproducibility of the switching event on microscopic length scales. The presence of magnetic domain walls has been found to limit the repeatability of the switching event, leading to nondeterministic switching in the vicinity of the domain walls . This ha26Fe74 film with out-of-plane easy axis of magnetization by magnetic imaging using photoemission electron microscopy (PEEM) with X-ray magnetic circular dichroism (XMCD) as magnetic contrast mechanism. We focus single ultrashort infrared laser pulses of 100 fs temporal width to the vicinity of magnetic domain walls and study the local deviations from a perfect, deterministic toggle switching and their relation to the domain-wall position for temperatures above and below the ferrimagnetic magnetization compensation temperature. Besides pulse-to-pulse variations in laser fluence, we identify two main mechanisms that can lead to non-deterministic switching on the nanoscale: (i) domain coarsening, that is, the reduction of domain-wall energy by avoiding sharp features created in a switching event, and (ii) laser-induced domain-wall motion. From our results, we cannot deduce an effect of the base temperature. Both effects can become important for optical writing of magnetic information.In this paper, we investigate nondeterministic all-optical toggle switching of a Gd2 incident fluence in the center for a sample temperature of 50 K, respectively, below the magnetization compensation temperature of the sample of about 120 K. The red ellipse indicates a line of constant fluence of 6.1 mJ/cm2 of the laser on the sample. Light and dark grey contrasts indicate the two opposite magnetization directions. 2 up to at least the maximum fluence of 6.8 mJ/cm2. Outside the ellipse, that is, at fluences below 6.1 mJ/cm2, no switching occurs except in the area marked by an arrow in 2 to the threshold for toggle switching in this sample at 50 K.Comparison of microscopic magnetic domain patterns, recorded in static XMCD-PEEM imaging before and after excitation of the sample by a single laser pulse in absence of any magnetic field reveals the lateral distribution of optically switched areas. The local microscopic deviation from a deterministic toggle switching defined by a uniform threshold fluence, as indicated by the arrow in 2 on the sample, dark and light gray contrast corresponds to opposite magnetization directions. The second row, labeled “B”, contains synthetic images sketching the magnetic domain images one would expect if everywhere inside the ellipse the magnetization direction had reversed, while it had remained constant outside. This would correspond to deterministic toggle switching with a threshold fluence marked by the ellipse. It was obtained by first discriminating the dark and bright contrast of the image from the previous column of row A into black and white and subsequently reversing this black-and-white contrast inside the red ellipse. The third row, labeled “C”, repeats the images from row “A” while indicating the deviation between the actually observed domain pattern and the one sketched in row “B”. White hatched areas indicate those parts of the sample that should have switched but did not and the black hatched areas those that should not have switched but did.To further investigate the effect of magnetic domain patterns on the toggle switching, different parts of the sample were excited by a sequence of individual laser pulses. 2 to place the threshold lines in From the images shown in Intrinsic effects concern properties of the sample itself. Such effects are seen in The same effect in the converse sense occurs at around the same position after the third pulse. Here the sharp tips expected at the end of the bright domains inside and outside the ellipse at the top left end are rounded off, leaving a rounded bulge of the dark domain 3. This aAfter the fourth pulse, the entire upper tip of the ellipse remained with dark contrast. This also saves domain-wall energy, since now the existing dark domain outside the top end of the ellipse connects to the dark domain resulting from the toggle switching in the center part of the ellipse. What is remarkable in Finally, after the fifth pulse, the situation around the top of the ellipse is similar to the pulses before: The dark domain that should remain outside the ellipse shrinks to a rounded shape on the left, reversing in some area outside the ellipse the magnetization from dark to bright. At the right edge of the ellipse, a uniform bright magnetization is observed and any dark domain that could have possibly been generated at the position where the bright domain at the right edge in These domain-wall motion events caused by domain-wall elasticity are observed independent of the temperature, at 50 K , 200 K , that is2 in the center. Here the sample is at a temperature of 200 K. The odd number of pulses switches the domain from dark to bright and the surrounding, within the threshold line for toggle switching, from bright to dark. The threshold for toggle switching is recognized from the two straight domain walls following the two long sides of the elliptic shape of the laser spot on the sample. The center of the laser pulse is about in the center of the image. We mentioned already that the boundaries between switched and unswitched regions of the sample are expected to be smooth and follow a line of constant fluence of the laser footprint for entirely deterministic switching. However, at some places the domain walls exhibit excursions from such a smooth shape, as for example in The bottom row of Continuing the series of laser pulses, these excursions get more pronounced. In addition, starting from the sixth pulse, a domain-wall movement towards the inside of the domain is observed at the lower left end of the domain, marked by the leftmost white “+” symbol in the last image. Except for this lower left end of the domain, the movement of the domain walls is towards the side of the higher fluence in the fluence gradient on the sample. The movement towards the right at the lower left end of the domain might as well be driven by the domain wall energy, since there the observed domain-wall motion reduces the overall domain wall length.25Fe75 bilayer [We interpret this domain-wall motion in accordance with our previous publications as laser-induced depinning and successive thermal domain-wall motion in the locally heated region of the sample –21. The  bilayer –24 or co bilayer . An alte bilayer . In the  bilayer ). Howeve bilayer , to diff26Fe74 film by single ultrashort laser pulses showed that the factors that cause this deviation can be distinguished as intrinsic and extrinsic effects. Variations in the fluence from pulse to pulse as an extrinsic effect is not fully avoidable and can lead to nondeterministic toggle switching close to the fluence threshold line in the footprint of the laser pulse. As intrinsic effects we can identify two different mechanisms: The first one is related to the shape of the magnetic domains as they are left directly after the all-optical switching and the force on the domain wall resulting from the balance of domain-wall and magnetostatic energy. Sharp domain features that would occur where domain walls are close to the boundary of the optically switched region in the laser spot are avoided by domain-wall motion events. This is observed independently of the sample temperature, below and above the magnetic compensation temperature. The second intrinsic effect is laser-induced domain-wall motion. This is clearly identified by local deviations in the position of existing domain walls after toggle-switching events. The direction of the laser-induced domain-wall motion is predominantly towards the higher laser fluence, but the data does not allow one to prove a unidirectional motion as it has been observed previously in a different sample [XMCD-PEEM imaging of the deviation from deterministic all-optical toggle switching of a Gdt sample . Our res26Fe74 rare-earth–transition-metal (RE–TM) alloy film with 15 nm thickness, deposited at room temperature using magnetron sputtering (base pressure <10−8 mbar) from elemental targets. The Ar sputter pressure was kept constant at 3.5 × 10−3 mbar during the deposition process. The film was prepared with 5 nm Pt as a seed layer on a Si(100) substrate with a 100 nm thick thermally oxidized SiOx layer and covered with a 3 nm Al capping layer to prevent oxidation. Composition and layer thicknesses were determined by calibrating the sputter rates with a quartz balance before the depositions. Thus, the sample structure was as follows: Al (3 nm)/Gd26Fe74 (15 nm)/Pt (5 nm)/SiOx (100 nm)/Si(100). Magnetic properties were characterized by superconducting quantum interference device vibrating sample magnetometry, which confirmed an out-of-plane easy axis of magnetization with rectangular hysteresis loops and a coercivity of about 5 mT at room temperature, see The sample was a GdM5 absorption edge at 1182.6 eV photon energy was used as magnetic contrast mechanism. A small electromagnet mounted inside the sample holder allows for applying a magnetic field to the sample for demagnetizing or creating domains. Before the start of each series of laser exposures, the electromagnet was used to saturate the sample. Magnetic domain walls have been created by subsequently applying magnetic fields of reversed polarity. All images presented here were recorded with positive helicity of circularly polarized X-rays. In those images, light (dark) gray contrast corresponds to magnetic domains having a positive (negative) projection of their magnetization direction on the X-ray incidence. Experiments were performed at a sample temperature of 50 K, which is below the magnetic compensation temperature (TM) of the ferrimagnet, as well as at 200 K and at room temperature, both being above TM.Magnetic domains were resolved using the photoemission electron microscope (PEEM) at the UE49-PGM SPEEM beamline of BESSY II. The acceleration potential between the sample and the first objective lens of the PEEM was set to 15 keV. X-ray magnetic circular dichroism (XMCD) at the Gd p-polarized laser pulses with a central wavelength of 800 nm were focused on the sample at a grazing incidence of 16° to a spot size of about 12 × 60 μm2 (at 1/e of the maximum intensity) by a lens inside the vacuum chamber. The combination of a polarizer and a half-wave liquid crystal plate constitutes a variable attenuator for tuning the fluence, which in the center of the spot ranged from 0 to 25 mJ/cm2 incident fluence. The numbers given for the fluence contain a 15% systematic error due to the uncertainty in the determination of the spot size on the sample and the attenuation of the laser light by the optical elements on the way to and into the vacuum chamber. The position of the laser spot on the sample was determined with an accuracy of 0.2 μm along the short axis and about 1 μm along the long axis of the elliptic footprint of the laser beam on the sample before and after acquisition of each series of magnetic domain images at a strongly reduced fluence and 1.25 MHz repetition rate, recording PEEM images of the intensity from three-photon photoemission processes at hot spots of the sample surface and fitting the resulting intensity to a two-dimensional elliptic Gaussian.The sample was excited by laser pulses from a Femtolasers Scientific XL Ti:sapphire oscillator set to about 100 fs temporal width. A Femtolasers Pulsfinder was used to deliver single laser pulses to the sample. The linearly Supporting information features temperature-dependent magnetization loops of the sample and the determination of the magnetic compensation temperature.File 1Magnetic characterization.


We observed that 473 putative orthocaspases were distributed among 62% cyanobacterial strains subsumed within all the taxonomical orders. However, high diversity among these orthocaspases was also evident as the conventional histidine–cysteine (HC) dyad was present only in 72.03% of orthocaspases (wild-type), whereas the rest 28.18% were pseudo-variants having substituted the catalytic dyad. Besides, the presence of various accessory functional domains with Peptidase C14 probably suggested the multifunctionality of the orthocaspases. Moreover, the early origin and emergence of wild-type orthocaspases were conferred by their presence in Gloeobacter; however, the complex phylogeny displayed by these caspase-homologs perhaps suggested horizontal a gene transfer for their acquisition. However, morpho-physiological advancements and larger genome size favored the acquisition of orthocaspases. Moreover, the conserved caspase hemoglobinase fold not only in the wild-type but also in the pseudo-orthocaspases in Nostoc sp. PCC 7120 ascertained the least effect of catalytic motifs in the protein tertiary structure. Further, the 100-ns molecular dynamic simulation and molecular mechanics/generalized born surface area exhibited stable binding of arginylarginine dipeptide with wild-type orthocaspase of Nostoc sp. PCC 7120, displaying arginine-P1 specificity of wild-type orthocaspases. This study deciphered the distribution, diversity, domain architecture, structure, and basic substrate specificity of putative cyanobacterial orthocaspases, which may aid in functional investigations in the future.The functionality of caspase homologs in prokaryotic cell execution has been perceived, yet the dimensions of their metabolic pertinence are still cryptic. Here, a detailed  Caspases are specialized cysteine-aspartate proteases, recognized to mediate apoptosis in metazoans. These peptidases are synthesized as inactive proenzymes, which upon activation attain dimeric configuration, having p20 and p10 sub-domains in each monomer . The pro2+-independent dimeric proteases  residues  motif in MCAs TbMC1 and TbMC4, probably rendering their proteolytic activity  composed of four parallel β-sheets surrounded by three α-helices . Furtherroteases , whereasresidues . Additioresidues . Owning residues . This 4%residues . Most ofresidues  and therresidues . An initresidues . MoreovePCC 7806 . Howeveractivity . The abuactivity .Nostoc sp. PCC 7120 was performed using homology modeling and compared elaborately with their pseudo-counterparts. Further, docking and molecular dynamic (MD) simulation of the protein-substrate complex was performed to ensure the substrate specificity of this OCA. In addition, the binding free energy (ΔGbind) calculated using the molecular mechanics/generalized born surface area (MMGBSA) also assured the stability of the complex. This study will provide an updated and holistic view on the attributes of putative OCAs and may pave new ways to answer the existence of OCA-mediated PCD in cyanobacteria.This study focuses on the functionality of wild-type OCAs  and their physiological congruity in cyanobacteria. Here a comprehensive bioinformatics study has been conducted to describe the distribution, diversity, abundance, domain configuration, and phylogeny of 473 putative cyanobacterial OCAs. Additionally, the structural analysis of a wild-type OCA from 1. The p20-like sub-domain (Peptidase C14)-containing protein sequences in 132 whole-genome-sequenced cyanobacterial strains were identified by performing domain enhanced lookup time accelerated (DELTA) BLAST (Trypanosoma brucei (Q585F3.1) as the query. Sequences with incomplete Peptidase C14, either at the N- or C-terminal, or lacking the domain, were manually eliminated. Finally, 473 putative OCA sequences from 82 cyanobacterial strains were further analyzed.The protein sequences of putative cyanobacterial OCAs were retrieved from the NCBI databaseA) BLAST , using 4All the protein sequences were manually analyzed and subsequently categorized into true (wild-type) and pseudo (mutated) OCAs, based on the catalytic dyad forming the active site. Only those proteins with classical HC dyad were considered as wild-type, whereas proteins having any other amino acid residues in the catalytic dyad were regarded as pseudo-OCAs. Further, the distribution and abundance of all OCA subtypes (true and pseudo OCAs) along the taxonomical order of these 82 cyanobacterial strains were compared against their genome sizes and morphological complexities.2  and phylogeny were performed using only the Peptidase C14 domain of all the putative OCAs. Additional domains associated with the proteases were not considered to avoid any possible uncertainty due to their varied origin and evolutionary history. Four hundred seventy-three Peptidase C14 domains were aligned by MUSCLE , in MEGA4 .Thalassiosira pseudonana CCMP1335 (XP_002295354.1) was used as the outgroup, since it is a marine diatom and possesses a closer evolutionary relationship with cyanobacteria (A rooted phylogenetic tree comprising 473 Peptidase C14 sequences was constructed by the neighbor-joining method  using MEbacteria . The brabacteria . The relbacteria –4. The bNostoc sp. PCC 7120 is a model cyanobacterium and not only harbored the wild-type (HC) OCAs but also consisted of other two abundant forms of pseudo OCAs, i.e., YS and YN types, therefore, three OCAs, i.e., WP_010999143.1 (HC), WP_010999263.1 (YS), and WP_010997820.1 (YN), of Nostoc sp. PCC 7120 were selected. Due to the lack of suitable templates, only the Peptidase C14 domains of WP_010999263.1 (YS) and WP_010997820.1 (YN) were modeled. High-quality homology models were constructed by Swiss-Model5 using default settings  and mutated (YS and YN) OCAs. Since settings . Furtherss-Model . The finModel  and aspartyl-glutamyl-valyl-aspartate (DEVD), were used to study the substrate specificity of wild-type OCA . While DEVD is a classical caspase substrate, RR was previously used as a substrate for cyanobacterial OCA, MaOC1 . For the+ and Cl– ions  at NPT. After equilibration, a production run of 100 ns was performed each at the NPT ensemble. The trajectory was visualized by VMD 1.9.3 and UCSF Chimera 1.13.1.bind) using the Molecular Mechanics Generalized Born Surface Area (MMGBSA) method implemented in Amber16. The calculation was performed at the dielectric constant of 1 and 80 for solute and solvent, respectively , Synechococcus sp. PCC 7502, Synechocystis sp. PCC 6714, Synechocystis sp. PCC 6803, Pleurocapsa sp. PCC 7327, Cyanothece sp. PCC 7425, and Nostoc azollae 0708 harbored only pseudo-OCAs . Out of 473 OCAs, 340 were wild-type (72.03%), having the HC dyad in their active sites, whereas the rest 133 (28.18%) were pseudo-variants. Among the pseudo-OCAs, YS- and YN-substituted catalytic motifs were predominant, accounting for approximately 47% and 31% of all pseudo-variants, respectively. Besides, 16 other pseudo-variants were also identified which comprised YG, YC, YQ, LG, RG, HN, HG, YH, YD, YR, NC, FS, QD, CN, QC, and CS motifs in their active sites . Among 8udo-OCAs . HoweverThe abundance of OCA was estimated to be highest in Nostocales and Oscillatoriales with an average occurrence of 0.0921 and 0.0723 true OCA per 100 proteins, respectively. Besides, an abundance of 0.0416 true OCA per 100 proteins was observed in Chroococcales and the lowest abundance of 0.0145 true OCA per 100 proteins was estimated in Synechococcales. The average abundance of pseudo-OCAs was comparable for Nostocales (0.0349/100 proteins), Oscillatoriales (0.0264/100 proteins), and Chroococcales (0.0261/100 proteins), while the lowest abundance was recorded again for Synechococcales (0.0027/100 proteins) . The actNostoc sphaeroides CCNUC1 having 14 true and 3 pseudo OCA-encoding genes , belonging to Gloeobacterales, Synechococcales, Chroococcales, Oscillatoriales, and Nostocales, displayed an exclusively high number of wild-type OCAs (≥5); however, the number of pseudo-OCAs ranged from 1 to 3 in 82 cyanobacteria. Nevertheless, the highest was observed in ng genes .Considerable variability in domain architecture among putative OCAs was observed due to the presence of multiple functional domains. Apart from Peptidase C14, 35 unique Pfam domains were identified in 473 OCAs . While tCrinalium epipsammum PCC 9333), WP_158517387.1 (Moorea producens PAL-8-15-08-1), and ABG51878.1 (Trichodesmium erythraeum IMS101) contained two TMs. Although the TM is generally located C-terminally to the Peptidase C14 domain, in AFZ48670.1 (C. stanieri PCC 7202), WP_015220159.1 (C. aponinum PCC 10605), AKV65607.1 (Microcystis panniformis FACHB-1757), WP_125731015.1 (Microcystis viridis NIES 102), WP_083305259.1 (M. producens PAL-8-15-08-1), and WP_167720235.1 (Tolypothrix sp. PCC 7910), TM was identified at the N-terminal with respect to Peptidase C14 , among which 30 and 37 were true and pseudo OCAs, respectively. The presence of one TM was fairly common among OCAs, but WP_015201768.1 , BAZ37947.1 , WP_015119030.1 (Rivularia sp. PCC 7116), and WP_011611472.1 (T. erythraeum IMS101). Furthermore, it was observed that the OCAs of strains belonging to Nostocales consisted of all the domain configurations , (G/A), and (L/I/M/F), (D), (X), (C/S/N), (H/F/Y/R) . Besides, G7 and D27 were also conserved in all the OCA subtypes, probably suggesting their essentiality.The sequence alignment of all OCA sequences displayed substantial diversity. The HMM logo exhibited a relatively conserved region surrounding the catalytic dyad . The conG. kilaueensis JS1 and G. violaceus PCC 7421  having only wild-type Peptidase C14, were included in HC III and HC VIII clades. Moreover, pseudo-OCAs from Chroococcales, Pleurocapsales, Chroococcidiopsidales, Oscillatoriales, and Nostocales were harbored within the YS clade, whereas the YN clade subsumed only filamentous and heterocytous strains from Synechococcales, Oscillatoriales, and Nostocales , WP_010999263.1 (YS), and WP_010997820.1 (YN) from ical CHF . Despiteical CHF . Howeverical CHF .bind (= −16.99 ± 5.99) of the HC–RR complex also exhibited high binding efficacy , further strengthened the previous assumption.The distribution of putative OCAs among cyanobacteria exhibited a clear relation with larger genome size and morphological complexities , since tpulation . Furthereobacter . Therefoifestyle . On the Trypanosoma brucei the role of TbMC4  in the cell cycle progression and virulence warranted their activity in cellular processes  the rest ∼28% was pseudo-OCAs. In general, these pseudo-OCAs in cyanobacteria are considered as catalytically inert due to lack of functional HC motif in the active site, but in rocesses . A recenynthesis . Additiobacteria .Being a rich repertoire of functional domains, apart from Peptidase C14, cyanobacterial OCAs presumably display multifunctionality, since these domains possess diverse catalytic capacities like protein–protein interactions (WD40), protein modification (FGE sulfatase), scaffold (HEAT_2), and signaling (GUN4). Mostly, these accessory domains are located C-terminally to Peptidase C14 but in some OCAs the C-terminally positioned Peptidase C14 domain was also observed, although these alterations in domain architecture may not have any functional amendments. In addition, the distribution of accessory domains was also uneven among cyanobacteria, since the OCAs from heterocytous strains with broader physiological abilities exhibited the presence of diverse accessory domains. The underlying reason is possibly the higher abundance of true OCAs in heterocytous strains. Additionally, the employment of these multidomain OCAs may benefit the physiologically robust life strategies. Furthermore, the presence of TM infers membrane localization of some OCAs, while others devoid of TM are probably cytosolic. The presence of putative cytosolic and membrane-bound true OCAs depicted the possibility of both intrinsic and extrinsic PCD in cyanobacteria , yet theMSA and phylogeny revealed significant sequence diversity and intermixing of cyanobacterial OCAs from different taxonomical orders. The wild-type OCAs were subsumed within nine distinct clades, whereas YS and YN motifs containing pseudo-variants formed two distinct clades, respectively; however, all other pseudo-OCAs were scattered throughout the tree. Mostly, the clades bracketed cyanobacterial strains with different morphological features and/or lifestyle preferences, suggesting the possibility of horizontal gene transfer (HGT) events for acquiring these genes . AdditioT. brucei) D95 and D211 exhibited their significance in binding basic substrates (Nicotiana tabacum L.), PCA MALT-1, and AtMC9 , respectively (The structural homology of three putative OCA variants with the HC, YS, and YN catalytic dyads is conferred by conserved CHF in their Peptidase C14 domain. Therefore, the substitution of catalytic motifs does not considerably amend the 3D conformation of these proteins ; howeverbstrates . Additioectively .Nostoc sp. PCC 7120 through detailed structural analysis, molecular docking, and MD simulation. Although the wild-type cyanobacterial OCAs possessed conserved fold and catalytic dyad, the protease activity and substrate specificity cannot be corroborated without experimental validations. However, delineating the functionality of the OCAs in cyanobacteria, progenitor of oxygenic photosynthesis, is crucial for understanding not only the evolution and acquisition of caspase homologs in the plant kingdom but also the physiological pertinence of these proteases in PCD.The identification of caspase homologs in prokaryotes advocated for the possibility of a protease-based cell death mechanism among these life forms. However, the involvement of all these caspase homologs in cell execution pathways is still uncertain. The present study exclusively describes the distribution, diversity, abundance, domain architectures, and phylogenetic relationship of putative cyanobacterial OCAs. Further, this work unraveled the conservation of CHF and Arg-P1 specificity of wild-type OCA in The original contributions presented in the study are included in the article/SB conceptualized the study and performed the computational analysis. SB and SK retrieved sequences from the database and performed the curation and analysis. AKM conceived and supervised the work. SB wrote the manuscript with contribution and thorough revision from SK and AKM. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


N-acetylglucosaminidase from V. campbellii reveal substrate specificity in chitin utilization.Crystal structures of a GH20 β- Vibrio species play a crucial role in maintaining the carbon and nitrogen balance between the oceans and the land through their ability to employ chitin as a sole source of energy. This study describes the structural basis for the action of the GH20 β-N-acetylglucosaminidase (VhGlcNAcase) in chitin metabolism by Vibrio campbellii (formerly V. harveyi) strain ATCC BAA-1116. Crystal structures of wild-type VhGlcNAcase in the absence and presence of the sugar ligand, and of the unliganded D437A mutant, were determined. VhGlcNAcase contains three distinct domains: an N-terminal carbohydrate-binding domain linked to a small α+β domain and a C-terminal (β/α)8 catalytic domain. The active site of VhGlcNAcase has a narrow, shallow pocket that is suitable for accommodating a small chitooligosaccharide. VhGlcNAcase is a monomeric enzyme of 74 kDa, but its crystal structures show two molecules of enzyme per asymmetric unit, in which Gln16 at the dimeric interface of the first molecule partially blocks the entrance to the active site of the neighboring molecule. The GlcNAc unit observed in subsite −1 makes exclusive hydrogen bonds to the conserved residues Arg274, Tyr530, Asp532 and Glu584, while Trp487, Trp546, Trp582 and Trp505 form a hydrophobic wall around the −1 GlcNAc. The catalytic mutants D437A/N and E438A/Q exhibited a drastic loss of GlcNAcase activity, confirming the catalytic role of the acidic pair (Asp437–Glu438). As reported in the CAZy database (http://www.cazy.org/GH20.html), other GH20 members include, for example, β-hexosaminidases (EC 3.2.1.52) and lacto-N-biosidases (EC 3.2.1.140). Human HexA  is a monomeric enzyme containing four functional domains, with the central (β/α)8 TIM-barrel domain acting as the catalytic domain  chitooligosaccharide chain from the nonreducing end, generating GlcNAc monomers as the final products . The enzyme can hydrolyze colloidal chitin, but its specific activity for the polymeric substrate is much lower than for chitin fragments  host. In this study, we report the first crystal structures of a GH20 exo-β-N-acetylglucosaminidase from the marine species V. campbellii in the absence and the presence of the natural substrate (GlcNAc)2, which was cleaved into GlcNAc during crystal soaking, so that one GlcNAc was bound in the active site. We also solved the structure of the D437A mutant of the same protein. Detailed crystallographic analysis revealed the important features of the enzyme for sugar binding and substrate specificity, and the roles of the active-site residues were elucidated by steady-state kinetic studies.2.2.1.nag2 gene encoding VhGlcNAcase (residues 1–642), with ten additional residues at the C-terminus (RSRS residues of the cloning site followed by the His6 tag), was cloned into the pQE-60 expression vector  as described in Suginta et al.  technique using the QuikChange Site-Directed Mutagenesis Kit  according to the manufacturer’s protocols. The full-length  al. 2010 and the 2.2.E. coli M15 (pREP4) host cells (Qiagen) transformed with the pQE60/GlcNAcase construct were picked and grown overnight at 37°C on Luria–Bertani (LB) agar plates containing 100 µg ml−1 ampicillin (Amp) and 25 µg ml−1 kanamycin (Kan). This overnight culture was used to inoculate a fresh 1 l culture using a 1:100 dilution. The fresh culture was Terrific Broth (TB) medium supplemented with 100 µg ml−1 Amp and 25 µg ml−1 Kan and was grown at 37°C with shaking at 200 rev min−1 until an OD600 of 0.6 was reached. To induce expression of recombinant VhGlcNAcase, isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to the culture medium to a final concentration of 0.4 mM. After 16 h of incubation at 20°C, the cell pellet was collected by centrifugation and resuspended in 20 ml extraction buffer  and then lysed on ice using a Sonopuls Ultrasonic homogenizer with a 6 mm diameter probe . Insoluble debris and unbroken cells were removed by centrifugation at 12 000g at 4°C for 1 h and the supernatant was immediately applied onto a polypropylene column packed with 5 ml TALON Superflow metal-affinity resin  operated under gravitational flow at 4°C. After sample application, unbound proteins were washed out with eight column volumes (CV) of equilibration buffer (20 mM Tris–HCl pH 8.0 containing 150 mM NaCl) followed by 7 CV of the same buffer supplemented with 5 mM imidazole.Single colonies of the VhGlcNAcase was eluted by applying 3 × 10 ml of 150 mM imidazole in the equilibration buffer at pH 8.0. Fractions containing active VhGlcNAcase were then pooled and concentrated using Vivaspin-20 ultrafiltration membrane concentrators  and further applied onto a HiPrep 16/60 Sephacryl S-200 column connected to an ÄKTAprime system . VhGlcNAcase-containing fractions were pooled and concentrated, and the protein purity was confirmed by 12% SDS–PAGE. The final concentration of VhGlcNAcase was determined from the absorbance at 280 nm using a molar extinction coefficient of 118 720 M−1 cm−1  was equilibrated with 20 mM Tris–HCl buffer pH 8.0 containing 150 mM NaCl and was operated at a flow rate of 2.0 ml min−1. The gel-phase distribution coefficient (Kav) of the analyte between the stationary and mobile phases was calculated asVe is the elution volume, Vo is the void volume and Vi is the volume of the stationary phase  was added as a control for the retention volume of each protein and also to determine the total volume of the column. A plot of the Kav of individual standard proteins  mixed with Nɛ-DNP-l-lysine was applied onto the HiPrep 26/60 Sephacryl S-300 gel-filtration column as specified. Fractions of 5 ml were collected, with VhGlcNAcase being eluted in two peaks: the first eluted close to the void peak, while the second eluted near the BSA peak. SDS–PAGE analysis and GlcNAcase activity assays showed that the first peak was aggregated and inactive enzyme, while the second peak was the active enzyme. Therefore, the MW of the active VhGlcNAcase was determined from the calibration curve as 76 kDa .The molecular weight (MW) of wild-type 2.4.VhGlcNAcase, both in the apo form and in complex with N-acetylglucosamine (GlcNAc), was performed as described elsewhere  was mixed with 1.5 µl reservoir solution consisting of 0.1 M sodium acetate pH 4.6, 1.4 M sodium malonate, while the D437A mutant was mixed with 0.1 M bis-Tris pH 7.5, 0.1 M sodium acetate, 20%(w/v) PEG 3350. 3D plate-shaped crystals of the wild type grew at 20°C within three days to dimensions of up to 400 × 200 × 20 µm. These wild-type crystals were subsequently successfully soaked with 10 mM (GlcNAc)2 in the corresponding mother liquor at 20°C for a period of approximately 30 min, as described previously 2. The D437A mutant grew in a condition consisting of 0.1 M bis-Tris pH 7.5, 0.1 M sodium acetate, 20%(w/v) PEG 3350 and was transferred into a cryoprotectant solution consisting of mother liquor supplemented with 25%(v/v) glycerol. X-ray diffraction data were collected from all crystals at 100 K on the PX-II beamline at the Swiss Light Source in Villigen, Switzerland using a PILATUS 6M detector. The data-collection strategy was determined with iMosflm  as a search model. The final model of wild-type VhGlcNAcase was subsequently used as a template to obtain the phases for the data sets for the VhGlcNAcase–substrate complex and the D437A mutant. Model building was performed by iterative cycles consisting of manual building in Coot  and then modeled into the corresponding 2Fo − Fc and Fo − Fc maps. The crystallographic data and refinement statistics of the finalized model of the VhGlcNAcase structures are summarized in Table 2PROCHECK  was employed to obtain phase information using 2.6.N-acetyl-β-d-glucosaminide  as a substrate. The reaction of 0.1–5 µg protein samples with 125 µMpNP-GlcNAc in 100 mM sodium phosphate buffer pH 7.0 in a total volume of 200 µl was carried out in triplicate in a 96-well microtiter plate at 37°C for 10 min with constant agitation in a ThermoMixer Comfort . The reaction was terminated by the addition of 100 µl 3 M sodium carbonate. The amount of 4-nitrophenol (4-NP) released was monitored optically at a wavelength of 405 nm using a Benchmark Plus microplate spectrophotometer . A calibration curve of a 4-NP standard varying from 0 to 20 nmol was constructed, allowing determination of the molar quantity of 4-NP liberated by the enzymatic reaction. The specific hydrolytic activity of the enzyme was expressed as nanomoles of 4-NP produced in 1 min at 37°C.GlcNAcase activity was determined by a colorimetric assay using 4-nitrophenyl 2.7.VhGlcNAcase were carried out using a colorimetric assay in a microtiter plate reader, as described above, with substrate concentrations varying from 0 to 500 µM. Briefly, a 200 µl reaction mixture consisting of 0–500 µMpNP-GlcNAc in 100 mM sodium phosphate buffer pH 7.0 and 0.1–30 µg enzyme was incubated at 37°C with constant shaking for 10 min. The enzymatic reactions were then terminated by adding 100 µl 3 M sodium carbonate. The amount of reaction product was measured at 405 nm and converted to molar quantities using a calibration curve for 4-NP as described previously. The kinetic parameters  were determined from triplicate assays using the Michaelis–Menten function in GraphPad Prism version 0.6.0 .Kinetic studies of wild-type and mutant 3.3.1.VhGlcNAcase and its catalytic mutant D437A  were expressed at high levels in E. coli M15 (pREP4) cells as C-terminally His6-tagged polypeptides and were purified to homogeneity on a cobalt-affinity column  followed by gel-filtration chromatography, giving a final yield of approximately 15–20 mg purified enzyme per litre of bacterial culture. Single crystals of apo WT and apo D437A were successfully grown by hanging-drop vapor diffusion under the optimized conditions described in Section 2VhGlcNAcase soaked with (GlcNAc)2 diffracted to a resolution of 2.50 Å  of bond lengths and angles of all crystals were refined to between 0.007 and 0.009 and between 1.20 and 1.34, respectively. The average B factors refined to 55.34 Å2 for apo WT, 36.35 Å2 for WT GlcNAc and 64.96 Å2 for apo D437A. The coordinates and structure factors were deposited in the Protein Data Bank with PDB codes 6ezr for apo WT, 6ezs for the WT–GlcNAc complex and 6ezt for apo D437A.Wild-type Å Table 2. All crya) shows the domain arrangement of VhGlcNAcases based on the 3D structure obtained from this study. The overall structure contains three distinct domains: an N-terminal carbohydrate-binding (CBD) domain  connected by a long linker to an α+β domain  followed by a C-terminal catalytic (Cat) domain   is a topology diagram showing details of the secondary-structural elements of VhGlcNAcase. The CBD domain consists primarily of eight antiparallel strands (β1–β8) that form an immunoglobulin-like fold, while the α+β domain is composed of two helices mixed with six strands flanked by three additional short strands that are aligned out of the main plane of this domain. The central Cat domain has a typical (β/α)8 TIM-barrel-like fold, in which the helices and strands alternate in an antiparallel fashion. Note that in the TIM-barrel domain the fifth helix (α5), which joins strands β5 and β6 of the canonical TIM-barrel fold, and the seventh helix (α7), which joins strands β7 and β8, are missing and are replaced by three short helical (η) segments. Helix η1 connects strands β5 and β6, and the η2 and η3 segments connect strands β7 and β8.Fig. 1SpHex  shows the structural architecture of VhGlcNAcase, in which the central Cat domain is flanked by the CBD domain and the α+β domain. This CBD domain is structurally related to the family 2 carbohydrate-binding module (CBM2) in the CAZy database . The α+β domain is similar to the α+β domain of β-Hex from A. aurescens . The function of this domain is unknown, but it may help to solubilize and stabilize the catalytic domain .We also observe an additional long helix at the end of helix α8, which is also seen in VhGlcNAcase 12 cysteine residues are distributed over the three protein domains. The previously reported structure of β-hexosaminidase from S. plicatus  suggested one disulfide bond in the Cat domain between Cys263 and Cys282 , these residues are not conserved in SpHex and their side chains are too far apart (4.5 Å) to form a disulfide bridge.In 3.2.6ezr) and holo (PDB entry 6ezs) VhGlcNAcase (in complex with GlcNAc) contained two identical (r.m.s.d. of 0.14–0.16 Å over 639 Cα atoms) molecules per asymmetric unit . Each holoenzyme molecule contains one GlcNAc unit (Fo − Fc density shown as an orange mesh with the sugar shown as sticks) at subsite −1 of the catalytic pocket (the Cat domain of Mol A is in green and that of Mol B in gray). The entrance to the substrate-binding cleft of the Cat domain is covered by the CBD domain (magenta for Mol A versus gray for Mol B) of the neighboring molecule. Notably, the WT crystals were soaked with the substrate (GlcNAc)2 to obtain the ligand-bound structure, but we observed only a single GlcNAc in the active site of the enzyme. Given that (GlcNAc)2 is a stable sugar, it was presumed that (GlcNAc)2 was hydrolyzed during crystal soaking, leaving a GlcNAc product in the high-affinity site (site −1). Fig. 2b) presents a surface representation of the molecular packing of Mol A and Mol B of WT VhGlcNAcase in complex with GlcNAc, with the residues in the dimer interface highlighted in orange. Fig. 2c) shows Mol A and Mol B separately in the same orientation as in Fig. 2a). The interface region (orange) covers 4054.87 Å2 of the total protein surface area (66 973.06 Å2). 19 residues  from the CBD domain, 12 residues  from the Cat domain and six residues  from the linker are involved in the dimer interface; all are mostly hydrophilic, consistent with the finding that VhGlcNAcase is a monomer in solution, as shown below.The crystallographic analysis showed that the single crystals of apo . The retention volume of 181 ml for VhGlcNAcase in the chromatographic profile was converted to the distribution coefficient (Kav), which corresponds to a MW of 76 kDa on the calibration curve plotted for Kav and log10 MW . Compared with the expected MW of VhGlcNAcase (74 kDa), the MW obtained from gel filtration clearly confirms that VhGlcNAcase is a monomer in solution. Thus, the dimer observed in the crystals is likely to be a crystallization artifact induced by the particularly high protein concentration needed for crystal formation. We also carried out a PISA analysis 2.The complex of WT 1) at the anomeric C atom of the −1 GlcNAc ring makes a hydrogen bond to the amide side chain of Gln16 on the loop belonging to Mol B. The side chain of Gln16 of Mol B also makes a second hydrogen bond to the O7 atom of the C2 acetamido group of −1 GlcNAc . The −1 GlcNAc adopted a 4C1 chair conformation based on the Cremer–Pople parameter calculator , the reducing end (C1 OH) of −1 GlcNAc makes two hydrogen bonds to the side chain of Gln16 of Mol B and Tyr530, and the C2 acetamido group is immobilized by the side chain of Gln16 of Mol B and a water molecule. The C3 OH forms two hydrogen bonds to nearby water molecules and a hydrogen bond to the side chain of Arg274. The nonreducing end (C4 OH) is immobilized by the side chains of Arg274 and Glu584, respectively. The C6 OH forms two hydrogen bonds to the side chains of Asp532 and the N atom of the Trp546 side chain. The sugar–enzyme interactions shown in Fig. 4b) are within a distance of 3.5 Å.In the holo structure, the glycosidic O atom  shows a surface representation of the empty subsite −1 of apo WT VhGlcNAcase (PDB entry 6ezr) that is surrounded by aromatic residues. Four conserved aromatic residues, Trp487, Trp505, Tyr530, Trp546 and Trp582, essentially create the pocket wall. Superimposition of Mol A of the apo VhGlcNAcase structure onto Mol A of the structure in complex with GlcNAc gives an r.m.s.d. of 0.90 Å over 1278 residues (two chains) and an r.m.s.d. of 0.26 Å2 over 639 residues, i.e. there are no significant differences between the CBD domain and the α+β domain of the two enzyme forms. When compared with the catalytic pocket of the unliganded enzyme , a small movement of the loop regions surroundings subsite −1 of the enzyme can be observed. The dimensions of the GlcNAc-fitted catalytic cleft (8.2 × 17.0 Å2) are slightly narrower than the empty pocket (9.4 × 17.6 Å2) . Four key side chains, Gln398 (part of loop L3), Asp437 and Glu438 (part of loop L4), and Trp505 (part of loop L6), are positioned close to the center of the catalytic pocket and are at an optimal distance to interact with the −1 GlcNAc.The predicted catalytic pair Asp437–Glu438 is located near the Cme Fig. 4c, a sma2) Fig. 4d. Four 3.5.VhGlcNAcase in complex with GlcNAc is similar to those of other GH20 β-N-acetylhexosaminidases, including the WT β-hexosaminidase SpHex from S. plicatus  shows the domain structures of the four GH20 GlcNAcase orthologs. As mentioned earlier, VhGlcNAcase contains three distinct domains, an N-terminal CDB domain (magenta), an α+β domain (cyan) and a C-terminal Cat domain (green), while SpHex contains only two domains: an α+β domain (cyan) connected to a C-terminal Cat domain (brown). The N-terminal CBD domain is missing in SpHex. SmChb consists of four domains designated as an N-terminal CBD domain (magenta) followed by an α+β domain (cyan), a TIM-barrel Cat domain (lilac) and a C-terminal immunoglobulin (IgG)-like domain (orange). The structure of insect OfHex is similar to that of SpHex, and contains two domains: an α+β domain (cyan) connected to a C-terminal Cat domain (red). Note that the Cat domain of the four enzymes has a common (β/α)8-barrel fold and is conserved among all GH20 GlcNAcases. Fig. 5b) shows the electrostatic surface around the active sites of VhGlcNAcase complexed with GlcNAc (PDB entry 6ezs) and NAG-thiazoline , SmChb with (GlcNAc)2 (PDB entry 1qbb) and OfHex1 E328A in complex with TMG-chitotriomycin (PDB entry 3vtr). The sugar-binding pockets of these GH20 GlcNAcases are highly polar due to six conserved proton-donating groups that form a strongly negatively charged surface (red) around subsite −1 of the active site of each enzyme [see also Fig. 6a) for the positions of these residues in the corresponding amino-acid sequences]. Further comparison of the substrate-binding pockets and the positions of the bound ligands of the above-mentioned GH20 enzymes suggests that the active sites of VhGlcNAcase and OfHex have elongated pockets with an open end towards the positive subsites , allowing the accommodation of a chitooligosaccharide chain of 2–4 units. The active sites of SpHex and SmChb, on the other hand, are small and rather short open pockets that are suitable for accommodating only one or two GlcNAc units. The sugar ligand of at least one unit is found at subsite −1, which is located at the closed end of the tunnel/pocket. In all enzymes, cleavage occurs between subsites −1 and +1 -d-Man β-1,4-N-GlcNAc} to investigate the substrate specificity of the enzyme. As shown in Fig. 7a), the long loops of VhGlcNAcase (in green and black) denoted L2 , L3  and L7  significantly differ from those of StrH , which has corresponding loops L2′ (residues 706–712), L3′ (residues 754–774) and L7′ (residues 898–914), respectively. Loop L3 has the highest thermal motion, with a B factor of around 40.6 Å2, followed by L2 and L7, which have B factors of approximately 34.3 and 26.9 Å2, respectively.Exo-β-VhGlcNAcase (in black) protrude into the region where the NGAB2 substrate of StrH would be, which causes the substrate-binding region of VhGlcNAcase to be narrower. Fig. 7b) emphasizes a severe clash between the side chains of Trp546 and Ala547 of loop L7 in VhGlcNAcase with the modeled NGA2B, suggesting that the active site of VhGlcNAcase cannot possibly accommodate a branched oligosaccharide.The long loops L2, L3 and L7 of 4.VhGlcNAcase variants: two apo structures, of the wild type and the D437A mutant, and one holo structure, of the wild type in complex with (GlcNAc)2. After soaking the apo wild-type VhGlcNAcase crystals with (GlcNAc)2, we observed a clear Fo − Fc density map corresponding to only one GlcNAc molecule in the −1 subsite. This GlcNAc is most likely to be the product of hydrolysis of (GlcNAc)2 by active WT enzyme on the surface of the crystal during the soaking time of 30 min. Soaking or co-crystallizing the D437A mutant with (GlcNAc)2 was not successful, i.e. no substrate was seen in the active site, probably owing to the entrance of the active site of one monomer in the asymmetric unit (Mol A) being blocked by the side chain of Gln16 of the last strand of the CBD domain of the second monomer (Mol B). Since the active site of VhGlcNAcase is almost completely blocked by the neighboring monomer in the crystal structure, we assume that this would render such a dimeric enzyme extremely inefficient. This suggests that the active form of VhGlcNAcase is a monomer in solution, which could be confirmed by a gel filtration experiment. In addition, another crystal structure of VhGlcNAcase in complex with NAG-thiazoline (NGT) shows a different space group  provides some ideas about substrate specificity and subsite preference. Essentially, the catalytic pocket of VhGlcNAcase is narrow and elongated, fitting a linear chitooligosaccharide chain of 2–4 GlcNAc units. The structure of the active site supports our previous quantitative HPLC and kinetic modeling of the enzymic reaction . Based on this subsite topology, the cleavage site is located between subsites −1 and +1. In a recent study, we examined the inhibitory effect of NAG-thiazoline (NGT), a reaction-intermediate analog and a common inhibitor of GH20 GlcNAcases, and found that NGT strongly inhibited the hydrolytic activity of VhGlcNAcase, with a Ki of 62 ± 3 µM and a Kd of 32 ± 1.2 µM 2 during crystallization. In both the apo and holo structures we observed an obstruction of the entrance of the active site by Gln16 from a neighboring molecule in the crystal. As shown by the SEC experiments, this is a crystallization artifact that would not occur in the monomeric enzyme in solution. The holo structure of VhGlcNAcase with GlcNAc provides a snapshot of the sugar ring in the 4C1 conformation, which signifies the most favorable form of the sugar. The same sugar conformation was also observed in the active site of the β-hexosaminidase Hex1T from Paenibacillus sp. TS12  × 1.2 Å (in width), while other parts of the active site remain unaltered. This induced-fit movement of Glu438 enables the bond cleavage of chitooligosacharide bound between subsites −1 and +1, while the positioning of Asp437 may help the enzyme to stabilize the transition state by interaction with the acetamido group of the substrate through a substrate-assisted catalysis mechanism , and the dissimilarities in the shape and the surface-charge properties of the sugar-binding pocket from those of the other enzymes highlights some novel features of this enzyme. Its elongated and shallow catalytic pocket is suited to accommodating a linear chitooligosaccharide chain of 2–4 units 8 TIM-barrel architecture, some GH20 enzymes, such as StrH, HexA and HexB, recognize a broad range of sugar substrates, including branched β(1–4), β(1–3), β(1–2) and β(1–6) glycosidic-linked GlcNAc-containing glycans that are components of glycolipids, glycoproteins or sulfated glycoconjugates  from the marine bacterium V. campbellii. VhGlcNAcase contains three domains: a carbohydrate-binding domain (CBD), an α+β domain and a conserved (β/α)8 TIM-barrel catalytic domain. Size-exclusion chromatography confirmed that VhGlcNAcase is a monomeric enzyme in solution, with an apparent MW of 74 kDa. In the complex with product, one GlcNAc was found at subsite −1 of the highly negatively charged catalytic pocket. Binding of GlcNAc induces local conformational changes around the −1 subsite, where the sugar makes contacts with polar side-chain residues through a network of hydrogen bonds and stacks against the side chain of Trp582. Site-directed mutagenesis and kinetic analysis suggest that the acidic pair Asp437 and Glu438 play an important role in catalysis. Docking of VhGlcNAcase with a branched GlcNAc-derived glycan reveals severe steric clashes between the branched sugar and the active-site surface, consistent with the fact that VhGlcNAcase prefers chitin-derived chitooligosaccharides as natural substrates. The structural insight into VhGlcNAcase provides a further understanding of how Vibrio bacteria can thrive in marine ecosystems using chitin as their sole carbon source. From a biotechnological point of view, chitinases and GlcNAcases from Vibrio species are chitin-degrading enzymes that are naturally responsible for the recycling of chitin, and a one-pot reaction of these two enzymes could act as a powerful biocatalyst for the complete conversion of chitin biomass into small sugar products that can be used as starting materials for further chemical modifications or for the organic synthesis of highly compatible chitin-derived functional biomaterials.This study reports the crystal structures of a GH20 β-VhGlcNAcase, wild type, apo, 6ezrPDB reference: wild type, complex with GlcNAc, 6ezsPDB reference: D437A mutant, apo, 6eztPDB reference: