Search Results (61)

α2-Macroglobulin (A2M), a large tetrameric glycoprotein with a molecular weight of approximately 720 kDa, is a key member of the α-macroglobulin superfamily. Its origin dates back 600–700 million years, positioning A2M as an evolutionary link within the α-macroglobulin family and complement components C3, C4, and C5. Structural predictions of A2M across different species reveal a remarkably high degree of conservation between invertebrates and vertebrates. A2M is abundantly present in the body fluids of both vertebrates and invertebrates, and its diverse biological functions are governed by five key functional domains within its molecular structure. The most well-established role of A2M is the entrapment and inhibition of proteases. Beyond that, it interacts with cytokines, growth factors, and membrane receptors, thereby playing a broad role in immune and inflammatory responses, hemostasis and coagulation, as well as in disease mechanisms and therapeutic processes. This review summarizes the origin and evolution of A2M, its molecular structure and functional domains, principal mechanisms of action, and research progress regarding its functions in both invertebrates and vertebrates. Our goal is to provide new insights and directions for further exploring the functional potential of A2M and its future applications in the treatment of clinical diseases. Full article

KCTD (K-potassium Channel Tetramerization Domain-containing) proteins form an emerging class of proteins involved in a wide range of physiological and pathological processes. The involvement of these proteins in diverse biological functions is often a characteristic shared within family clades. Among them, the multifunctional roles of the cluster members 1B, which include KCTD1 and KCTD15, are particularly intriguing. Several studies have shown that these proteins participate in various signaling pathways and are linked to diseases such as cancer, genetic disorders, and obesity. In this proceedings paper, after a brief review of recent findings on the various (mis)functional roles of these proteins, we analyze available structural data and illustrate how such data have helped uncover structure-function relationships. A summary of the key open questions in the field is also included. Full article

Phylogenetic studies of the pyruvate kinase family reveal two clusters: the K⁺-dependent and -independent enzymes. Thermoplasma acidophilum pyruvate kinase belongs to the latter but possesses the conserved signature of those K⁺-dependent. Recently, we found two distinct ways for these groups to catalyze. It is interesting to elucidate how the T. acidophilum enzyme achieves its active conformation. A structural model of this enzyme revealed the presence of an extra C-terminal sequence (ECTS). To understand its role, an enzyme lacking this sequence from T. acidophilum was constructed. We then compared the kinetic parameters, far-UV CD spectra, thermal stability, molecular dynamics simulations, and oligomeric states of both the wild-type and truncated enzymes. We found that the truncated enzyme is aggregated and almost inactive, with residual 20% of the total interactions, and it exhibits a soluble fraction of smaller oligomeric states than the wild-type enzyme. These findings suggest that ECTS plays a crucial role in maintaining its active tetrameric state. This sequence is the first reported in an archaeal pyruvate kinase and is also found in other archaea and bacteria. Phylogenetic analysis of ECTS in pyruvate kinases exhibits a sparse distribution that might be explained if ECTS represents an ancient domain prone to loss. Full article

Tongue squamous cell carcinoma (TSCC), a subtype of head and neck squamous cell carcinoma, is characterized by frequent chemoresistance. Genetic mutations commonly observed in TSCC play a critical role in malignant progression; thus, elucidating their functional significance is essential for developing effective treatment strategies. To more accurately investigate the relationship between mutations and chemoresistance, we established low-passage TSCC cells, CTSC-1, obtained from a chemoresistant patient, and CTSC-2, from a treatment-naïve patient. Sanger sequencing revealed a specific TP53 mutation (Q331*) in CTSC-1, leading to the loss of the tetramerization and C-terminal regulatory domains. Notably, CTSC-1 cells harboring TP53-Q331* and CTSC-2 cells with TP53 knockout that have been engineered to ectopically express TP53-Q331* exhibit enhanced chemoresistance and increased cancer stem cell-like properties. Mechanistically, TP53-Q331* upregulates the expression of inhibitor of DNA binding 2 (ID2), which is crucial for maintaining the stemness of TSCC cells. Subsequently, ID2 activates the expression of nucleotide excision repair (NER) pathway-related genes ERCC4 and ERCC8, thereby enhancing the chemoresistance in TSCC. In conclusion, our study demonstrates that the TP53-Q331* mutation enhances TSCC chemoresistance through an ID2-mediated NER pathway, providing a potential prognostic marker and therapeutic target for TSCC chemotherapy resistance. Full article

Cry toxins from Bacillus thuringiensis are effective biopesticides that kill lepidopteran pests, replacing chemical pesticides that indiscriminately attack both target and non-target organisms. However, resistance in susceptible pests is an emerging problem. B. thuringiensis also produces vegetative insecticidal protein (Vip3A), which can kill insect targets in the same group as Cry toxins but using different host receptors, making the combined application of Cry and Vip3A an exciting possibility. Vip3A toxicity requires the formation of a homotetramer. Hence, screening of Vip3A mutants for increased stability requires orthogonal biophysical assays that can test both tetrameric integrity and monomeric robustness. For this purpose, we have used herein for the first time a combination of analytical ultracentrifugation (AUC), mass photometry (MP), differential static light scattering (DSLS) and differential scanning fluorimetry (DSF) to test five mutants at domains I and II. Although all mutants appeared more stable than the wild type (WT) in DSLS, mutants that showed more dissociation into dimers in MP and AUC experiments also showed earlier thermal unfolding by DSF at domains IV–V. All of the mutants were less toxic than the WT, but toxicity was highest for domain II mutations N242C and F229Y. Activation of the protoxin was complete and resulted in a form with a lower sedimentation coefficient. Future high-resolution structural data may lead to a deeper understanding of the increased stability that will help with rational design while retaining native toxicity. Full article

Yellow fever virus (YFV) infections can cause severe diseases in humans, resulting in mass casualties in Africa and the Americas each year. Secretory NS1 (sNS1) is thought to be used as a diagnostic marker of flavivirus infections, playing an essential role in the flavivirus life cycle, but little is known about the composition and structure of YFV sNS1. Here, we present that the recombinant YFV sNS1 exists in a heterogeneous mixture of oligomerizations, predominantly in the tetrameric form. The cryoEM structures show that the YFV tetramer of sNS1 is stacked by the hydrophobic interaction between β-roll domains and greasy fingers. According to the 3D variability analysis, the tetramer is in a semi-stable state that may contain multiple conformations with dynamic changes. We believe that our study provides critical insights into the oligomerization of NS1 and will aid the development of NS1-based diagnoses and therapies. Full article

Regulatory cystathionine β-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of the underlying regulation mechanism. Tetrameric microbial inorganic pyrophosphatase containing such domains (CBS-PPase) is allosterically inhibited by AMP and ADP and activated by ATP and cell alarmones diadenosine polyphosphates. Each CBS-PPase subunit contains a pair of CBS domains but binds cooperatively to only one molecule of the mono-adenosine derivatives. We used site-directed mutagenesis of Desulfitobacterium hafniense CBS-PPase to identify the key elements determining the direction of the effect (activation or inhibition) and the “half-of-the-sites” ligand binding stoichiometry. Seven amino acid residues were selected in the CBS1 domain, based on the available X-ray structure of the regulatory domains, and substituted by alanine and other residues. The interaction of 11 CBS-PPase variants with the regulating ligands was characterized by activity measurements and isothermal titration calorimetry. Lys100 replacement reversed the effect of ADP from inhibition to activation, whereas Lys95 and Gly118 replacements made ADP an activator at low concentrations but an inhibitor at high concentrations. Replacement of these residues for alanine increased the stoichiometry of mono-adenosine phosphate binding by twofold. These findings identified several key protein residues and suggested a “two non-interacting pairs of interacting regulatory sites” concept in CBS-PPase regulation. Full article

Xylose isomerase (XI, also known as glucose isomerase) is an oxidoreductase that interconverts aldoses and ketoses. XI catalyzes the reversible isomerization of D-glucose and D-xylose into D-fructose and D-xylulose, respectively. The molecular function of XI is widely applied in producing high-fructose corn syrup (HFCS) in the food industry and bioethanol from hemicellulose in the biofuel industry. The structural information of XI from diverse strains is important for understanding molecular properties that can provide insights into protein engineering to improve enzyme efficiency. To extend the knowledge of the structural information on XI, the crystal structure of XI from Streptomyces avermitilis (SavXI) was determined at a 2.81 Å resolution. SavXI containing TIM barrel and extended α-helix domains formed the tetrameric assembly. The two metal-binding sites and their coordinating residues showed diverse conformations, providing the structural flexibility of the active site of SavXI. The structural comparison of SavXI and XI homologs exhibited unique metal-binding sites and conformations of the C-terminal α-helix domain. These structural results extend our knowledge of the molecular flexibility and mechanism of the XI family. Full article

We explored the functional redundancy of three structurally related KCTD (Potassium Channel Tetramerization Domain) proteins, KCTD2, KCTD5, and KCTD17, by progressively knocking them out in HEK 293 cells using CRISPR/Cas9 genome editing. After validating the knockout, we assessed the effects of progressive knockout on cell growth and gene expression. We noted that the progressive effects of knockout of KCTD isoforms on cell growth were most pervasive when all three isoforms were deleted, suggesting some functions were conserved between them. This was also reflected in progressive changes in gene expression. Our previous work indicated that Gβ1 was involved in the transcriptional control of gene expression, so we compared the gene expression patterns between GNB1 and KCTD KO. Knockout of GNB1 led to numerous changes in the expression levels of other G protein subunit genes, while knockout of KCTD isoforms had the opposite effect, presumably because of their role in regulating levels of Gβ1. Our work demonstrates a unique relationship between KCTD proteins and Gβ1 and a global role for this subfamily of KCTD proteins in maintaining the ability of cells to survive and proliferate. Full article

Bacillus thuringiensis Vip3 toxins form a tetrameric structure crucial for their insecticidal activity. Each Vip3Aa monomer comprises five domains. Interaction of the first four α-helices in domain I with the target cellular membrane was proposed to be a key step before pore formation. In this study, four N-terminal α-helix-deleted truncations of Vip3Aa were produced and, it was found that they lost both liposome permeability and insecticidal activity against Spodoptera litura. To further probe the role of domain I in membrane permeation, the full-length domain I and the fragments of N-terminal α-helix-truncated domain I were fused to green fluorescent protein (GFP), respectively. Only the fusion carrying the full-length domain I exhibited permeability against artificial liposomes. In addition, seven Vip3Aa-Cry1Ac fusions were also constructed by combination of α-helices from Vip3Aa domains I and II with the domains II and III of Cry1Ac. Five of the seven combinations were determined to show membrane permeability in artificial liposomes. However, none of the Vip3Aa-Cry1Ac combinations exhibited insecticidal activity due to the significant reduction in proteolytic stability. These results indicated that the N-terminal helix α1 in the Vip3Aa domain I is essential for both insecticidal activity and liposome permeability and that domain I of Vip3Aa preserved a high liposome permeability independently from domains II–V. Full article

KCTD ((K)potassium Channel Tetramerization Domain-containing) proteins constitute an emerging class of proteins involved in fundamental physio-pathological processes. In these proteins, the BTB domain, which represents the defining element of the family, may have the dual role of promoting oligomerization and favoring functionally important partnerships with different interactors. Here, by exploiting the potential of recently developed methodologies for protein structure prediction, we report a comprehensive analysis of the interactions of all KCTD proteins with their most common partner Cullin 3 (Cul3). The data here presented demonstrate the impressive ability of this approach to discriminate between KCTDs that interact with Cul3 and those that do not. Indeed, reliable and stable models of the complexes were only obtained for the 15 members of the family that are known to interact with Cul3. The generation of three-dimensional models for all KCTD–Cul3 complexes provides interesting clues on the determinants of the structural basis of this partnership as clear structural differences emerged between KCTDs that bind or do not bind Cul3. Finally, the availability of accurate three-dimensional models for KCTD–Cul3 interactions may be valuable for the ad hoc design and development of compounds targeting specific KCTDs that are involved in several common diseases. Full article

The serine/threonine protein kinase CK2 is implicated in the regulation of fundamental processes in eukaryotic cells. CK2 consists of two catalytic α or α’ isoforms and two regulatory CK2β subunits. These three proteins exist in a free form, bound to other cellular proteins, as tetrameric holoenzymes composed of CK2α₂/β₂, CK2αα’/β₂, or CK2α’₂/β₂ as well as in higher molecular forms of the tetramers. The catalytic domains of CK2α and CK2α’ share a 90% identity. As CK2α contains a unique C-terminal sequence. Both proteins function as protein kinases. These properties raised the question of whether both isoforms are just backups of each other or whether they are regulated differently and may then function in an isoform-specific manner. The present review provides observations that the regulation of both CK2α isoforms is partly different concerning the subcellular localization, post-translational modifications, and aggregation. Up to now, there are only a few isoform-specific cellular binding partners. The expression of both CK2α isoforms seems to vary in different cell lines, in tissues, in the cell cycle, and with differentiation. There are different reports about the expression and the functions of the CK2α isoforms in tumor cells and tissues. In many cases, a cell-type-specific expression and function is known, which raises the question about cell-specific regulators of both isoforms. Another future challenge is the identification or design of CK2α’-specific inhibitors. Full article

Regulatory adenine nucleotide-binding cystathionine β-synthase (CBS) domains are widespread in proteins; however, information on the mechanism of their modulating effects on protein function is scarce. The difficulty in obtaining structural data for such proteins is ascribed to their unusual flexibility and propensity to form higher-order oligomeric structures. In this study, we deleted the most movable domain from the catalytic part of a CBS domain-containing bacterial inorganic pyrophosphatase (CBS-PPase) and characterized the deletion variant both structurally and functionally. The truncated CBS-PPase was inactive but retained the homotetrameric structure of the full-size enzyme and its ability to bind a fluorescent AMP analog (inhibitor) and diadenosine tetraphosphate (activator) with the same or greater affinity. The deletion stabilized the protein structure against thermal unfolding, suggesting that the deleted domain destabilizes the structure in the full-size protein. A “linear” 3D structure with an unusual type of domain swapping predicted for the truncated CBS-PPase by Alphafold2 was confirmed by single-particle electron microscopy. The results suggest a dual role for the CBS domains in CBS-PPase regulation: they allow for enzyme tetramerization, which impedes the motion of one catalytic domain, and bind adenine nucleotides to mitigate or aggravate this effect. Full article

Bacillus thuringiensis (Bt) strains produce pore-forming toxins (PFTs) that attack insect pests. Information for pre-pore and pore structures of some of these Bt toxins is available. However, for the three-domain (I-III) crystal (Cry) toxins, the most used Bt toxins in pest control, this crucial information is still missing. In these Cry toxins, biochemical data have shown that 7-helix domain I is involved in insertion in membranes, oligomerization and formation of a channel lined mainly by helix α4, whereas helices α1 to α3 seem to have a dynamic role during insertion. In the case of Cry1Aa, toxic against Manduca sexta larvae, a tetrameric oligomer seems to precede membrane insertion. Given the experimental difficulty in the elucidation of the membrane insertion steps, we used Alphafold-2 (AF2) to shed light on possible oligomeric structural intermediates in the membrane insertion of this toxin. AF2 very accurately (<1 Å RMSD) predicted the crystal monomeric and trimeric structures of Cry1Aa and Cry4Ba. The prediction of a tetramer of Cry1Aa, but not Cry4Ba, produced an ‘extended model’ where domain I helices α3 and α2b form a continuous helix and where hydrophobic helices α1 and α2 cluster at the tip of the bundle. We hypothesize that this represents an intermediate that binds the membrane and precedes α4/α5 hairpin insertion, together with helices α6 and α7. Another Cry1Aa tetrameric model was predicted after deleting helices α1 to α3, where domain I produced a central cavity consistent with an ion channel, lined by polar and charged residues in helix α4. We propose that this second model corresponds to the ‘membrane-inserted’ structure. AF2 also predicted larger α4/α5 hairpin n-mers (14 ≤n ≤ 17) with high confidence, which formed even larger (~5 nm) pores. The plausibility of these models is discussed in the context of available experimental data and current paradigms. Full article

The p53 protein is a transcriptional regulatory factor and many of its functions require that it forms a tetrameric structure. Although the tetramerization domain of mammalian p53 proteins (p53TD) share significant sequence similarities, it was recently shown that the tree shrew p53TD is considerably more thermostable than the human p53TD. To determine whether other mammalian species display differences in this domain, we used biophysical, functional, and structural studies to compare the properties of the p53TDs from six mammalian model organisms (human, tree shrew, guinea pig, Chinese hamster, sheep, and opossum). The results indicate that the p53TD from the opossum and tree shrew are significantly more stable than the human p53TD, and there is a correlation between the thermostability of the p53TDs and their ability to activate transcription. Structural analysis of the tree shrew and opossum p53TDs indicated that amino acid substitutions within two distinct regions of their p53TDs can dramatically alter hydrophobic packing of the tetramer, and in particular substitutions at positions corresponding to F341 and Q354 of the human p53TD. Together, the results suggest that subtle changes in the sequence of the p53TD can dramatically alter the stability, and potentially lead to important changes in the functional activity, of the p53 protein. Full article