The monodomain Kunitz protein EgKU-7 from the dog tapeworm Echinococcus granulosus is a high-affinity trypsin inhibitor with two interaction sites.

Typical Kunitz proteins (I2 family of the MEROPS database, Kunitz-A family) are metazoan competitive inhibitors of serine peptidases that form tight complexes of 1:1 stoichiometry, mimicking substrates. The cestode Echinococcus granulosus, the dog tapeworm causing cystic echinococcosis in humans and livestock, encodes an expanded family of monodomain Kunitz proteins, some of which are secreted to the dog host interface. The Kunitz protein EgKU-7 contains, in addition to the Kunitz domain with the anti-peptidase loop comprising a critical arginine, a C-terminal extension of ∼20 amino acids. Kinetic, electrophoretic, and mass spectrometry studies using EgKU-7, a C-terminally truncated variant, and a mutant in which the critical arginine was substituted by alanine, show that EgKU-7 is a tight inhibitor of bovine and canine trypsins with the unusual property of possessing two instead of one site of interaction with the peptidases. One site resides in the anti-peptidase loop and is partially hydrolyzed by bovine but not canine trypsins, suggesting specificity for the target enzymes. The other site is located in the C-terminal extension. This extension can be hydrolyzed in a particular arginine by cationic bovine and canine trypsins but not by anionic canine trypsin. This is the first time to our knowledge that a monodomain Kunitz-A protein is reported to have two interaction sites with its target. Considering that putative orthologs of EgKU-7 are present in other cestodes, our finding unveils a novel piece in the repertoire of peptidase-inhibitor interactions and adds new notes to the evolutionary host-parasite concerto.

1H, 15N and 13C backbone and side chain solution NMR assignments of the TPM domain-containing protein of the thermophilic bacterium Rhodothermus marinus.

The InterPro family IPR007621 TPM_phosphatase is a widely conserved family of protein domains found in prokaryotes, plants and invertebrates. Despite similar predicted protein folding, members of this family are involved in different cellular processes. In recent years, the structural and biochemical characterization of evolutionarily divergent TPM domains has shown their ability to hydrolyze phosphate groups of different substrates. However, there are still inaccurate functional annotations and uncertain relationships between the structure and function of this domain family. We here report the 1H, 13C, and 15N backbone and sidechain resonances of the TPM domain of a predicted TPM domain-containing protein of the thermophilic bacterium Rhodothermus marinus. These data will lay the groundwork for future NMR-based investigations, contributing to a thorough comprehension of the intricate aspects governing the interplay between structure and function of TPM domains. Additionally, they will unlock opportunities to explore dynamic structural changes, providing valuable insights into the molecular mechanisms underlying the evolutionary adaptations to extreme environmental conditions within this protein family.

Molten Globule Driven and Self-downmodulated Phase Separation of a Viral Factory Scaffold.

Viral factories of liquid-like nature serve as sites for transcription and replication in most viruses. The respiratory syncytial virus factories include replication proteins, brought together by the phosphoprotein (P) RNA polymerase cofactor, present across non-segmented negative stranded RNA viruses. Homotypic liquid-liquid phase separation of RSV-P is governed by an α-helical molten globule domain, and strongly self-downmodulated by adjacent sequences. Condensation of P with the nucleoprotein N is stoichiometrically tuned, defining aggregate-droplet and droplet-dissolution boundaries. Time course analysis show small N-P nuclei gradually coalescing into large granules in transfected cells. This behavior is recapitulated in infection, with small puncta evolving to large viral factories, strongly suggesting that P-N nucleation-condensation sequentially drives viral factories. Thus, the tendency of P to undergo phase separation is moderate and latent in the full-length protein but unleashed in the presence of N or when neighboring disordered sequences are deleted. This, together with its capacity to rescue nucleoprotein-RNA aggregates suggests a role as a "solvent-protein".

MliR, a novel MerR-like regulator of iron homeostasis, impacts metabolism, membrane remodeling, and cell adhesion in the marine Bacteroidetes Bizionia argentinensis.

The MerR family is a group of transcriptional activators with conserved N-terminal helix-turn-helix DNA binding domains and variable C-terminal effector binding regions. In most MerR proteins the effector binding domain (EBD) contains a cysteine center suited for metal binding and mediates the response to environmental stimuli, such as oxidative stress, heavy metals or antibiotics. We here present a novel transcriptional regulator classified in the MerR superfamily that lacks an EBD domain and has neither conserved metal binding sites nor cysteine residues. This regulator from the psychrotolerant bacteria Bizionia argentinensis JUB59 is involved in iron homeostasis and was named MliR (MerR-like iron responsive Regulator). In silico analysis revealed that homologs of the MliR protein are widely distributed among different bacterial species. Deletion of the mliR gene led to decreased cell growth, increased cell adhesion and filamentation. Genome-wide transcriptomic analysis showed that genes associated with iron homeostasis were downregulated in mliR-deletion mutant. Through nuclear magnetic resonance-based metabolomics, ICP-MS, fluorescence microscopy and biochemical analysis we evaluated metabolic and phenotypic changes associated with mliR deletion. This work provides the first evidence of a MerR-family regulator involved in iron homeostasis and contributes to expanding our current knowledge on relevant metabolic pathways and cell remodeling mechanisms underlying in the adaptive response to iron availability in bacteria.

Structure of the putative long tail fiber receptor-binding tip of a novel temperate bacteriophage from the Antarctic bacterium Bizionia argentinensis JUB59.

Tailed bacteriophages are one of the most widespread biological entities on Earth. Their singular structures, such as spikes or fibers are of special interest given their potential use in a wide range of biotechnological applications. In particular, the long fibers present at the termini of the T4 phage tail have been studied in detail and are important for host recognition and adsorption. Although significant progress has been made in elucidating structural mechanisms of model phages, the high-resolution structural description of the vast population of marine phages is still unexplored. In this context, we present here the crystal structure of C24, a putative receptor-binding tip-like protein from Bizionia argentinensis JUB59, a psychrotolerant bacterium isolated from the marine surface waters of Potter Cove, Antarctica. The structure resembles the receptor-binding tip from the bacteriophage T4 long tail fiber yet showing marked differences in its domain organization, size, sequence identity and metal binding nature. We confirmed the viral origin of C24 by induction experiments using mitomycin C. Our results reveal the presence of a novel uncharacterized prophage in the genome of B. argentinensis JUB59, whose morphology is compatible with the order Caudovirales and that carries the nucleotide sequence of C24 in its genome. This work provides valuable information to expand our current knowledge on the viral machinery prevalent in the oceans.

Codon usage clusters correlation: towards protein solubility prediction in heterologous expression systems in E. coli.

Production of soluble recombinant proteins is crucial to the development of industry and basic research. However, the aggregation due to the incorrect folding of the nascent polypeptides is still a mayor bottleneck. Understanding the factors governing protein solubility is important to grasp the underlying mechanisms and improve the design of recombinant proteins. Here we show a quantitative study of the expression and solubility of a set of proteins from Bizionia argentinensis. Through the analysis of different features known to modulate protein production, we defined two parameters based on the %MinMax algorithm to compare codon usage clusters between the host and the target genes. We demonstrate that the absolute difference between all %MinMax frequencies of the host and the target gene is significantly negatively correlated with protein expression levels. But most importantly, a strong positive correlation between solubility and the degree of conservation of codons usage clusters is observed for two independent datasets. Moreover, we evince that this correlation is higher in codon usage clusters involved in less compact protein secondary structure regions. Our results provide important tools for protein design and support the notion that codon usage may dictate translation rate and modulate co-translational folding.

Functional diversity of secreted cestode Kunitz proteins: Inhibition of serine peptidases and blockade of cation channels.

We previously reported a multigene family of monodomain Kunitz proteins from Echinococcus granulosus (EgKU-1-EgKU-8), and provided evidence that some EgKUs are secreted by larval worms to the host interface. In addition, functional studies and homology modeling suggested that, similar to monodomain Kunitz families present in animal venoms, the E. granulosus family could include peptidase inhibitors as well as channel blockers. Using enzyme kinetics and whole-cell patch-clamp, we now demonstrate that the EgKUs are indeed functionally diverse. In fact, most of them behaved as high affinity inhibitors of either chymotrypsin (EgKU-2-EgKU-3) or trypsin (EgKU-5-EgKU-8). In contrast, the close paralogs EgKU-1 and EgKU-4 blocked voltage-dependent potassium channels (Kv); and also pH-dependent sodium channels (ASICs), while showing null (EgKU-1) or marginal (EgKU-4) peptidase inhibitory activity. We also confirmed the presence of EgKUs in secretions from other parasite stages, notably from adult worms and metacestodes. Interestingly, data from genome projects reveal that at least eight additional monodomain Kunitz proteins are encoded in the genome; that particular EgKUs are up-regulated in various stages; and that analogous Kunitz families exist in other medically important cestodes, but not in trematodes. Members of this expanded family of secreted cestode proteins thus have the potential to block, through high affinity interactions, the function of host counterparts (either peptidases or cation channels) and contribute to the establishment and persistence of infection. From a more general perspective, our results confirm that multigene families of Kunitz inhibitors from parasite secretions and animal venoms display a similar functional diversity and thus, that host-parasite co-evolution may also drive the emergence of a new function associated with the Kunitz scaffold.

Structural and functional characterization of a cold adapted TPM-domain with ATPase/ADPase activity.

The Pfam PF04536 TPM_phosphatase family is a broadly conserved family of domains found across prokaryotes, plants and invertebrates. Despite having a similar protein fold, members of this family have been implicated in diverse cellular processes and found in varied subcellular localizations. Very recently, the biochemical characterization of two evolutionary divergent TPM domains has shown that they are able to hydrolyze phosphate groups from different substrates. However, there are still incorrect functional annotations and uncertain relationships between the structure and function of this family of domains. BA41 is an uncharacterized single-pass transmembrane protein from the Antarctic psychrotolerant bacterium Bizionia argentinensis with a predicted compact extracytoplasmic TPM domain and a C-terminal cytoplasmic low complexity region. To shed light on the structural properties that enable TPM domains to adopt divergent roles, we here accomplish a comprehensive structural and functional characterization of the central TPM domain of BA41 (BA41-TPM). Contrary to its predicted function as a beta-propeller methanol dehydrogenase, light scattering and crystallographic studies showed that BA41-TPM behaves as a globular monomeric protein and adopts a conserved Rossmann fold, typically observed in other TPM domain structures. Although the crystal structure reveals the conservation of residues involved in substrate binding, no putative catalytic or intramolecular metal ions were detected. Most important, however, extensive biochemical studies demonstrated that BA41-TPM has hydrolase activity against ADP, ATP, and other di- and triphosphate nucleotides and shares properties of cold-adapted enzymes. The role of BA41 in extracellular ATP-mediated signaling pathways and its occurrence in environmental and pathogenic microorganisms is discussed.

Structural and functional characterization of a cold-adapted stand-alone TPM domain reveals a relationship between dynamics and phosphatase activity.

The TPM domain constitutes a family of recently characterized protein domains that are present in most living organisms. Although some progress has been made in understanding the cellular role of TPM-containing proteins, the relationship between structure and function is not clear yet. We have recently solved the solution and crystal structure of one TPM domain (BA42) from the Antarctic bacterium Bizionia argentinensis. In this work, we demonstrate that BA42 has phosphoric-monoester hydrolase activity. The activity of BA42 is strictly dependent on the binding of divalent metals and retains nearly 70% of the maximum at 4 °C, a typical characteristic of cold-adapted enzymes. From HSQC, 15 N relaxation measurements, and molecular dynamics studies, we determine that the flexibility of the crossing loops was associated to the protein activity. Thermal unfolding experiments showed that the local increment in flexibility of Mg2+ -bound BA42, when compared with Ca2+ -bound BA42, is associated to a decrease in global protein stability. Finally, through mutagenesis experiments, we unambiguously demonstrate that the region comprising the metal-binding site participates in the catalytic mechanism. The results shown here contribute to the understanding of the relationship between structure and function of this new family of TPM domains providing important cues on the regulatory role of Mg2+ and Ca2+ and the molecular mechanism underlying enzyme activity at low temperatures.

Solution and crystal structure of BA42, a protein from the Antarctic bacterium Bizionia argentinensis comprised of a stand-alone TPM domain.

The structure of the BA42 protein belonging to the Antarctic flavobacterium Bizionia argentinensis was determined by nuclear magnetic resonance and X-ray crystallography. This is the first structure of a member of the PF04536 family comprised of a stand-alone TPM domain. The structure reveals a new topological variant of the four ß-strands constituting the central ß-sheet of the αßα architecture and a double metal binding site stabilizing a pair of crossing loops, not observed in previous structures of proteins belonging to this family. BA42 shows differences in structure and dynamics in the presence or absence of bound metals. The affinity for divalent metal ions is close to that observed in proteins that modulate their activity as a function of metal concentration, anticipating a possible role for BA42.

Draft genome sequence of Bizionia argentinensis, isolated from Antarctic surface water.

A psychrotolerant marine bacterial strain, designated JUB59(T), was isolated from Antarctic surface seawater and classified as a new species of the genus Bizionia. Here, we present the first draft genome sequence for this genus, which suggests interesting features such as UV resistance, hydrolytic exoenzymes, and nitrogen metabolism.

Tuned Escherichia coli as a host for the expression of disulfide-rich proteins.

Disulfide-bond formation is a major post-translational modification and is essential for protein folding, stability, and function. This is especially true for secreted proteins, many of which possess great potential for biotechnological applications. Focusing on the use of Escherichia coli for the production of this class of proteins, we describe the mechanisms that maintain redox compartmentalization in the cell, with an emphasis on those that promote the formation and isomerization of disulfide bonds in the bacterial periplasm, while presenting parallel pathways in the eukaryotic endoplasmic reticulum. Based on these concepts, we review the use of E. coli as a cell factory for the production of heterologous disulfide-containing proteins using either peri- or cytoplasmic expression and, in particular, how these compartments can be tuned to improve the yield of correctly folded recombinant proteins. Finally, we describe a few examples of the production of small disulfide-rich proteins (protease inhibitors) to illustrate how soluble, active, and fully oxidized recombinants may be successfully obtained upon peri- or cytoplasmic expression in E. coli.