Uncharacterized Domain Research Articles

The protein structurome of Orthornavirae and its dark matter.

Advanced methods for protein structure prediction, such as AlphaFold2, greatly expand our capability to identify protein domains and infer their likely functions and evolutionary relationships. This is particularly pertinent for proteins encoded by viruses that are known to evolve rapidly and as a result often cannot be adequately characterized by analysis of the protein sequences. We performed an exhaustive structure prediction and comparative analysis for uncharacterized proteins and domains ("dark matter") encoded by viruses with RNA genomes. The results show the dark matter of RNA virus proteome consists mostly of disordered and all-α-helical domains that cannot be readily assigned a specific function and that likely mediate various interactions between viral proteins and between viral and host proteins. The great majority of globular proteins and domains of RNA viruses are already known although we identified several unexpected domains represented in individual viral families.

Loss of GATAD1 in cardiomyocyte does not cause cardiomyopathy in mice.

GATA zinc finger domain containing 1 (GATAD1) is an as-yet uncharacterized zinc finger domain protein, which was initially identified as a histone 3 trimethylated at lysine 4 (H3K4me3) interactor. A recessive mutation in GATAD1 is associated with adult-onset dilated cardiomyopathy and heart failure, suggesting that GATAD1 is critical for maintaining normal cardiac structure and function. However, little is known as to the specific role of GATAD1 in cardiomyocytes. A mammalian Gatad1 knockout model has yet to be generated for investigating its specific role in the heart. To address this, we generated a Gatad1 cardiomyocyte-specific knockout (cKO) mouse model. Gatad1 cKO mutants exhibited normal cardiac function during the aging process up to 18 months of age. Unlike the abnormal nuclei shape observed in patients carrying GATAD1 mutations, the nuclei shape of cardiomyocytes remained unaffected by the loss of Gatad1. Furthermore, Gatad1 cKO mice responded normally to pressure overload induced by transverse aortic constriction (TAC) surgery. Together, these observations suggest that deletion of Gatad1 in cardiomyocytes does not induce cardiomyopathy during aging or affect the response to pressure overload stress in mice.

Structural basis for Rab6 activation by the Ric1-Rgp1 complex

Rab GTPases act as molecular switches to regulate organelle homeostasis and membrane trafficking. Rab6 plays a central role in regulating cargo flux through the Golgi and is activated via nucleotide exchange by the Ric1-Rgp1 protein complex. Ric1-Rgp1 is conserved throughout eukaryotes but the structural and mechanistic basis for its function has not been established. Here we report the cryoEM structure of a Ric1-Rgp1‐Rab6 complex representing a key intermediate of the nucleotide exchange reaction. Ric1-Rgp1 interacts with the nucleotide-binding domain of Rab6 using an uncharacterized helical domain, which we establish as a RabGEF domain by identifying residues required for Rab6 activation. Unexpectedly, the complex uses an arrestin fold to interact with the Rab6 hypervariable domain, indicating that interactions with the unstructured C-terminal regions of Rab GTPases may be a common binding mechanism used by their activators. Collectively, our findings provide a detailed mechanistic understanding of regulated Rab6 activation at the Golgi.

LARP1 binds ribosomes and TOP mRNAs in repressed complexes

Terminal oligopyrimidine motif-containing mRNAs (TOPs) encode all ribosomal proteins in mammals and are regulated to tune ribosome synthesis to cell state. Previous studies have implicated LARP1 in 40S- or 80S-ribosome complexes that are thought to repress and stabilize TOPs. However, a molecular understanding of how LARP1 and TOPs interact with these ribosome complexes is lacking. Here, we show that LARP1 directly binds non-translating ribosomal subunits. Cryo-EM structures reveal a previously uncharacterized domain of LARP1 bound to and occluding the mRNA channel of the 40S subunit. Increased availability of free ribosomal subunits downstream of various stresses promote 60S joining at the same interface to form LARP1-80S complexes. Simultaneously, LARP1 engages the TOP via its previously characterized La/PAM2 and DM15 domains. Contrary to expectations, ribosome binding within these complexes is not required for LARP1-mediated TOP repression or stabilization, two canonical LARP1 functions. Together, this work provides molecular insight into how LARP1 directly binds ribosomal subunits and challenges existing models describing the function of repressed LARP1-40S/80S-TOP complexes.

Structure of a Putative Terminal Amidation Domain in Natural Product Biosynthesis.

Bacteria are rich sources of pharmaceutically valuable natural products, many crafted by modular polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS). PKS and NRPS systems typically contain a thioesterase (TE) to offload a linear or cyclized product from a carrier protein, but alternative chemistry is needed for products with a terminal amide. Several pathways with amidated products also possess an uncharacterized 400-amino acid terminal domain. We present the characterization and structure of this putative terminal amidation domain (TAD). TAD binds NAD with the nicotinamide near an invariant cysteine that is also accessible to an intermediate on a carrier protein, indicating a catalytic role. The TAD structure resembles cyanobacterial acyl-ACP reductase (AAR), which binds NADPH near an analogous catalytic cysteine. Bioinformatic analysis reveals that TADs are broadly distributed across bacterial phyla and often occur at the end of terminal NRPS modules, suggesting many amidated products may yet be discovered.

Annotation-Based Study on Hypothetical Proteins in Bacteria Using Classification Features

Background: Hypothetical proteins (HPs) are those proteins whose functions are unknown; therefore, the present study was carried out to predict similarity-based functionality of HPs in selected bacteria Streptomyces coelicolor A3(2) and Neisseria meningitidis. Methods: Annotation-based approaches using Pfam, orthology, String, Bi-directional Best Blast Hit, PSLpred, Subloc, Cello, homology modeling, and computational tools were used in evaluating the functionality of HPs. Results: Thirty-one domains in both bacterial species were retrieved based on the E-value score and compared with bacterial species already existing in databases. Statistical analysis was duly done to check which features performed well Conclusion: Out of 31 HPs found in Streptomyces coleicolor strain A3(2), 14 domains were found to be uncharacterized in their functionality, while 2 uncharacterized domains in the case of Neisseria meningitidis were assigned a function on similarity-based approaches. The annotation of HPs is a challenge in bacteria as these are based on the similarity of proteins in other species.

Arabidopsis Dph4 is an Hsp70 Cochaperone with Iron-Binding Properties.

J-domain proteins (JDPs) are obligate cochaperones of Hsp70s with a wide range of functions in protein homeostasis. Although the J-domain is required for the stimulation of Hsp70s ATPase activity, the functional specificity of JDPs is governed by domains or regions other than the J-domain. Jjj3/Dph4, a class III JDP, is required for diphthamide (DPH) biosynthesis in eukaryotes, including yeast and mammals. Dph4 has a conserved N-terminal J-domain and an uncharacterized C-terminal domain containing a signature CSL zinc finger motif. Previously, we showed that the Dph4 ortholog in Arabidopsis thaliana (atDjC13/AtJjj3/AtDph4) could restore DPH biosynthesis in yeast jjj3Δ mutant in a J-domain-dependent manner. Here, we characterize the C-terminal CSL motif of AtDph4 using yeast genetic and biochemical approaches. The CSL motif of AtDph4 is essential for DPH biosynthesis, and like human Dph4, AtDph4 showed distinct iron-binding activity, which is not present in its yeast counterpart. ScDph4 and AtDph4 proteins exhibit distinct iron-binding capabilities, as evidenced by UV-vis spectrophotometry, SEM-EDS (energy-dispersive spectroscopy function on the scanning electron microscope) and electron paramagnetic resonance (EPR) spectra analyses. Collectively, our data suggests that beyond their role as an Hsp70 cochaperone, Dph4 homologues in complex eukaryotes may have iron-binding abilities, indicating a potential role in iron-sulfur cluster assembly and iron homeostasis.

Structural insights into RNA cleavage by a novel family of bacterial RNases.

Processing of RNA is a key regulatory mechanism for all living systems. Escherichia coli protein YicC belongs to the well-conserved YicC family and has been identified as a novel ribonuclease. Here, we report a 2.8-Å-resolution crystal structure of the E. coli YicC apo protein and a 3.2-Å-cryo-EM structure of YicC bound to an RNA substrate. The apo YicC forms a dimer of trimers with a large open channel. In the RNA-bound form, the top trimer of YicC rotates nearly 70°and closes the RNA substrate inside the cavity to form a clamshell-pearl conformation that resembles no other known RNases. The structural information combined with mass spectrometry and biochemical data identified cleavage on the upstream side of an RNA hairpin. Mutagenesis studies demonstrated that the previously uncharacterized domain, DUF1732, is critical in both RNA binding and catalysis. These studies shed light on the mechanism of the previously unexplored YicC RNase family.

Characterization of the Arabidopsis thaliana chromatin remodeler DEK3 for its interaction with histones and DNA

Chromatin structure and dynamics regulate all DNA-templated processes, such as transcription, replication, and repair. Chromatin binding factors, chromatin architectural proteins, and nucleosome remodelers modulate chromatin structure and dynamics and, thereby, the various DNA-dependent processes. Arabidopsis thaliana DEK3, a member of the evolutionarily conserved DEK domain-containing chromatin architectural proteins, is an important factor for chromatin structure and function, involved in transcriptional programming to regulate flowering time and abiotic stress tolerance. AtDEK3 contains an uncharacterized N-terminal domain, a middle SAF domain (winged helix-like domain), and a C-terminal DEK domain, but their role in the interaction of AtDEK3 with histones and DNA remained poorly understood. Using biochemical and biophysical analyses, we provide a comprehensive in vitro characterization of the different AtDEK3 domains for their interaction with histone H3/H4 and DNA. AtDEK3 directly interacts with histone H3/H4 tetramers through its N-terminal domain and the C-terminal DEK domain in a 1:1 stoichiometry. Upon interaction with H3/H4, the unstructured N-terminal domain of AtDEK3 undergoes a conformational change and adopts an alpha-helical conformation. In addition, the in-solution envelope structures of the AtDEK3 domains and their complex with H3/H4 have been characterized. The SAF and DEK domains associate with double-stranded and four-way junction DNA. As DEK3 possesses a histone-interacting domain at the N- and the C-terminus and a DNA-binding domain in the middle and at the C-terminus, the protein might play a complex role as a chromatin remodeler.

Angiopoietin-like 4 protects against endothelial dysfunction during bacterial sepsis.

Loss of endothelial integrity and vascular leakage are central features of sepsis pathogenesis; however, no effective therapeutic mechanisms for preserving endothelial integrity are available. Here we show that, compared to dermal microvessels, brain microvessels resist infection by Neisseria meningitidis, a bacterial pathogen that causes sepsis and meningitis. By comparing the transcriptional responses to infection in dermal and brain endothelial cells, we identified angiopoietin-like 4 as a key factor produced by the brain endothelium that preserves blood-brain barrier integrity during bacterial sepsis. Conversely, angiopoietin-like 4 is produced at lower levels in the peripheral endothelium. Treatment with recombinant angiopoietin-like 4 reduced vascular leakage, organ failure and death in mouse models of lethal sepsis and N. meningitidis infection. Protection was conferred by a previously uncharacterized domain of angiopoietin-like 4, through binding to the heparan proteoglycan, syndecan-4. These findings reveal a potential strategy to prevent endothelial dysfunction and improve outcomes in patients with sepsis.

MUT-7 exoribonuclease activity and localization are mediated by an ancient domain.

The MUT-7 family of 3'-5' exoribonucleases is evolutionarily conserved across the animal kingdom and plays essential roles in small RNA production in the germline. Most MUT-7 homologues carry a C-terminal domain of unknown function named MUT7-C appended to the exoribonuclease domain. Our analysis shows that the MUT7-C is evolutionary ancient, as a minimal version of the domain exists as an individual protein in prokaryotes. In animals, MUT7-C has acquired an insertion that diverged during evolution, expanding its functions. Caenorhabditis elegans MUT-7 contains a specific insertion within MUT7-C, which allows binding to MUT-8 and, consequently, MUT-7 recruitment to germ granules. In addition, in C. elegans and human MUT-7, the MUT7-C domain contributes to RNA binding and is thereby crucial for ribonuclease activity. This RNA-binding function most likely represents the ancestral function of the MUT7-C domain. Overall, this study sheds light on MUT7-C and assigns two functions to this previously uncharacterized domain.

Uncovering the BIN1-SH3 interactome underpinning centronuclear myopathy.

Truncation of the protein-protein interaction SH3 domain of the membrane remodeling Bridging Integrator 1 (BIN1, Amphiphysin 2) protein leads to centronuclear myopathy. Here, we assessed the impact of a set of naturally observed, previously uncharacterized BIN1 SH3 domain variants using conventional in vitro and cell-based assays monitoring the BIN1 interaction with dynamin 2 (DNM2) and identified potentially harmful ones that can be also tentatively connected to neuromuscular disorders. However, SH3 domains are typically promiscuous and it is expected that other, so far unknown partners of BIN1 exist besides DNM2, that also participate in the development of centronuclear myopathy. In order to shed light on these other relevant interaction partners and to get a holistic picture of the pathomechanism behind BIN1 SH3 domain variants, we used affinity interactomics. We identified hundreds of new BIN1 interaction partners proteome-wide, among which many appear to participate in cell division, suggesting a critical role of BIN1 in the regulation of mitosis. Finally, we show that the identified BIN1 mutations indeed cause proteome-wide affinity perturbation, signifying the importance of employing unbiased affinity interactomic approaches.

PARG Protein Regulation Roles in Drosophila Longevity Control.

Aging, marked by a gradual decline in physiological function and heightened vulnerability to age-related diseases, remains a complex biological process with multifaceted regulatory mechanisms. Our study elucidates the critical role of poly(ADP-ribose) glycohydrolase (PARG), responsible for catabolizing poly(ADP-ribose) (pADPr) in the aging process by modulating the expression of age-related genes in Drosophila melanogaster. Specifically, we uncover the regulatory function of the uncharacterized PARG C-terminal domain in controlling PARG activity. Flies lacking this domain exhibit a significantly reduced lifespan compared to wild-type counterparts. Furthermore, we observe progressive dysregulation of age-related gene expression during aging, accelerated in the absence of PARG activity, culminating in a premature aging phenotype. Our findings reveal the critical involvement of the pADPr pathway as a key player in the aging process, highlighting its potential as a therapeutic target for mitigating age-related effects.

Structural dissection of two redox proteins from the shipworm symbiont Teredinibacter turnerae.

The discovery of lytic polysaccharide monooxygenases (LPMOs), a family of copper-dependent enzymes that play a major role in polysaccharide degradation, has revealed the importance of oxidoreductases in the biological utilization of biomass. In fungi, a range of redox proteins have been implicated as working in harness with LPMOs to bring about polysaccharide oxidation. In bacteria, less is known about the interplay between redox proteins and LPMOs, or how the interaction between the two contributes to polysaccharide degradation. We therefore set out to characterize two previously unstudied proteins from the shipworm symbiont Teredinibacter turnerae that were initially identified by the presence of carbohydrate binding domains appended to uncharacterized domains with probable redox functions. Here, X-ray crystal structures of several domains from these proteins are presented together with initial efforts to characterize their functions. The analysis suggests that the target proteins are unlikely to function as LPMO electron donors, raising new questions as to the potential redox functions that these large extracellular multi-haem-containing c-type cytochromes may perform in these bacteria.

Dual membrane-spanning anti-sigma factors regulate vesiculation in Bacteroides thetaiotaomicron

Bacteroidota are abundant members of the human gut microbiota that shape the enteric landscape by modulating host immunity and degrading dietary- and host-derived glycans. These processes are mediated in part by Outer Membrane Vesicles (OMVs). Here, we developed a high-throughput screen to identify genes required for OMV biogenesis and its regulation in Bacteroides thetaiotaomicron (Bt). We identified a family of Dual membrane-spanning anti-sigma factors (Dma) that control OMV biogenesis. We conducted molecular and multiomic analyses to demonstrate that deletion of Dma1, the founding member of the Dma family, modulates OMV production by controlling the activity of the ECF21 family sigma factor, Das1, and its downstream regulon. Dma1 has a previously uncharacterized domain organization that enables Dma1 to span both the inner and outer membrane of Bt. Phylogenetic analyses reveal that this common feature of the Dma family is restricted to the phylum Bacteroidota. This study provides mechanistic insights into the regulation of OMV biogenesis in human gut bacteria.

A CYBDOM protein impacts iron homeostasis and primary root growth under phosphate deficiency in Arabidopsis

Arabidopsis primary root growth response to phosphate (Pi) deficiency is mainly controlled by changes in apoplastic iron (Fe). Upon Pi deficiency, apoplastic Fe deposition in the root apical meristem activates pathways leading to the arrest of meristem maintenance and inhibition of cell elongation. Here, we report that a member of the uncharacterized cytochrome b561 and DOMON domain (CYBDOM) protein family, named CRR, promotes iron reduction in an ascorbate-dependent manner and controls apoplastic iron deposition. Under low Pi, the crr mutant shows an enhanced reduction of primary root growth associated with increased apoplastic Fe in the root meristem and a reduction in meristematic cell division. Conversely, CRR overexpression abolishes apoplastic Fe deposition rendering primary root growth insensitive to low Pi. The crr single mutant and crr hyp1 double mutant, harboring a null allele in another member of the CYDOM family, shows increased tolerance to high-Fe stress upon germination and seedling growth. Conversely, CRR overexpression is associated with increased uptake and translocation of Fe to the shoot and results in plants highly sensitive to Fe excess. Our results identify a ferric reductase implicated in Fe homeostasis and developmental responses to abiotic stress, and reveal a biological role for CYBDOM proteins in plants.

Reappraisal of the DNA phosphorothioate modification machinery: uncovering neglected functional modalities and identification of new counter-invader defense systems.

The DndABCDE systems catalysing the unusual phosphorothioate (PT) DNA backbone modification, and the DndFGH systems, which restrict invasive DNA, have enigmatic and paradoxical features. Using comparative genomics and sequence-structure analyses, we show that the DndABCDE module is commonly functionally decoupled from the DndFGH module. However, the modification gene-neighborhoods encode other nucleases, potentially acting as the actual restriction components or suicide effectors limiting propagation of the selfish elements. The modification module's core consists of a coevolving gene-pair encoding the DNA-scanning apparatus - a DndD/CxC-clade ABC ATPase and DndE with two ribbon-helix-helix (MetJ/Arc) DNA-binding domains. Diversification of DndE's DNA-binding interface suggests a multiplicity of target specificities. Additionally, many systems feature DNA cytosine methylase genes instead of PT modification, indicating the DndDE core can recruit other nucleobase modifications. We show that DndFGH is a distinct counter-invader system with several previously uncharacterized domains, including a nucleotide kinase. These likely trigger its restriction endonuclease domain in response to multiple stimuli, like nucleotides, while blocking protective modifications by invader methylases. Remarkably, different DndH variants contain a HerA/FtsK ATPase domain acquired from multiple sources, including cellular genome-segregation systems and mobile elements. Thus, we uncovered novel HerA/FtsK-dependent defense systems that might intercept invasive DNA during replication, conjugation, or packaging.

Addition of α-1,3-glucan-binding domains to α-1,3-glucanase Agn1p from Schizosaccharomyces pombe enhances hydrolytic activity of insoluble α-1,3-glucan.

The glycoside hydrolase (GH) 71 α-1,3-glucanase (Agn1p) from Schizosaccharomyces pombe consists of an N-terminal signal sequence and a catalytic domain. Meanwhile, the GH87 α-1,3-glucanase (Agl-KA) from Bacillus circulans KA-304 consists of an N-terminal signal sequence, a first discoidin domain (DS1), a carbohydrate-binding module family 6 (CBM6), a threonine and proline repeat linker (TP), a second discoidin domain (DS2), an uncharacterized domain, and a catalytic domain. DS1, CBM6, and DS2 exhibit α-1,3-glucan binding activity. This study involved genetically fusing TP, DS1, CBM6, TP, and DS2 to the C-terminus of Agn1p, generating the fusion enzyme Agn1p-DCD. The fusion enzyme was then expressed in Escherichia coli and purified from the cell-free extract. Agn1p-DCD and Agn1p exhibited similar characteristics, such as optimal pH, optimal temperature, pH stability, and thermostability. Insoluble α-1,3-glucan (1%) hydrolyzing assay showed that Agn1p-DCD and Agn1p released approximately 7.6 and 5.0 mM of reducing sugars, respectively, after 48 h of reaction. Kinetic analysis and an α-1,3-glucan binding assay indicated that the addition of DS1, CBM6, and DS2 enhanced the affinity of Agn1p for α-1,3-glucan. Moreover, Agn1p-DCD contributed to enhancing the fungal growth inhibition activity when combined with a mixture of GH19 chitinase and GH16 β-1,3-glucanase.

Toxoplasma membrane inositol phospholipid binding protein TgREMIND is essential for secretory organelle function and host infection

Apicomplexan parasites possess specialized secretory organelles called rhoptries, micronemes, and dense granules that play a vital role in host infection. In this study, we demonstrate that TgREMIND, a protein found in Toxoplasma gondii, is necessary for the biogenesis of rhoptries and dense granules. TgREMIND contains a Fes-CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain, which binds to membrane phospholipids, as well as a novel uncharacterized domain that we have named REMIND (regulator of membrane-interacting domain). Both the F-BAR domain and the REMIND are crucial for TgREMIND functions. When TgREMIND is depleted, there is a significant decrease in the abundance of dense granules and abnormal transparency of rhoptries, leading to a reduction in protein secretion from these organelles. The absence of TgREMIND inhibits host invasion and parasite dissemination, demonstrating that TgREMIND is essential for the proper function of critical secretory organelles required for successful infection by Toxoplasma.

Membrane-specific and calcium-dependent binding of the Arabidopsis C2 domain protein CaLB revealed by ATR-FTIR spectroscopy

C2 domain-containing proteins bind to cellular membranes and mediate diverse cellular processes. Although many of these membrane-interacting proteins have been identified, the molecular mechanisms of protein-membrane interactions and conformational dynamics are often poorly understood and remain to be investigated with appropriate methods. Here, we used attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy and biomimetic membrane systems to analyse CalB, a yet uncharacterized Arabidopsis C2 domain protein. We studied membrane binding, lipid specificity and calcium dependency with solid-supported lipid membranes (SSLB) and small unilamellar lipid vesicles (SUVs). Membranes were composed of pure POPC lipids or of POPC/PI(3)P lipid mixtures. A significantly increased protein binding affinity was observed with membranes containing 1% PI(3)P indicating the high binding specificity of CaLB for PI(3)P. Furthermore, membrane binding occurs in a calcium-dependent manner with a higher calcium concentration increasing the binding of CaLB to the POPC/PI(3)P membrane. Secondary structure analysis of IR-spectra reveals that only minor conformational changes take place upon binding with a slight increase in the helical and disordered regions of CaLB.