Asymmetric Structure of the Native Rhodobacter sphaeroides Dimeric LH1-RC Complex

The light-harvesting-reaction center (LH1-RC) core complex of purple photosynthetic bacterium Rhodobacter (Rba.) sphaeroides is characterized by the presence of both a dimeric form and a monomeric form. Following structure determination of the monomeric LH1-RC including its previously unrecognized component designated protein-U (Nat. Common.12, 6300, 2021), here we present cryo-EM structures of the dimeric LH1-RC from native Rba. sphaeroides IL106 at 2.75 Å resolution and from an LH1-RC monomer lacking protein-U (ΔU) at 2.64 Å resolution. The native dimeric core complex reveals many asymmetric features in the arrangement of its two monomeric components including the structural integrity of protein-U, the overall LH1 organization, and the rigidities of the proteins and pigments that form the complex. PufX polypeptides play a critical role in connecting two monomers, with one PufX interacting at its N-terminus with another PufX and an LH1 β-polypeptide in another monomer, in good agreement with biochemical analyses. One of the proteins-U was only partially identified in the dimeric structure, signaling significantly different degrees of disorder in the two monomers. The ΔU LH1-RC monomer revealed a half-moon-shaped structure containing 11 α- and 10 β-polypeptides (compared with 14 of each in the wild type), indicating a critical role for protein-U in controlling the number of αβ-subunits required for correct assembly and stabilization of the LH1-RC dimer. The structural features are discussed in relation to the unusual topology of intracytoplasmic photosynthetic membranes and an assembly model proposed for the native Rba. sphaeroides dimeric LH1-RC complex in membranes of wild-type cells.

Structural exploration with AlphaFold2-generated STAT3α structure reveals selective elements in STAT3α-GRIM-19 interactions involved in negative regulation

STAT3, an important transcription factor constitutively activated in cancers, is bound specifically by GRIM-19 and this interaction inhibits STAT3-dependent gene expression. GRIM-19 is therefore, considered as an inhibitor of STAT3 and may be an effective anti-cancer therapeutic target. While STAT3 exists in a dimeric form in the cytoplasm and nucleus, it is mostly present in a monomeric form in the mitochondria. Although GRIM-19-binding domains of STAT3 have been identified in independent experiments, yet the identified domains are not the same, and hence, discrepancies exist. Human STAT3-GRIM-19 complex has not been crystallised yet. Dictated by fundamental biophysical principles, the binding region, interactions and effects of hotspot mutations can provide us a clue to the negative regulatory mechanisms of GRIM-19. Prompted by the very nature of STAT3 being a challenging molecule, and to understand the structural basis of binding and interactions in STAT3α-GRIM-19 complex, we performed homology modelling and ab-initio modelling with evolutionary information using I-TASSER and avant-garde AlphaFold2, respectively, to generate monomeric, and subsequently, dimeric STAT3α structures. The dimeric form of STAT3α structure was observed to potentially exist in an anti-parallel orientation of monomers. We demonstrate that during the interactions with both unphosphorylated and phosphorylated STAT3α, the NTD of GRIM-19 binds most strongly to the NTD of STAT3α, in direct contrast to the earlier works. Key arginine residues at positions 57, 58 and 68 of GRIM-19 are mainly involved in the hydrogen-bonded interactions. An intriguing feature of these arginine residues is that these display a consistent interaction pattern across unphosphorylated and phosphorylated monomers as well as unphosphorylated dimers in STAT3α-GRIM-19 complexes. MD studies verified the stability of these complexes. Analysing the binding affinity and stability through free energy changes upon mutation, we found GRIM-19 mutations Y33P and Q61L and among GRIM-19 arginines, R68P and R57M, to be one of the top-most major and minor disruptors of binding, respectively. The proportionate increase in average change in binding affinity upon mutation was inclined more towards GRIM-19 mutants, leading to the surmise that GRIM-19 may play a greater role in the complex formation. These studies propound a novel structural perspective of STAT3α-GRIM-19 binding and inhibitory mechanisms in both the monomeric and dimeric forms of STAT3α as compared to that observed from the earlier experiments, these experimental observations being inconsistent among each other.

Biochemical characterization of a family IV esterase with R-form enantioselectivity from a compost metagenomic library

A novel family IV esterase (hormone-sensitive lipase, HSL) gene, est15L, was isolated from a compost metagenomic library. Encoded Est15L comprised 328 amino acids with a molecular weight of 34,770 kDa and was an intracellular esterase without a signal peptide. The multiple sequence alignment (MSA) of Est15L with other family IV esterases showed conserved regions such as HGG, DYR, GXSXG, DPL, and GXIH. Native Est15L was a dimeric form from the results of size exclusion chromatography. It was optimally active at 50 ℃ and pH 9.0, indicating alkaline esterase. However, it showed a low thermostability with half-lives of 30.3 at 30 ℃ and 2.7 min at 40 ℃. It preferred p-nitrophenyl butyrate (C4) with Km and Vmax values of 0.28 mM and 270.8 U/mg, respectively. Est15L was inhibited by organic solvents such as 30% methanol, isopropanol, and acetonitrile with residual activities of 12.5, 0.9, and 0.3%, respectively. It was also inhibited by 1% SDS and 1% PMSF; however, Est15L maintained its activity at 1% Triton X-100 and EDTA. Est15L was inhibited by Cu2+, Zn2+, Mn2+, Co2+, Fe2+, and Na+. In addition, Est15L hydrolyzed glyceryl tributyrate with a residual substrate amount of 43.7% at 60 min but could not hydrolyze the oils (fish and olive) and glyceryl trioleate. Interestingly, Est15L showed significant enantioselectivity toward the R-form with a residual substrate amount of 44.6%, lower than that of the S-form (83.5%). Considering its properties, Est15L can be a potential candidate for chemical reactions, such as the synthesis of pharmaceutical compounds.

β-Barrels and Amyloids: Structural Transitions, Biological Functions, and Pathogenesis

Insoluble protein aggregates with fibrillar morphology called amyloids and β-barrel proteins both share a β-sheet-rich structure. Correctly folded β-barrel proteins can not only function in monomeric (dimeric) form, but also tend to interact with one another—followed, in several cases, by formation of higher order oligomers or even aggregates. In recent years, findings proving that β-barrel proteins can adopt cross-β amyloid folds have emerged. Different β-barrel proteins were shown to form amyloid fibrils in vitro. The formation of functional amyloids in vivo by β-barrel proteins for which the amyloid state is native was also discovered. In particular, several prokaryotic and eukaryotic proteins with β-barrel domains were demonstrated to form amyloids in vivo, where they participate in interspecies interactions and nutrient storage, respectively. According to recent observations, despite the variety of primary structures of amyloid-forming proteins, most of them can adopt a conformational state with the β-barrel topology. This state can be intermediate on the pathway of fibrillogenesis (“on-pathway state”), or can be formed as a result of an alternative assembly of partially unfolded monomers (“off-pathway state”). The β-barrel oligomers formed by amyloid proteins possess toxicity, and are likely to be involved in the development of amyloidoses, thus representing promising targets for potential therapy of these incurable diseases. Considering rapidly growing discoveries of the amyloid-forming β-barrels, we may suggest that their real number and diversity of functions are significantly higher than identified to date, and represent only “the tip of the iceberg”. Here, we summarize the data on the amyloid-forming β-barrel proteins, their physicochemical properties, and their biological functions, and discuss probable means and consequences of the amyloidogenesis of these proteins, along with structural relationships between these two widespread types of β-folds.

A Novel Sesquiterpene Lactone Xanthatin-13-(pyrrolidine-2-carboxylic acid) Isolated from Burdock Leaf Up-Regulates Cells’ Oxidative Stress Defense Pathway

The aim of our study was to identify novel molecules able to induce an adaptative response against oxidative stress during the first stages of metabolic syndrome. A cellular survival in vitro test against H2O2-based test was applied after pretreatment with various natural bitter Asteraceae extracts. This screening revealed potent protection from burdock leaf extract. Using chromatography and LC-MS—RMN, we then isolated and identified an original sesquiterpene lactone bioactive molecule: the Xanthatin-13-(pyrrolidine-2-carboxylic acid) (XPc). A real-time RT-qPCR experiment was carried out on three essential genes involved in oxidative stress protection: GPx, SOD, and G6PD. In presence of XPc, an over-expression of the G6PD gene was recorded, whereas no modification of the two others genes could be observed. A biochemical docking approach demonstrated that XPc had a high probability to directly interact with G6PD at different positions. One of the most probable docking sites corresponds precisely to the binding site of AG1, known to stabilize the G6PD dimeric form and enhance its activity. In conclusion, this novel sesquiterpene lactone XPc might be a promising prophylactic bioactive agent against oxidative stress and inflammation in chronic diseases such as metabolic syndrome or type 2 diabetes.

Allergic Inflammation Caused by Dimerized Translationally Controlled Tumor Protein is Attenuated by Cardamonin

We demonstrated in our previous reports that dimeric form of translationally controlled tumor protein (dTCTP) initiates a variety of allergic phenomena. In the present study, we examined whether and how dTCTP’s role in allergic inflammation can be modulated or negated. The possible potential of cardamonin as an anti-allergic agent was assessed by ELISA using BEAS-2B cells and OVA-challenged allergic mouse model. The interaction between cardamonin and dTCTP was confirmed by SPR assay. Cardamonin was found to reduce the secretion of IL-8 caused by dTCTP in BEAS-2B cells by interacting with dTCTP. This interaction between dTCTP and cardamonin was confirmed through kinetic analysis (KD = 4.72 ± 0.07 μM). Also, cardamonin reduced the migration of various inflammatory cells in the bronchoalveolar lavage fluid (BALF), inhibited OVA specific IgE secretion and bronchial remodeling. In addition, cardamonin was observed to have an anti-allergic response by inhibiting the activity of NF-κB. Cardamonin exerts anti-allergic anti-inflammatory effect by inhibiting dTCTP, suggesting that it may be useful in the therapy of allergic diseases.

Structural architecture of a dimeric paramyxovirus polymerase complex

Human parainfluenza virus type 3 (hPIV3), a member of non-segmented, negative-strand RNA viruses (nsNSVs), is the second most common cause of severe respiratory disease in pediatrics. The transcription and replication processes of nsNSVs are catalyzed by a multi-functional RNA-dependent RNA polymerase (RdRp) complex composed of the large protein (L) and the phosphoprotein (P). Previous studies have shown that the polymerase can adopt a dimeric form, however, the structure of the dimer and how it functions are not understood. Here we determined the cryo-EM structure of hPIV3 L-P complex at 2.7 Å with substantial structural details. A putative catalytic magnesium ion could be built in our structure, and structural comparison revealed atomic features conserved with other RNA viruses. Interactions identified between the two priming and intrusion loops and the connector domain potentially trigger the spatial movement of three C-terminal L domains for different steps of transcription and replication. Structural comparison with other nsNSV RdRps suggests common features of L-P binding. Furthermore, we report for the first time the structural basis of the L-L interaction in the partially modelled dimeric L-P structure, in which the connector domain of one L is positioned at the putative RNA template entry of the other L. Based on these findings, we propose a model by which L dimerization promotes efficient conversion of nascent RNA into a template.

The structure of a dimeric form of SARS-CoV-2 polymerase

The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe its genome. Previous structures of the RdRp revealed a monomeric enzyme composed of the catalytic subunit nsp12, two copies of subunit nsp8, and one copy of subunit nsp7. Here we report an alternative, dimeric form of the enzyme and resolve its structure at 5.5 Å resolution. In this structure, the two RdRps contain only one copy of nsp8 each and dimerize via their nsp7 subunits to adopt an antiparallel arrangement. We speculate that the RdRp dimer facilitates template switching during production of sub-genomic RNAs.

Crystal Structure and Cellular Functions of uPAR Dimer

Receptor dimerization of urokinase-type plasminogen activator receptor (uPAR) was previously identified at protein level and on the cell surface. Recently, a dimeric form of mouse uPAR isoform 2 was proposed, which induced kidney disease. Here, we report the crystal structure of human uPAR dimer at 2.96 Å. The structure reveals enormous conformational changes of the dimer compared to the monomeric structure: D1 of uPAR opens up into a large expanded loop that captures a β-hairpin loop of a neighboring uPAR to form an expanded β-sheet, leading to an elongated, highly intertwined dimeric uPAR. Based on the structure, we identify the E49P mutation promoting dimer formation. The mutation increases receptor binding to amino terminal fragment (ATF) of its primary ligand uPA, induces the receptor to distribute to the basal membrane, promotes cell proliferation, and alters cell morphology via the ERK activation of β1 integrin signaling. These results reveal the structural basis for uPAR dimerization, its effect on cell function, and provide new insight and tools to study this multifunctional receptor.

The dimeric form of CXCL12 binds to atypical chemokine receptor 1

The pleiotropic chemokine CXCL12 is involved in diverse physiological and pathophysiological processes, including embryogenesis, hematopoiesis, leukocyte migration, and tumor metastasis. It is known to engage the classical receptor CXCR4 and the atypical receptor ACKR3. Differential receptor engagement can transduce distinct cellular signals and effects as well as alter the amount of free, extracellular chemokine. CXCR4 binds both monomeric and the more commonly found dimeric forms of CXCL12, whereas ACKR3 binds monomeric forms. Here, we found that CXCL12 also bound to the atypical receptor ACKR1 (previously known as Duffy antigen/receptor for chemokines or DARC). In vitro nuclear magnetic resonance spectroscopy and isothermal titration calorimetry revealed that dimeric CXCL12 bound to the extracellular N terminus of ACKR1 with low nanomolar affinity, whereas the binding affinity of monomeric CXCL12 was orders of magnitude lower. In transfected MDCK cells and primary human Duffy-positive erythrocytes, a dimeric, but not a monomeric, construct of CXCL12 efficiently bound to and internalized with ACKR1. This interaction between CXCL12 and ACKR1 provides another layer of regulation of the multiple biological functions of CXCL12. The findings also raise the possibility that ACKR1 can bind other dimeric chemokines, thus potentially further expanding the role of ACKR1 in chemokine retention and presentation.