Domain Exchange Research Articles

Didepside Formation by the Nonreducing Polyketide Synthase Preu6 of Preussia isomera Requires Interaction of Starter Acyl Transferase and Thioesterase Domains

AbstractOrsellinic acid (OA) derivatives are produced by filamentous fungi using nonreducing polyketide synthases (nrPKSs). The chain‐releasing thioesterase (TE) domains of such nrPKSs were proposed to also catalyze dimerization to yield didepsides, such as lecanoric acid. Here, we use combinatorial domain exchanges, domain dissections and reconstitutions to reveal that the TE domain of the lecanoric acid synthase Preu6 of Preussia isomera must collaborate with the starter acyl transferase (SAT) domain from the same nrPKS. We show that artificial SAT‐TE fusion proteins are highly effective catalysts and reprogram the ketide homologation chassis to form didepsides. We also demonstrate that dissected SAT and TE domains of Preu6 physically interact, and SAT and TE domains of OA‐synthesizing nrPKSs may co‐evolve. Our work highlights an unexpected domain–domain interaction in nrPKSs that must be considered for the combinatorial biosynthesis of unnatural didepsides, depsidones, and diphenyl ethers.

Didepside Formation by the Nonreducing Polyketide Synthase Preu6 of Preussia isomera Requires Interaction of Starter Acyl Transferase and Thioesterase Domains.

Orsellinic acid (OA) derivatives are produced by filamentous fungi using nonreducing polyketide synthases (nrPKSs). The chain-releasing thioesterase (TE) domains of such nrPKSs were proposed to also catalyze dimerization to yield didepsides, such as lecanoric acid. Here, we use combinatorial domain exchanges, domain dissections and reconstitutions to reveal that the TE domain of the lecanoric acid synthase Preu6 of Preussia isomera must collaborate with the starter acyl transferase (SAT) domain from the same nrPKS. We show that artificial SAT-TE fusion proteins are highly effective catalysts and reprogram the ketide homologation chassis to form didepsides. We also demonstrate that dissected SAT and TE domains of Preu6 physically interact, and SAT and TE domains of OA-synthesizing nrPKSs may co-evolve. Our work highlights an unexpected domain-domain interaction in nrPKSs that must be considered for the combinatorial biosynthesis of unnatural didepsides, depsidones, and diphenyl ethers.

‘The Good, the Bad and the Ugly’ of terminology in Cross Domain Fusion

This article is a contribution to the Informatik Spektrum special issue „Cross-Domain Fusion“ – Heft 2. Terminologies are paramount to establish robust communication within interdisciplinary working groups inside and outside academia. To find the “common language” is hence essential and sometimes a long way to go. Within the idea of Cross Domain Fusion, we want to tackle this issue from the very beginning. Therefore, we set up a database based on the open source MediaWiki content management system. In this dictionary, a dedicated consortium from different disciplines evaluates terminologies used in Cross Domain Fusion and provides them within the Dialogue:Wiki. The aim is to provide accessible insight into commonalities and differences between different domain-specific terminologies to foster cross domain exchange.

Exclusive production of ρ meson in gamma-proton collisions: dσ/dt and the role of helicity flip processes

We calculate the differential cross section dσ/dt for the diffractive photoproduction process γp→ρp and compare to recent data extracted by the CMS collaboration from ultraperipheral proton-lead collisions. Our model is based on two-gluon exchange in the nonperturbative domain. We take into account both helicity conserving and often neglected helicity-flip amplitudes in the γ→V transition. The latter can contribute at finite t. The shape of the differential cross section as well as the role of helicity flip processes is strongly related to the dependence of the unintegrated gluon distribution on transverse momenta in the nonperturbative region.

Loss of function of the nuclear envelope protein LEMD2 causes DNA damage-dependent cardiomyopathy.

Mutations in nuclear envelope proteins (NEPs) cause devastating genetic diseases, known as envelopathies, that primarily affect the heart and skeletal muscle. A mutation in the NEP LEM domain-containing protein 2 (LEMD2) causes severe cardiomyopathy in humans. However, the roles of LEMD2 in the heart and the pathological mechanisms responsible for its association with cardiac disease are unknown. We generated knockin (KI) mice carrying the human c.T38>G Lemd2 mutation, which causes a missense amino acid exchange (p.L13>R) in the LEM domain of the protein. These mice represent a preclinical model that phenocopies the human disease, as they developed severe dilated cardiomyopathy and cardiac fibrosis leading to premature death. At the cellular level, KI/KI cardiomyocytes exhibited disorganization of the transcriptionally silent heterochromatin associated with the nuclear envelope. Moreover, mice with cardiac-specific deletion of Lemd2 also died shortly after birth due to heart abnormalities. Cardiomyocytes lacking Lemd2 displayed nuclear envelope deformations and extensive DNA damage and apoptosis linked to p53 activation. Importantly, cardiomyocyte-specific Lemd2 gene therapy via adeno-associated virus rescued cardiac function in KI/KI mice. Together, our results reveal the essentiality of LEMD2 for genome stability and cardiac function and unveil its mechanistic association with human disease.

Influence of NaCl and pH on lysostaphin catalytic activity, cell binding, and bacteriolytic activity.

Peptidoglycan-degrading enzymes are a group of proteins intensively studied as novel antibacterials, with some of them having reached pre-clinical and clinical stages of research. Many peptidoglycan-degrading enzymes have modular organization and consist of a catalytic and a cell wall binding domain. This property has been exploited in enzyme engineering efforts, and many new peptidoglycan-degrading enzymes were generated through domain exchange. However, rational combination of domains from different enzymes is still challenging since relative contribution of every domain to the cumulative bacteriolytic activity is not yet clearly understood. In this work, we investigated the influence of ionic strength and pH on the catalytic efficiency and cell binding of peptidoglycan-degrading enzyme lysostaphin and how this influence is reflected in the lysostaphin bacteriolytic activity. Contrary to generally accepted view, lysostaphin domains are not completely independent and their combination within one protein leads to increased bacteriolytic activity with increasing NaCl concentration, despite both catalysis and cell binding being inhibited by NaCl. This effect is likely mediated by changes in conformation of bacterial cell wall peptidoglycan rather than the physical inter-domain interaction. KEY POINTS: • NaCl enhances bacteriolytic activity of lysostaphin but not of its catalytic domain. • Catalytic activity and cell binding of lysostaphin are inhibited by NaCl. • Peptidoglycan conformation likely affects lysostaphin bacteriolytic activity.

Characterization of 2S albumin allergenic proteins for anaphylaxis in common buckwheat

2S albumin (2SA) is responsible for anaphylaxis following consumption of buckwheat in allergic individuals. To reduce allergen incidents, characterization of 2SA polypeptides is prerequisite, thus was analyzed in this study. Of the five 2S albumin genes (g03, g11, g13, g14, and g28), g03 was seemingly non-functional. The g14 content was 3- and 40-fold higher than that of g11/g28 and g13, respectively. The g11/g28 were more processed to a ∼8 kDa band from a 16 kDa band than g14 in seeds, agreeing with that g11/g28 have high similarity with Fag e 8kD. Meanwhile, anti-g13 produced only a single ∼10 kDa band. Modification of g13 and domain exchange between g13 and g14 suggested that the hydrophobicity of the first domain and the nature of some amino acids in g13 contributed, at least in part, to the lower apparent molecular weight of g13 than expected. Thus, g13 might be an unexplored and noteworthy allergen.

Prodrug-Activating Chain Exchange (PACE) converts targeted prodrug derivatives to functional bi- or multispecific antibodies.

Driven by the potential to broaden the target space of conventional monospecific antibodies, the field of multi-specific antibody derivatives is growing rapidly. The production and screening of these artificial proteins entails a high combinatorial complexity. Antibody-domain exchange was previously shown to be a versatile strategy to produce bispecific antibodies in a robust and efficient manner. Here, we show that the domain exchange reaction to generate hybrid antibodies also functions under physiological conditions. Accordingly, we modified the exchange partners for use in therapeutic applications, in which two inactive prodrugs convert into a product with additional functionalities. We exemplarily show the feasibility for generating active T cell bispecific antibodies from two inactive prodrugs, which per se do not activate T cells alone. The two complementary prodrugs harbor antigen-targeting Fabs and non-functional anti-CD3 Fvs fused to IgG-CH3 domains engineered to drive chain-exchange reactions between them. Importantly, Prodrug-Activating Chain Exchange (PACE) could be an attractive option to conditionally activate therapeutics at the target site. Several examples are provided that demonstrate the efficacy of PACE as a new principle of cancer immunotherapy in vitro and in a human xenograft model.

Structural insights into composition design of Li-rich layered cathode materials for high-energy rechargeable battery

The Li-rich layered oxide is considered as one of the most promising cathode materials for high energy density batteries, due to its ultrahigh capacity derived from oxygen redox. Although incorporating over-stoichiometric Li into layered structure can generate Li2MnO3-like domain and enhance the oxygen redox activity thermodynamically, the fast and complete activation of the Li2MnO3-like domain remains challenging. Herein, we performed a systematic study on structural characteristics of Li-rich cathode materials to decipher the factors accounting for activation of oxygen redox. We reveal that the activation of Li-rich cathode materials is susceptible to local Co coordination environments. The Co ions can intrude into Li2MnO3-like domain and modulate the electronic structure, thereby facilitating the activation of Li-rich layered cathode materials upon first charging, leading to higher reversible capacity. In contrast, Li2MnO3-like domain hardly contains any Ni ions which contribute little to the activation process. The optimum composition design of this class of materials is discussed and we demonstrate a small amount of Co/Mn exchange in Li2MnO3-like domain can significantly promote the oxygen redox activation. Our findings highlight the vital role of Co ions in the activation of oxygen redox Li-rich layered cathode materials and provide new insights into the pathway toward achieving high-capacity Li-rich layered cathode materials.

Origin and Isoform Specific Functions of Exchange Proteins Directly Activated by cAMP: A Phylogenetic Analysis.

Exchange proteins directly activated by cAMP (EPAC1 and EPAC2) are one of the several families of cellular effectors of the prototypical second messenger cAMP. To understand the origin and molecular evolution of EPAC proteins, we performed a comprehensive phylogenetic analysis of EPAC1 and EPAC2. Our study demonstrates that unlike its cousin PKA, EPAC proteins are only present in multicellular Metazoa. Within the EPAC family, EPAC1 is only associated with chordates, while EPAC2 spans the entire animal kingdom. Despite a much more contemporary origin, EPAC1 proteins show much more sequence diversity among species, suggesting that EPAC1 has undergone more selection and evolved faster than EPAC2. Phylogenetic analyses of the individual cAMP binding domain (CBD) and guanine nucleotide exchange (GEF) domain of EPACs, two most conserved regions between the two isoforms, further reveal that EPAC1 and EPAC2 are closely clustered together within both the larger cyclic nucleotide receptor and RAPGEF families. These results support the notion that EPAC1 and EPAC2 share a common ancestor resulting from a fusion between the CBD of PKA and the GEF from RAPGEF1. On the other hand, the two terminal extremities and the RAS-association (RA) domains show the most sequence diversity between the two isoforms. Sequence diversities within these regions contribute significantly to the isoform-specific functions of EPACs. Importantly, unique isoform-specific sequence motifs within the RA domain have been identified.

Conformational exchange of the Zα domain of human RNA editing enzyme ADAR1 studied by NMR spectroscopy

Z-DNA binding proteins (ZBPs) play important roles in RNA editing, innate immune responses, and viral infections. Numerous studies have implicated a role for conformational motions during ZBPs binding upon DNA, but the quantitative intrinsic conformational exchanges of ZBP have not been elucidated. To understand the correlation between the biological function and dynamic feature of the Zα domains of human ADAR1 (hZαADAR1), we have performed the 15N backbone amide Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments on the free hZαADAR1 at two different magnetic fields at 35 °C. The robust inter-dependence of parameters in the global fitting process using multi-magnetic field CPMG profiles allows us characterizing the dynamic properties of conformational changes in hZαADAR1. This study found that free hZαADAR1 exhibited the conformational exchange with a kex of 5784 s−1 between the states “A” (89% population) and “B” (11% population). The different hydrophobic interactions among helices α1, α2, and α3 between these two states might correlate with efficient Z-DNA binding achieved by the hydrogen bonding interactions between its side-chains and the phosphate backbone of Z-DNA.

EvoProDom: evolutionary modeling of protein families by assessing translocations of protein domains.

Here, we introduce a novel ‘evolution of protein domains’ (EvoProDom) model for describing the evolution of proteins based on the ‘mix and merge’ of protein domains. We assembled and integrated genomic and proteomic data comprising protein domain content and orthologous proteins from 109 organisms. In EvoProDom, we characterized evolutionary events, particularly, translocations, as reciprocal exchanges of protein domains between orthologous proteins in different organisms. We showed that protein domains that translocate with highly frequency are generated by transcripts enriched in trans‐splicing events, that is, the generation of novel transcripts from the fusion of two distinct genes. In EvoProDom, we describe a general method to collate orthologous protein annotation from KEGG, and protein domain content from protein sequences using tools such as KoFamKOAL and Pfam. To summarize, EvoProDom presents a novel model for protein evolution based on the ‘mix and merge’ of protein domains rather than DNA‐based evolution models. This confers the advantage of considering chromosomal alterations as drivers of protein evolutionary events.

Modeling processive motion of kinesin‐13 MCAK and kinesin‐14 Cik1‐Kar3 molecular motors

Kinesin-13 MCAK, which is composed of two identical motor domains, can undergo unbiased one-dimensional diffusion on microtubules. Kinesin-14 Cik1-Kar3, which is composed of a Kar3 motor domain and a Cik1 motor homology domain with no ATPase activity, can move processively toward the minus end of microtubules. Here, we present a model for the diffusion of MCAK homodimer and a model for the processive motion of Cik1-Kar3 heterodimer. Although the two dimeric motors show different domain composition, in the models it is proposed that the two motors use the similar physical mechanism to move processively. With the models, the dynamics of the two dimers is studied analytically. The theoretical results for MCAK reproduce quantitatively the available experimental data about diffusion constant and lifetime of the motor bound to microtubule in different nucleotide states. The theoretical results for Cik1-Kar3 reproduce quantitatively the available experimental data about load dependence of velocity and explain consistently the available experimental data about effects of the exchange and mutation of the motor homology domain on the velocity of the heterodimer. Moreover, predicted results for other aspects of the dynamics of the two dimers are provided.

The Use of Metamodel-based Approach for Designing Healthcare Applications

Recently, the use of Model-Driven Engineering (MDE) via metamodeling approach is gaining more attention for software applications development. The community from the healthcare domain also attempts to employ the metamodel approach for producing quality healthcare applications. Healthcare applications have become an imperative in every attempt to improve healthcare management. Numerous studies reported that the healthcare domain is seen as a complex and unique domain, which involves dynamic characteristics. In addition, it is widely recognized that the increase of information exchange in the healthcare domain is caused by the diversity of healthcare data. This has led to the increase use of information technologies in the healthcare industry so as to enhance the healthcare delivery process via healthcare applications. However, the complexity of healthcare information leads to ineffective models and design of healthcare applications. Modeling the healthcare processes and developing healthcare applications are challenging tasks. Hence, the advances of MDE have influenced the use of the metamodeling technique in the development of healthcare applications. Various metamodels are developed as a solution to provide a clear healthcare process model and a correct healthcare application. The aim of this paper is to analyse the use of the metamodel-based approach in designing healthcare applications. We believe that the metamodel-based approach would improve the development of healthcare applications.

Arabidopsis thaliana G3BP Ortholog Rescues Mammalian Stress Granule Phenotype across Kingdoms.

Stress granules (SGs) are dynamic RNA–protein complexes localized in the cytoplasm that rapidly form under stress conditions and disperse when normal conditions are restored. The formation of SGs depends on the Ras-GAP SH3 domain-binding protein (G3BP). Formations, interactions and functions of plant and human SGs are strikingly similar, suggesting a conserved mechanism. However, functional analyses of plant G3BPs are missing. Thus, members of the Arabidopsis thaliana G3BP (AtG3BP) protein family were investigated in a complementation assay in a human G3BP knock-out cell line. It was shown that two out of seven AtG3BPs were able to complement the function of their human homolog. GFP-AtG3BP fusion proteins co-localized with human SG marker proteins Caprin-1 and eIF4G1 and restored SG formation in G3BP double KO cells. Interaction between AtG3BP-1 and -7 and known human G3BP interaction partners such as Caprin-1 and USP10 was also demonstrated by co-immunoprecipitation. In addition, an RG/RGG domain exchange from Arabidopsis G3BP into the human G3BP background showed the ability for complementation. In summary, our results support a conserved mechanism of SG function over the kingdoms, which will help to further elucidate the biological function of the Arabidopsis G3BP protein family.

Testing the ‘two water worlds’ hypothesis under variable preferential flow conditions

AbstractWidespread observations of ecohydrological separation are interpreted by suggesting that water flowing through highly conductive soil pores resists mixing with matrix storage over periods of days to months (i.e., two ‘water worlds’ exist). These interpretations imply that heterogeneous flow can produce ecohydrological separation in soils, yet little mechanistic evidence exists to explain this phenomenon. We quantified the separation between mobile water moving through preferential flow paths versus less mobile water remaining in the soil matrix after free‐drainage to identify the amount of preferential flow necessary to maintain a two water world's scenario. Soil columns of varying macropore structure were subjected to simulated rainfall of increasing rainfall intensity (26 mm h−1, 60 mm h−1, and 110 mm h−1) whose stable isotope signatures oscillated around known baseline values. Prior to rainfall, soil matrix water δ2H nearly matched the known value used to initially wet the pore space whereas soil δ18O deviated from this value by up to 3.4‰, suggesting that soils may strongly fractionate 18O. All treatments had up to 100% mixing between rain and matrix water under the lowest (26 mm h−1) and medium (60 mm h−1) rainfall intensities. The highest rainfall intensity (110 mm h−1), however, reduced mixing of rain and matrix water for all treatments and produced significantly different preferential flow estimates between columns with intact soil structure compared to columns with reduced soil structure. Further, artificially limiting exchange between preferential flow paths and matrix water reduced bypass flow under the most intense rainfall. We show that (1) precipitation offset metrics such as lc‐excess and d‐excess may yield questionable interpretations when used to identify ecohydrological separation, (2) distinct domain separation may require extreme rainfall intensities and (3) domain exchange is an important component of macropore flow.

The domain exchange between 3‐domain Cry toxins contributes to their diversity and specificity

Spore-forming bacteria Bacillus thuringiensis (Bt) can infect a wide range of invertebrate hosts efficiently and specifically; therefore, it is extensively implemented in ecological biopesticide production. This phenomenon is primarily explained by the presence of highly diversified crystalliferous Cry-toxins embedded in spores; however, the exact underlying causes of these toxins' divergence remains speculative. Nonetheless, understanding these natural mechanisms could provide insights into constructing artificial insecticidal moieties, which is highly demanded due to the sporadic occurrence of resistance in pests. Three-domain Cry toxins (3-D) represent the most abundant class of Cry toxins sharing similar structural features yet are immensely diverse in sequence identity. The latter leads to the absence of thorough phylogenetic studies involving these toxins on a large-scale, and we have attempted to fill this gap. We started with gathering Cry toxin sequences from all publicly available data sources, including Bt- genome assemblies, the Genbank database, and the Bt-nomenclature. After searching for 3-D toxins followed by deduplication, we obtained 370 toxins, for which phylogenetic trees were reconstructed based on individual domains and full-length toxins. A striking dissimilarity between their topologies (which was most evident for the third domain) has allowed us to infer that the domains' evolution is somehow independent and also supported the hypothesis that the emergence of the new toxins is orchestrated by shifts of the third domain between sequences. We next ran the RDP (Recombination Detection Protocol) program, searching for recombination events. As a result, we observed more than 70 events. In other words, almost every fourth toxin is formed after the onset of the recombination event. Two-third of the domain exchanges related to the third domain; nevertheless, exchanges affected all three domains. That observation substantially extends the former hypothesis. Finally, we assessed detected events for functional significance. Not only were the events characterized by a remarkable effect on the evolutionary selection (with significant signals of positive selection), but recombination led to alterations in host specificity for several events. All things considered, we have drawn a conclusion that the domain exchange is one of the primary mechanisms underlying the emergence and specificity of Cry toxins. This observation has a potential practical implementation in using in silico experiments to construct novel artificial biopesticides.

Experimental exchange of paralogous domains in the MLH family provides evidence of sub-functionalization after gene duplication.

Baker’s yeast contains a large number of duplicated genes; some function redundantly, whereas others have more specialized roles. We used the MLH family of DNA mismatch repair (MMR) proteins as a model to better understand the steps that lead to gene specialization following a gene duplication event. We focused on two highly conserved yeast MLH proteins, Pms1 and Mlh3, with Pms1 having a major role in the repair of misincorporation events during DNA replication and Mlh3 acting to resolve recombination intermediates in meiosis to form crossovers. The baker’s yeast Mlh3 and Pms1 proteins are significantly diverged (19% overall identity), suggesting that an extensive number of evolutionary steps, some major, others involving subtle refinements, took place to diversify the MLH proteins. Using phylogenetic and molecular approaches, we provide evidence that all three domains (N-terminal ATP binding, linker, C-terminal endonuclease/MLH interaction) in the MLH protein family are critical for conferring pathway specificity. Importantly, mlh3 alleles in the ATP binding and endonuclease domains improved MMR functions in strains lacking the Pms1 protein and did not disrupt Mlh3 meiotic functions. This ability for mlh3 alleles to complement the loss of Pms1 suggests that an ancestral Pms1/Mlh3 protein was capable of performing both MMR and crossover functions. Our strategy for analyzing MLH pathway specificity provides an approach to understand how paralogs have evolved to support distinct cellular processes.

Vortex Domain Wall Structures and Exchange Coupled Magnetization Switching in Bit Patterned Media

After twenty years study of the exchange spring effect, this effect has shown strong potential in application on the perpendicular record process. It satisfies all the necessary properties required by the perpendicular record process. The exchange coupled patterned media also provide a new way to further increase the data storage in the future. Although studies involve many aspects of the exchange spring effect are taken by experiments and theoretical simulations, a systematic study on a magnetic island perpendicular switching for patterned media is not present. We studied the in-plane and out-of-plane switching process with domain wall movement coupling with the finite boundary condition. Many details like magnetic domain wall, coercive field, remanent magnetization and hysteresis loops are discussed to enhance the magnetic device design. An in-plane vortex domain wall was reported in the simulation and the magnetic loops can be artificially designed.

Rescue of Infectious Rotavirus Reassortants by a Reverse Genetics System Is Restricted by the Receptor-Binding Region of VP4.

The rotavirus species A (RVA) capsid contains the spike protein VP4, which interacts with VP6 and VP7 and is involved in cellular receptor binding. The capsid encloses the genome consisting of eleven dsRNA segments. Reassortment events can result in novel strains with changed properties. Using a plasmid-based reverse genetics system based on simian RVA strain SA11, we previously showed that the rescue of viable reassortants containing a heterologous VP4-encoding genome segment was strain-dependent. In order to unravel the reasons for the reassortment restrictions, we designed here a series of plasmids encoding chimeric VP4s. Exchange of the VP4 domains interacting with VP6 and VP7 was not sufficient for rescue of viable viruses. In contrast, the exchange of fragments encoding the receptor-binding region of VP4 resulted in virus rescue. All parent strains and the rescued reassortants replicated efficiently in MA-104 cells used for virus propagation. In contrast, replication in BSR T7/5 cells used for plasmid transfection was only efficient for the SA11 strain, whereas the rescued reassortants replicated slowly, and the parent strains failing to produce reassortants did not replicate. While future research in this area is necessary, replication in BSR T7/5 cells may be one factor that affects the rescue of RVAs.