Domain Architecture Research Articles

Molecular Characterization of Germin-like Protein Genes in Zea mays (ZmGLPs) Using Various In Silico Approaches.

Germin (GER) and germin-like proteins (GLPs) play an important role in various plant processes. Zea mays contains 26 germin-like protein genes (ZmGLPs) located on chromosomes 2, 4, and 10; most of which are functionally unexplored. The present study aimed to characterize all ZmGLPs using the latest computational tools. All of them were studied at a physicochemical, subcellular, structural, and functional level, and their expression was predicted in plant development, against biotic and abiotic stresses using various in silico approaches. Overall, ZmGLPs showed greater similarity in their physicochemical properties, domain architecture, and structure, mostly localized in the cytoplasmic or extracellular regions. Phylogenetically, they have a narrow genetic background with a recent history of gene duplication events on chromosome 4. Functional analysis revealed novel enzymatic activities of phosphoglycolate phosphatase, adenosylhomocysteinase, phosphoglycolate phosphatase-like, osmotin/thaumatin-like, and acetohydroxy acid isomeroreductase largely mediated by disulfide bonding. Expression analysis revealed their crucial role in the root, root tips, crown root, elongation and maturation zones, radicle, and cortex with the highest expression being observed during germination and at the maturity levels. Further, ZmGLPs showed strong expression against biotic (Aspergillus flavus, Colletotrichum graminicola, Cercospora zeina, Fusarium verticillioides, and Fusarium virguliforme) while limited expression was noted against abiotic stresses. Concisely, our results provide a platform for additional functional exploration of the ZmGLP genes against various environmental stresses.

Comparative analysis of three families of hygrophilid snails shows that the egg mass fluid protein haemocyanin-like 1 (Hcl-1) is unique to planorbids

ABSTRACT The egg mass fluid (EMF) of the freshwater snail Biomphalaria glabrata (Hygrophila: Planorbidae) contains haemocyanin-like 1 (Hcl-1) protein, distinct from respiratory haemocyanins. The distribution of Hcl-1 was investigated among major families of Hygrophila, Physidae and Lymnaeidae, both of which employ respiratory haemocyanins, and Planorbidae, a group that evolved haemoglobin as a respiratory pigment. Immunoblotting detected c. 150 kDa protein (molecular weight of Hcl-1) cross-reactive with anti-keyhole limpet haemocyanin antiserum in the EMF of planorbids Bulinus globosus and Planorbella duryi (from a genus closely related to Biomphalaria), but not Physella acuta (Physidae) and Ladislavella elodes (Lymnaeidae). High throughput sequence data revealed Hcl-1 homologs from Bulinus globosus and Planorbella duryi, representative species that span the range of planorbid phylogeny, but not from Physella acuta (Physidae) and Lymnaea stagnalis (Lymnaeidae). A domain architecture comprising only three functional units (FUs) and predicted secondary structures within the C-terminal FU distinguish planorbid Hcl-1 protein from molluscan respiratory haemocyanins that are natively assembled as functional didecamers. Immunoblotting confirmed a monomeric configuration of native Hcl-1. Molecular clock analysis estimated divergence of Hcl-1 proteins from gastropod respiratory haemocyanins at 267 ± 143 Ma. It is hypothesized that Hcl proteins originated in the ancestor of the planorbid lineage when evolution of respiratory haemoglobin altered selective pressures for maintaining original function, facilitating mutation and refunctionalization of the ancestral respiratory haemocyanin in Planorbidae.

Real or fake? Measuring the impact of protein annotation errors on estimates of domain gain and loss events.

Protein annotation errors can have significant consequences in a wide range of fields, ranging from protein structure and function prediction to biomedical research, drug discovery, and biotechnology. By comparing the domains of different proteins, scientists can identify common domains, classify proteins based on their domain architecture, and highlight proteins that have evolved differently in one or more species or clades. However, genome-wide identification of different protein domain architectures involves a complex error-prone pipeline that includes genome sequencing, prediction of gene exon/intron structures, and inference of protein sequences and domain annotations. Here we developed an automated fact-checking approach to distinguish true domain loss/gain events from false events caused by errors that occur during the annotation process. Using genome-wide ortholog sets and taking advantage of the high-quality human and Saccharomyces cerevisiae genome annotations, we analyzed the domain gain and loss events in the predicted proteomes of 9 non-human primates (NHP) and 20 non-S. cerevisiae fungi (NSF) as annotated in the Uniprot and Interpro databases. Our approach allowed us to quantify the impact of errors on estimates of protein domain gains and losses, and we show that domain losses are over-estimated ten-fold and three-fold in the NHP and NSF proteins respectively. This is in line with previous studies of gene-level losses, where issues with genome sequencing or gene annotation led to genes being falsely inferred as absent. In addition, we show that insistent protein domain annotations are a major factor contributing to the false events. For the first time, to our knowledge, we show that domain gains are also over-estimated by three-fold and two-fold respectively in NHP and NSF proteins. Based on our more accurate estimates, we infer that true domain losses and gains in NHP with respect to humans are observed at similar rates, while domain gains in the more divergent NSF are observed twice as frequently as domain losses with respect to S. cerevisiae. This study highlights the need to critically examine the scientific validity of protein annotations, and represents a significant step toward scalable computational fact-checking methods that may 1day mitigate the propagation of wrong information in protein databases.

A Genomic and Transcriptomic Analysis of the C-Type Lectin Gene Family Reveals Highly Expanded and Diversified Repertoires in Bivalves

C-type lectins belong to a widely conserved family of lectins characterized in Metazoa. They show important functional diversity and immune implications, mainly as pathogen recognition receptors. In this work, C-type lectin-like proteins (CTLs) of a set of metazoan species were analyzed, revealing an important expansion in bivalve mollusks, which contrasted with the reduced repertoires of other mollusks, such as cephalopods. Orthology relationships demonstrated that these expanded repertoires consisted of CTL subfamilies conserved within Mollusca or Bivalvia and of lineage-specific subfamilies with orthology only between closely related species. Transcriptomic analyses revealed the importance of the bivalve subfamilies in mucosal immunity, as they were mainly expressed in the digestive gland and gills and modulated with specific stimuli. CTL domain-containing proteins that had additional domains (CTLDcps) were also studied, revealing interesting gene families with different conservation degrees of the CTL domain across orthologs from different taxa. Unique bivalve CTLDcps with specific domain architectures were revealed, corresponding to uncharacterized bivalve proteins with putative immune function according to their transcriptomic modulation, which could constitute interesting targets for functional characterization.

Crystal structure of a highly conserved enteroviral 5′ cloverleaf RNA replication element

The extreme 5′-end of the enterovirus RNA genome contains a conserved cloverleaf-like domain that recruits 3CD and PCBP proteins required for initiating genome replication. Here, we report the crystal structure at 1.9 Å resolution of this domain from the CVB3 genome in complex with an antibody chaperone. The RNA folds into an antiparallel H-type four-way junction comprising four subdomains with co-axially stacked sA-sD and sB-sC helices. Long-range interactions between a conserved A40 in the sC-loop and Py-Py helix within the sD subdomain organize near-parallel orientations of the sA-sB and sC-sD helices. Our NMR studies confirm that these long-range interactions occur in solution and without the chaperone. The phylogenetic analyses indicate that our crystal structure represents a conserved architecture of enteroviral cloverleaf-like domains, including the A40 and Py-Py interactions. The protein binding studies further suggest that the H-shape architecture provides a ready-made platform to recruit 3CD and PCBP2 for viral replication.

All DACs in a Row: Domain Architectures of Bacterial and Archaeal Diadenylate Cyclases.

Cyclic dimeric AMP (c-di-AMP) is a widespread second messenger that controls such key functions as osmotic homeostasis, peptidoglycan biosynthesis, and response to various stresses. C-di-AMP is synthesized by diadenylate cyclases that contain the DAC (DisA_N) domain, which was originally characterized as the N-terminal domain in the DNA integrity scanning protein DisA. In other experimentally studied diadenylate cyclases, DAC domain is typically located at the protein C termini and its enzymatic activity is controlled by one or more N-terminal domains. As in other bacterial signal transduction proteins, these N-terminal modules appear to sense environmental or intracellular signals through ligand binding and/or protein-protein interactions. Studies of bacterial and archaeal diadenylate cyclases also revealed numerous sequences with uncharacterized N-terminal regions. This work provides a comprehensive review of the N-terminal domains of bacterial and archaeal diadenylate cyclases, including the description of five previously undefined domains and three PK_C-related domains of the DacZ_N superfamily. These data are used to classify diadenylate cyclases into 22 families, based on their conserved domain architectures and the phylogeny of their DAC domains. Although the nature of the regulatory signals remains obscure, the association of certain dac genes with anti-phage defense CBASS systems and other phage-resistance genes suggests that c-di-AMP might also be involved in the signaling of phage infection.

Abstract 878: In silico insights from NEK10 modeling, its interactome, and protein-protein interactions

Abstract Cancer is the second-leading cause of death worldwide and by 2040, it is projected that the global burden is expected to grow to 27.5 million new cases. Proteins that directly regulate cell cycle progression are attractive targets in cancer therapy since aberrant regulation of the cell cycle, especially at checkpoints, allows the cells to accumulate DNA errors and continue to grow/divide. A subset of protein kinases functionally involved in regulating the cell cycle and its checkpoints are the eleven members of the human NIMA-related kinases (NEKs). NEK10 is the most divergent member of the NEK family and is linked to melanoma, breast cancer, and various ciliopathies. It is unique in having a centrally positioned coiled-coil flanked catalytic domain, in contrast to the N-terminal catalytic domains of other NEKs. Despite its unique domain architecture and biological relevance, very little is known about its complete structural information and structure-function relationships. To address this gap in knowledge we have modeled and characterized the full-length NEK10 protein, including its unique catalytic domain flanked by two coiled-coil domains, a region with armadillo-type repeats, an ATP binding site, and two unreported putative sites of interaction with ubiquitin (UBA domains). Additionally, we present a comprehensive interactome of NEK10. From this interactome we selected 2 unique and previously unreported NEK10 interactions with the known cancer-causing proteins, Mitogen-activated protein kinase kinase kinase 1 (MAP3K1) and Heat Shock Factor Binding Protein 1 (HSPB1). MAP3K1, a serine/threonine kinase and ubiquitin ligase that plays a pivotal role in integrating cellular responses to mitogenic/metabolic stimuli and mutated in a significant number of cancers, most prominently in luminal breast cancer, contains a protein kinase domain, a PHD finger with E3 ubiquitin ligase activity, and scaffold regions that mediate protein-protein interactions. Our docking analysis shows that I693 of NEK10 located within its kinase domain interacts with well-known phosphosite S275 of MAP3K1. We speculate a scenario in which, upon UV irradiation, NEK10 phosphorylates and activates MAP3K1, which in turn phosphorylates MAP2K1/2-ERK1/2 promoting cell survival. HSPB1, is a molecular chaperone, characterized by a common alpha-crystallin domain in its C-terminus and its overexpression is linked with poor clinical outcome in gastric, uterine, breast, prostate, ovarian, kidney and head/neck cancers. Our docking analysis suggests that the distinctive armadillo repeats of NEK10 may be responsible for its interaction with HSPB1 where H301 of NEK10 forms a salt bridge with I179 of HSPB1, within a known protein-protein hotspot of HSPB1. Our results establish the framework for elucidating the detailed molecular mechanisms of NEK10 interactions with other proteins to further explore its potential as a therapeutic target. Citation Format: Andriele Silva Eichner, Igor Semchenkov Pastukhov, Shaneen Singh. In silico insights from NEK10 modeling, its interactome, and protein-protein interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 878.

Rhodanese-Fold Containing Proteins in Humans: Not Just Key Players in Sulfur Trafficking.

The Rhodanese-fold is a ubiquitous structural domain present in various protein subfamilies associated with different physiological functions or pathophysiological conditions in humans. Proteins harboring a Rhodanese domain are diverse in terms of domain architecture, with some representatives exhibiting one or several Rhodanese domains, fused or not to other structural domains. The most famous Rhodanese domains are catalytically active, thanks to an active-site loop containing an essential cysteine residue which allows for catalyzing sulfur transfer reactions involved in sulfur trafficking, hydrogen sulfide metabolism, biosynthesis of molybdenum cofactor, thio-modification of tRNAs or protein urmylation. In addition, they also catalyse phosphatase reactions linked to cell cycle regulation, and recent advances proposed a new role into tRNA hydroxylation, illustrating the catalytic versatility of Rhodanese domain. To date, no exhaustive analysis of Rhodanese containing protein equipment from humans is available. In this review, we focus on structural and biochemical properties of human-active Rhodanese-containing proteins, in order to provide a picture of their established or putative key roles in many essential biological functions.

Block copolymer interfaces investigated by means of NMR, atomic force microscopy and dielectric spectroscopy

Self-assembly of block copolymers (BC) is a key feature for bottom-up patterning that is applied in the fabrication of templates for nanostructured membranes, photonic nanodevices, or solar cells. The wide range of possible BC applications is because the dissimilar blocks can be appropriately selected to form distinct domains with controllable dimensions and functionalities. An important representative of these materials is polystyrene-b-poly(ethylene oxide) (PS-b-PEO) diblock copolymer. This copolymer is of considerable academic interest and therefore it is examined by a plethora of experimental techniques to elucidate its structure and molecular dynamics. Particularly worth investigating is the interphase, consisting of polymer chains located at the interface of crystallite-amorphous and inter-domain regions, which codetermines the properties of such a system. It is shown that below the melting point of the PEO, the majority of the amorphous PEO are occupying interfacial sites and are represented by the rigid amorphous fraction (RAF) which can be monitored by dielectric and nuclear magnetic resonance spectroscopies. Moreover, two independent NMR spin-diffusion experiments reveal the asymmetrical nature of PS-PEO interface indicating a significant fraction of the PEO phase under the influence of stiff, adjacent polystyrene. An analysis of complementary thermal calorimetry and X-ray scattering data confirms the presence of microphase-separated morphology as well as the semicrystalline nature of PEO component. Lamelar domain architecture was confirmed by atomic force microscopy data acquired in the case of thin copolymer film.

Broad-host-range lytic Erwinia phage Key with exopolysaccharide degrading activity

In this study, the genome of the lytic broad-host-range phage Key infecting Erwinia amylovora, Erwinia horticola, and Pantoea agglomerans strains was characterized. Key phage has a 115,651 bp long double-stranded DNA genome with the G + C ratio of 39.03%, encoding 182 proteins and 27 tRNA genes. The majority (69%) of predicted coding sequences (CDSs) encode proteins with unknown functions. The protein products of 57 annotated genes were found to have probable functions in nucleotide metabolism, DNA replication, recombination, repair, and packaging, virion morphogenesis, phage-host interaction and lysis. Furthermore, the product of gene 141 shared amino acid sequence similarity and conserved domain architecture with the exopolysaccharide (EPS) degrading proteins of Erwinia and Pantoea infecting phages as well as bacterial EPS biosynthesis proteins. Due to the genome synteny and similarity to the proteins of T5-related phages, phage Key, together with its closest relative, Pantoea phage AAS21, was suggested to represent a novel genus within the Demerecviridae family, for which we tentatively propose the name “Keyvirus”.

A systematic survey of LU domain-containing proteins reveals a novel human gene, LY6A, which encodes the candidate ortholog of mouse Ly-6A/Sca-1 and is aberrantly expressed in pituitary tumors.

The Ly-6 and uPAR (LU) domain-containing proteins represent a large family of cell-surface markers. In particular, mouse Ly-6A/Sca-1 is a widely used marker for various stem cells; however, its human ortholog is missing. In this study, based on a systematic survey and comparative genomic study of mouse and human LU domain-containing proteins, we identified a previously unannotated human gene encoding the candidate ortholog of mouse Ly-6A/Sca-1. This gene, hereby named LY6A, reversely overlaps with a lncRNA gene in the majority of exonic sequences. We found that LY6A is aberrantly expressed in pituitary tumors, but not in normal pituitary tissues, and may contribute to tumorigenesis. Similar to mouse Ly-6A/Sca-1, human LY6A is also upregulated by interferon, suggesting a conserved transcriptional regulatory mechanism between humans and mice. We cloned the full-length LY6A cDNA, whose encoded protein sequence, domain architecture, and exon-intron structures are all well conserved with mouse Ly-6A/Sca-1. Ectopic expression of the LY6A protein in cells demonstrates that it acts the same as mouse Ly-6A/Sca-1 in their processing and glycosylphosphatidylinositol anchoring to the cell membrane. Collectively, these studies unveil a novel human gene encoding a candidate biomarker and provide an interesting model gene for studying gene regulatory and evolutionary mechanisms.

Domain Architecture of the Nonreceptor Tyrosine Kinase Ack1.

The nonreceptor tyrosine kinase (NRTK) Ack1 comprises a distinct arrangement of non-catalytic modules. Its SH3 domain has a C-terminal to the kinase domain (SH1), in contrast to the typical SH3-SH2-SH1 layout in NRTKs. The Ack1 is the only protein that shares a region of high homology to the tumor suppressor protein Mig6, a modulator of EGFR. The vertebrate Acks make up the only tyrosine kinase (TK) family known to carry a UBA domain. The GTPase binding and SAM domains are also uncommon in the NRTKs. In addition to being a downstream effector of receptor tyrosine kinases (RTKs) and integrins, Ack1 can act as an epigenetic regulator, modulate the degradation of the epidermal growth factor receptor (EGFR), confer drug resistance, and mediate the progression of hormone-sensitive tumors. In this review, we discuss the domain architecture of Ack1 in relation to other protein kinases that possess such defined regulatory domains.

Identification, characterization and spatiotemporal expression analysis of the FKBP family genes in Locusta migratoria

FK506 binding proteins (FKBPs) are a highly-conserved group of proteins known to bind to FK506, an immunosuppressive drug. They play different physiological roles, including transcription regulation, protein folding, signal transduction and immunosuppression. A number of FKBP genes have been identified in eukaryotes; however, very little information about these genes has been reported in Locusta migratoria. Here, we identified and characterized 10 FKBP genes from L. migratoria. Phylogenetic analysis and comparison of domain architectures indicated that the LmFKBP family can be divided into two subfamilies and five subclasses. Developmental and tissue expression pattern analysis revealed that all LmFKBPs transcripts, including LmFKBP46, LmFKBP12, LmFKBP47, LmFKBP79, LmFKBP16, LmFKBP24, LmFKBP44b, LmFKBP53, were periodically expressed during different developmental stages and mainly expressed in the fat body, hemolymph, testis, and ovary. In brief, our work depicts a outline but panoramic picture of LmFKBP family in L. migratoria, and provides a solid foundation to further investigate the molecular functions of LmFKBPs.

Genomic Analysis, Evolution and Characterization of E3 Ubiquitin Protein Ligase (TRIM) Gene Family in Common Carp (Cyprinus carpio)

Tripartite motifs (TRIM) is a large family of E3 ubiquitin ligases that play an important role in ubiquitylation. TRIM proteins regulate a wide range of biological processes from cellular response to viral infection and are implicated in various pathologies, from Mendelian disease to cancer. Although the TRIM family has been identified and characterized in tetrapods, but the knowledge about common carp and other teleost species is limited. The genes and proteins in the TRIM family of common carp were analyzed for evolutionary relationships, characterization, and functional annotation. Phylogenetic analysis was used to elucidate the evolutionary relationship of TRIM protein among teleost and higher vertebrate species. The results show that the TRIM orthologs of highly distant vertebrates have conserved sequences and domain architectures. The pairwise distance was calculated among teleost species of TRIMs, and the result exhibits very few mismatches at aligned position thus, indicating that the members are not distant from each other. Furthermore, TRIM family of common carp clustered into six groups on the basis of phylogenetic analysis. Additionally, the analysis revealed conserved motifs and functional domains in the subfamily members. The difference in functional domains and motifs is attributed to the evolution of these groups from different ancestors, thus validating the accuracy of clusters in the phylogenetic tree. However, the intron-exon organization is not precisely similar, which suggests duplication of genes and complex alternative splicing. The percentage of secondary structural elements is comparable for members of the same group, but the tertiary conformation is varied and dominated by coiled-coil segments required for catalytic activity. Gene ontology analysis revealed that these proteins are mainly associated with the catalytic activity of ubiquitination, immune system, zinc ion binding, positive regulation of transcription, ligase activity, and cell cycle regulation. Moreover, the biological pathway analyses identified four KEGG and 22 Reactome pathways. The predicted pathways correspond to functional domains, and gene ontology which proposes that proteins with similar structures might perform the same functions.

Nitrate reductase enzymes in alga Chattonella subsalsa are regulated by environmental cues at the translational and post-translational levels

Nitrate reductase (NR) catalyzes the rate-limiting step in nitrate assimilation. Plant and algal NRs have a highly conserved domain architecture but differ in regulation. In plants, NR activity is regulated by reversible phosphorylation and subsequent binding of 14-3-3 proteins at a conserved serine residue. Algal NRs typically lack 14-3-3 binding motifs, which have only recently been identified in a few algal species. Previous research indicates that the alga, Chattonella subsalsa, possesses a novel NR, NR2-2/2HbN (NR2), which incorporates a 2/2 hemoglobin domain. A second NR (NR3) in C. subsalsa lacks the cytochrome b5 (heme-Fe) domain but includes a putative binding motif for 14-3-3 proteins. The expression of NR2 and NR3 genes indicates that NR2 transcript abundance was regulated by light, nitrogen source, and temperature, while NR3 transcript levels were only regulated by light. Here, we measured total NR activity in C. subsalsa and the potential for regulation of NR activity by putative 14-3-3 binding proteins. Results indicate that NR activity in C. subsalsa was regulated by light, nitrogen source, and temperature at the translational level. NR activity was also regulated by endogenous rhythm and temperature at the post-translational level, supporting the hypothesis that NR3 is regulated by 14-3-3 binding proteins. Together with a previous report describing the regulation of NR gene expression in C. subsalsa, results suggest that C. subsalsa responds to environmental conditions by differential regulation of NRs at transcriptional, translational, and post-translational levels. This flexibility may provide a competitive advantage for this species in the environment. To date, this is the first report which provides evidence for the potential post-translational regulation of NR by 14-3-3 proteins in algal species and suggests that regulatory mechanisms for NR activity may be shared between plants and some algal species.

Identification and Characterization of Phytocyanin Family Genes in Cotton Genomes

Phytocyanins (PCs) are a class of plant-specific blue copper proteins that have been demonstrated to play a role in electron transport and plant development. Through analysis of the copper ligand residues, spectroscopic properties, and domain architecture of the protein, PCs have been grouped into four subfamilies: uclacyanins (UCs), stellacyanins (SCs), plantacyanins (PLCs), and early nodulin-like proteins (ENODLs). The present study aimed to identify and characterise the PCs present in three distinct cotton species (Gossypium hirsutum, Gossyium arboreum, and Gossypium raimondii) through the identification of 98, 63, and 69 genes respectively. We grouped PCs into four clades by using bioinformatics analysis and sequence alignment, which exhibit variations in gene structure and motif distribution. PCs are distributed across all chromosomes in each of the three species, with varying numbers of exons per gene and multiple conserved motifs, and with a minimum of 1 and maximum of 11 exons found on one gene. Transcriptomic data and qRT-PCR analysis revealed that two highly differentiated PC genes were expressed at the fibre initiation stage, while three highly differentiated PCs were expressed at the fibre elongation stage. These findings serve as a foundation for further investigations aimed at understanding the contribution of this gene family in cotton fibre production.

ATP binding by an F1Fo ATP synthase ε subunit is pH dependent, suggesting a diversity of ε subunit functional regulation in bacteria.

It is a conjecture that the ε subunit regulates ATP hydrolytic function of the F1Fo ATP synthase in bacteria. This has been proposed by the ε subunit taking an extended conformation, with a terminal helix probing into the central architecture of the hexameric catalytic domain, preventing ATP hydrolysis. The ε subunit takes a contracted conformation when bound to ATP, thus would not interfere with catalysis. A recent crystallographic study has disputed this; the Caldalkalibacillus thermarum TA2.A1 F1Fo ATP synthase cannot natively hydrolyse ATP, yet studies have demonstrated that the loss of the ε subunit terminal helix results in an ATP synthase capable of ATP hydrolysis, supporting ε subunit function. Analysis of sequence and crystallographic data of the C. thermarum F1Fo ATP synthase revealed two unique histidine residues. Molecular dynamics simulations suggested that the protonation state of these residues may influence ATP binding site stability. Yet these residues lie outside the ATP/Mg2+ binding site of the ε subunit. We then probed the effect of pH on the ATP binding affinity of the ε subunit from the C. thermarum F1Fo ATP synthase at various physiologically relevant pH values. We show that binding affinity changes 5.9 fold between pH 7.0, where binding is weakest, to pH 8.5 where it is strongest. Since the C. thermarum cytoplasm is pH 8.0 when it grows optimally, this correlates to the ε subunit being down due to ATP/Mg2+ affinity, and not being involved in blocking ATP hydrolysis. Here, we have experimentally correlated that the pH of the bacterial cytoplasm is of critical importance for ε subunit ATP affinity regulated by second-shell residues thus the function of the ε subunit changes with growth conditions.

Conservation of land plant-specific receptor-like cytoplasmic kinase subfamily XI possessing a unique kinase insert domain.

The number of genes encoding receptor-like kinases (RLKs) has expanded in the plant lineage. Their expansion has resulted in the emergence of diverse domain architectures that function in signaling cascades related to growth, development, and stress response. In this study, we focused on receptor-like cytoplasmic kinase subfamily XI (RLCK XI) in plants. We discovered an exceptionally long kinase insert domain (KID), averaging 280 amino acids, between subdomains VII and VIII of the conserved protein kinase domain. Using sequence homology search, we identified members of RLCK XI with the unique KID architecture in terrestrial plants, up to a single copy in several hornwort and liverwort species. The KID shows a high propensity for being disordered, resembling the activation segment in the model kinase domain. Several conserved sequence motifs were annotated along the length of the KID. Of note, the KID harbors repetitive nuclear localization signals capable of mediating RLCK XI translocation from the plasma membrane to the nucleus. The possible physiological implication of dual localization of RLCK XI members is discussed. The presence of a KID in RLCK XI represents a unique domain architecture among RLKs specific to land plants.

Exon shuffling potentiates a diverse repertoire of brown algal NB-ARC-TPR candidate immune receptor proteins via alternative splicing.

Like other organisms, brown algae are subject to diseases caused by bacteria, fungi, and viruses. Brown algal immunity mechanisms are not well characterized; however, there is evidence suggesting that pathogen receptors exist in brown algae. One key protein family likely associated with brown algal innate immunity possesses an NB-ARC domain analogous to innate immune proteins in plants and animals. In this study, we conducted an extensive survey of NB-ARC genes in brown algae and obtained insights into the domain organization and evolutionary history of the encoded proteins. Our data show that brown algae possess an ancient NB-ARC-tetratricopeptide repeat (NB-TPR) domain architecture. We identified an N-terminal effector domain, the four-helix bundle, which was not previously found associated with NB-ARC domains. The phylogenetic tree including NB-ARC domains from all kingdoms of life suggests the three clades of brown algal NB-TPRs are likely monophyletic, whereas their TPRs seem to have distinct origins. One group of TPRs exhibit intense exon shuffling, with various alternative splicing and diversifying selection acting on them, suggesting exon shuffling is an important mechanism for evolving ligand-binding specificities. The reconciliation of gene duplication and loss events of the NB-ARC genes reveals that more independent gene gains than losses have occurred during brown algal evolution, and that tandem duplication has played a major role in the expansion of NB-ARC genes. Our results substantially enhance our understanding of the evolutionary history and exon shuffling mechanisms of the candidate innate immune repertoire of brown algae.

Enterococcal bacteriophage: A survey of the tail associated lysin landscape

Bacteriophages are viruses that exclusively infect bacteria which require local degradation of cell barriers. This degradation is accomplished by various lysins located mainly within the phage tail structure. In this paper we surveyed and analysed the genomes of 506 isolated bacteriophage and prophage infecting or harboured within the genomes of the medically important Enterococcus faecalis and faecium. We highlight and characterise the major features of the genomes of phage in the morphological groups podovirus, siphovirus and myovirus, and explore their categorisation according to the new ICTV classifications, with a focus on putative extracellular lysins chiefly within tail modules. Our analysis reveals a range of potential cell-wall targeting enzyme domains that are part of tail, tape measure or other predicted base structures of these phages or prophages. These largely fall into protein domains targeting pentapeptide or glycosidic linkages within peptidoglycan but also potentially the enterococcal polysaccharide antigen (EPA) and wall teichoic acids of these species (i.e., Pectinesterases and Phosphodiesterases). Notably, there is a great variety of domain architectures that reveal the diversity of evolutionary solutions to attack the Enterococcus cell wall. Despite this variety, most phage and prophage possess a putative endopeptidase (70%), reflecting the ubiquity of this cell surface barrier. We also identified a predicted lytic transglycosylase domain belonging to the glycosyl hydrolase (GH) family 23 and present exclusively within tape measure proteins. Our data also reveal distinct features of the genomes of podo-, sipho- and myo-type viruses that most likely relate to their size and complexity. Overall, we lay a foundation for expression of recombinant TAL proteins and engineering of enterococcal and other phage that will be invaluable for researchers in this field.