Tetrameric Complex Research Articles

Study of p-(3-carboxymethyl-1-adamantyl)calix[4]arene and tetrapropoxy-p-(3-carboxymethyl-1-adamantyl)calix[4]arene by vibrational spectroscopy and DFT

The IR and Raman spectra of p-(3-carboxymethyl-1-adamantyl)calix[4]arene (1) and tetrapropoxy-p-(3-carboxymethyl-1-adamantyl)calix[4]arene (2) were studied. In the IR spectra of crystals and solutions of calixarenes, there is no band of stretching vibrations of free OH groups. In the IR spectrum of compound 1 in the crystalline state, an intense wide band is observed at 3187 cm−1. In the IR spectrum of a dilute solution in CCl4, this band shifts to 3118 cm−1. The carboxyl groups form cyclic dimer or tetramer complexes.The formation of dimers of carboxyl groups is more favorable than for cyclic tetramers. The energy difference is 33.8 and 53.0 kJ/mol in compounds 1 and 2, respectively. The calculation showed that molecules 1 and 2 of dimeric and tetrameric complexes assume the cone conformation. The molecules of compound 2 are in the cone conformation even in the absence of hydrogen bonds along the lower rim.

Abstract IA-022: Lysine 68 acetylation directs MnSOD as a tetrameric detoxification complex versus a monomeric tumor promoter

Abstract Manganese superoxide dismutase (MnSOD) functions as a tumor suppressor; however, once tumorigenesis occurs, clinical data suggest MnSOD levels correlate with more aggressive human tumors, implying a potential dual function of MnSOD in the regulation of metabolism. Here we show, using in vitro transformation and xenograft growth assays that the MnSOD-K68 acetylation (Ac) mimic mutant (MnSODK68Q) functions as a tumor promoter. Interestingly, in various breast cancer and primary cell types the expression of MnSODK68Q is accompanied with a change of MnSOD’s stoichiometry from a known homotetramer complex to a monomeric form. Biochemical experiments using the MnSOD-K68Q Ac-mimic, or physically K68-Ac (MnSOD-K68-Ac), suggest that these monomers function as a peroxidase, distinct from the established MnSOD superoxide dismutase activity. MnSODK68Q expressing cells exhibit resistance to tamoxifen (Tam) and cells selected for Tam resistance exhibited increased K68-Ac and monomeric MnSOD. These results suggest a MnSOD-K68-Ac metabolic pathway for Tam resistance, carcinogenesis and tumor progression. Citation Format: David Gius. Lysine 68 acetylation directs MnSOD as a tetrameric detoxification complex versus a monomeric tumor promoter [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-022.

Direct Assembly in Aqueous Solutions of Stable Chlorophyllide Complexes with Type II Water-soluble Chlorophyll Proteins.

Water-soluble chlorophyll-binding proteins (WSCPs) from Brassicaceae constitute a small family of non-photosynthetic proteins that may provide a useful benchmark and model system for studying molecular aspects of chlorophyll-protein interactions such as the tuning of absorption and emission spectra, and binding selectivity. WSCP apo-proteins are readily expressed by recombinant DNA techniques and can be assembled in vitro with natural and synthetic chlorophyll derivatives. The complexes with native chlorophylls are exceptionally stable toward thermal dissociation and protein denaturation due to hydrophobic interactions with the chlorophyll's phytyl chains that stabilize the core of the WSCP tetrameric complexes. However, assembly requires the use of detergents or water-in-oil emulsions to introduce the hydrophobic pigments into the water-soluble apo-proteins. Here, we explore the direct assembly of recombinant WSCPs with the water-soluble phytyl-free chlorophyll analogue chlorophyllide a in aqueous solutions. We show that the complexes formed by mixing chlorophyllide and WSCP apo-proteins are exclusively tetrameric, and while they lack the extreme thermostability of the respective chlorophyll complexes, they are still thermostable up to around 60°C. Their absorption and CD spectra are very similar to the chlorophyll complexes albeit slight peak shifts and broadening of the bands indicate variations in pigment and protein conformations, and less rigid structures. Simplifying the assembly process of WSCPs opens new possibilities for their use in modelling natural chlorophyll-protein complexes, and as templates for designing novel artificial protein-pigment complexes.

A Resource of High-Quality Nanobodies for Drug Delivery

Therapeutic and diagnostic efficacies of numerous small biomolecules and chemical compounds are hampered by poor pharmacokinetics. Here we developed a repertoire of distinct and high-affinity albumin-nanobodies (NbHSA) to facilitate drug delivery. Using biophysics and hybrid structural methods, we have systematically characterized the Nb repertoire, mapped the epitopes, and resolved the architecture of a tetrameric Nb-albumin complex. We employed quantitative proteomics for accurate and multiplex pharmacokinetic analysis of dozens of diverse and high-quality NbHSA and confirmed the most stable construct has a 771-fold T1/2 improvement compared to non-albumin binding Nbs. Interestingly, the pharmacokinetics of NbHSA is related to their biophysical and structural properties. To demonstrate the utility of NbHSA, we developed stable NbHSA-cytokine conjugates “Duraleukins” and confirmed the high anticancer efficacy of a Duraluekin in vivo. This high-quality Nb resource may help advance research into novel biotherapeutics.

The Use of Enhanced Sampling Molecular Dynamics (ES-MD) to Determine PIP2 Binding in SK2-Calmodulin Complex

Small conductance Ca2+-activated K+ (SK) channels are unique since they are gated solely by changes in intracellular Ca2+ and are known to be upregulated in heart failure and cardiac arrhythmias. SK channels are regulated via the ubiquitous Ca2+ sensing protein, calmodulin (CaM). New evidence suggests that the anionic lipid phosphatidylinositol 4,5-bisphosphate (PIP2) is also essential for the activation for SK channels. Recent cryo-EM structures with a nearly complete tetrameric SK4-CaM complex (PDB: 6CNM, 6CNN, 6CNO) allowed us to generate nearly full-length SK2-CaM homology models (fSK2-CaM). In addition to PIP2 binding sites previously predicted in a crystal structure of SK2 peptide complexed with CaM (PDB: 4QNH), we have identified areas of basic amino acid clustering in the fSK2-CaM structure around the lower leaflet of the surrounding lipid bilayer. Dockings of PIP2 onto fSK2-CaM were performed to assess possible PIP2 binding sites. We further used all-atom enhanced sampling molecular dynamics (ES-MD) simulations on Frontera CPU nodes to refine the proposed binding sites for 3 separate states of fSK2-CaM (closed, open/inactivated, and open/activated). Orientation of PIP2 in the binding sites, corresponding binding affinities and mechanisms of channel activation were determined from ES-MD runs and further tested biochemically. The use of ES-MD has provided novel insights into PIP2 binding and regulation of SK2-CaM complexes.

Abstract PR009: The functional genomic landscape of recurrent glioblastoma

Abstract Glioblastoma (GBM) is a highly fatal brain cancer where mortality is predominantly caused by disease recurrence and lack of second-line therapies. Here, utilizing genome-wide CRISPR-Cas9 screening, we uncover treatment-based conditional genetic interactions modulating sensitivity to combined radiation therapy (RT) and temozolomide (TMZ), the standard of care treatment administered to over 70% of glioblastoma patients. To investigate therapy-induced changes in the functional landscape of glioblastoma stem cells (GSCs) without confounding patient-to-patient differences, we systematically mapped genetic dependencies in patient-matched pre-treatment primary and post-treatment recurrent GSCs. This comparison highlights a remodelling of genes and pathways regulating tumor cell survival at disease relapse, arming recurrent GSCs with newly acquired genetic drivers and further loss of tumor suppressors. Among these genes, we identify PTP4A2, encoding protein tyrosine phosphatase 4A2, as a major regulator of stemness and tumorigenicity in recurrent GSCs. Genetic perturbation or pharmacological inhibition of PTP4A2 influences the phosphorylation status and expression of interacting proteins belonging to axonal guidance pathways, as phosphoproteomic studies showed specific enrichment in axon guidance regulator Roundabout Guidance Receptor 1 (ROBO1) in recurrent GSCs. We therefore designed and developed efficacious Camelid biotinylated single-domain antibody (bio-sdAb) and tetrameric complexes targeting human ROBO1 (MKRo-20). Subsequently, we orthotopically engrafted recurrent GSCs into immunocompromised mice and administered local treatments of tetrameric MKRo-20. All mice treated with tetrameric MKRo-20 were rendered tumor free, suggesting that modulation of ROBO1 with bio-sdAbs presents a new immunotherapeutic strategy to target recurrent glioblastoma. Our work reveals a context-specific dependence on axonal guidance through a PTP4A2-ROBO axis in recurrent glioblastoma, highlighting new avenues of therapeutic intervention for second-line therapy in GBM. This abstract is also being presented as PO025. Citation Format: Chirayu Chokshi, David Tieu, Kevin Brown, Chitra Venugopal, Lina Liu, Laura Kuhlman, Katherine Chan, Amy Tong, Neil Savage, Dillon McKenna, Nikoo Aghaei, Minomi Subapanditha, Joseph M Salamoun, Martin Rossotti, Peter Wipf, Elizabeth Sharlow, John S. Lazo, Thomas Kislinger, Kevin Henry, Yu Lu, Jason Moffat, Sheila K. Singh. The functional genomic landscape of recurrent glioblastoma [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR009.

On the Oxidation of [Ge9]4− – Crystal Structures and Raman Spectroscopic Investigation of Linked Ge9 Clusters

AbstractOxidation of Ge9 Zintl clusters can afford condensed cluster species in the form of dimers, oligomers and polymers. In this work, we present a new route for the synthesis of Ge9 tetramers present in the compound [K(18‐crown‐6)]8[Ge9=Ge9=Ge9=Ge9] ⋅ 4 pyr. The reaction of a pyridine solution of K4Ge9 with 18‐crown‐6 first leads to the formation of green needles of yet unknown composition, which convert into the reported tetramer over time. In this reaction pyridine acts as oxidation reagent. Raman studies on the green needles as well as on tetrameric and polymeric Ge9 feature complex vibrational patterns. Furthermore, we present the crystal structure of a new type of Ge9 dimer in the compound K4[K(2.2.2‐crypt)]2[Ge9−Ge9] ⋅ 4 en ⋅ 2 thf, which represents an intermediate between the two established conformers.

Allosteric effects of SSB C-terminal tail on assembly of E. coli RecOR proteins.

Escherichia coli RecO is a recombination mediator protein that functions in the RecF pathway of homologous recombination, in concert with RecR, and interacts with E. coli single stranded (ss) DNA binding (SSB) protein via the last 9 amino acids of the C-terminal tails (SSB-Ct). Structures of the E. coli RecR and RecOR complexes are unavailable; however, crystal structures from other organisms show differences in RecR oligomeric state and RecO stoichiometry. We report analytical ultracentrifugation studies of E. coli RecR assembly and its interaction with RecO for a range of solution conditions using both sedimentation velocity and equilibrium approaches. We find that RecR exists in a pH-dependent dimer-tetramer equilibrium that explains the different assembly states reported in previous studies. RecO binds with positive cooperativity to a RecR tetramer, forming both RecR4O and RecR4O2 complexes. We find no evidence of a stable RecO complex with RecR dimers. However, binding of RecO to SSB-Ct peptides elicits an allosteric effect, eliminating the positive cooperativity and shifting the equilibrium to favor a RecR4O complex. These studies suggest a mechanism for how SSB binding to RecO influences the distribution of RecOR complexes to facilitate loading of RecA onto SSB coated ssDNA to initiate homologous recombination.

Temperature effect on water dynamics in tetramer phosphofructokinase matrix and the super-arrhenius respiration rate

Advances in understanding the temperature effect on water dynamics in cellular respiration are important for the modeling of integrated energy processes and metabolic rates. For more than half a century, experimental studies have contributed to the understanding of the catalytic role of water in respiration combustion, yet the detailed water dynamics remains elusive. We combine a super-Arrhenius model that links the temperature-dependent exponential growth rate of a population of plant cells to respiration, and an experiment on isotope labeled 18O2 uptake to H218O transport role and to a rate-limiting step of cellular respiration. We use Phosphofructokinase (PFK-1) as a prototype because this enzyme is known to be a pacemaker (a rate-limiting enzyme) in the glycolysis process of respiration. The characterization shows that PFK-1 water matrix dynamics are crucial for examining how respiration (PFK-1 tetramer complex breathing) rates respond to temperature change through a water and nano-channel network created by the enzyme folding surfaces, at both short and long (evolutionary) timescales. We not only reveal the nano-channel water network of PFK-1 tetramer hydration topography but also clarify how temperature drives the underlying respiration rates by mapping the channels of water diffusion with distinct dynamics in space and time. The results show that the PFK-1 assembly tetramer possesses a sustainable capacity in the regulation of the water network toward metabolic rates. The implications and limitations of the reciprocal-activation–reciprocal-temperature relationship for interpreting PFK-1 tetramer mechanisms are briefly discussed.

Distance-based measurement determines the coexistence of B protein hetero- and homodimers in lily tepal and stamen tetrameric complexes.

The floral quartet model proposes that plant MADS box proteins function as higher order tetrameric complexes. However, in planta evidence for MADS box tetramers remains scarce. Here, we applied a strategy using in vivo fluorescence resonance energy transfer (FRET) based on thedistance change and distance symmetry of stable tetrameric complexes in tobacco (Nicotiana benthamiana) leaf cells to improve the accuracy of the estimation of heterotetrameric complex formation. This measuring system precisely verified the stable state of Arabidopsis petal (AP3/PI/SEP3/AP1) and stamen (AP3/PI/SEP3/AG) complexes and showed that the lily (Lilium longiflorum) PI co-orthologs LMADS8 and LMADS9 likely formed heterotetrameric petal complexes with Arabidopsis AP3/SEP3/AP1, which rescued petal defects of pi mutants. However, L8/L9 did not form heterotetrameric stamen complexes with Arabidopsis AP3/SEP3/AG to rescue the stamen defects of the pi mutants. Importantly, this system was applied successfully to find complicated tepal and stamen heterotetrameric complexes in lily. We found that heterodimers of B function AP3/PI orthologs (L1/L8) likely coexist with the homodimers of PI orthologs (L8/L8, L9/L9) to form five (two most stable and three stable) tepal- and four (one most stable and three stable) stamen-related heterotetrameric complexes with A/E and C/E function proteins in lily. Among these combinations, L1 preferentially interacted with L8 to form the most stable heterotetrameric complexes, and the importance of the L8/L8 and L9/L9 homodimers in tepal/stamen formation in lily likely decreased to a minor part during evolution. The system provides substantial improvements for successfully estimating the existence of unknown tetrameric complexes in plants.

Synthesis, oligomerization and catalytic studies of a redox-active Ni4-cubane: a detailed mechanistic investigation.

A robust tetrameric nickel complex [Ni4((Oal−)2L-Me)4(s)4] (3) (s = solvent) with cubane-like Ni4O4 core topology was isolated as a light greenish-orange crystalline solid in excellent yield. The mechanism of formation of 3 involving the two chloride-containing precursors [Ni4((Oal−)2L-Me)4(s)4]·2MeOH (1) and [Ni4((O−)2L-Me)3((Oal−)(OH)L-Me)Cl] (2) was studied by ESI mass spectrometry and confirmed by the solid state isolation and single-crystal X-ray diffraction. The challenging ligand fields containing mono/di-anionic O2N donating atoms and/or chloride ions stabilized the pentacoordinate Ni(ii) ions in 1–2 upon controlling the experimental conditions. Complexes 1–3 have been characterized by NMR, UV-Vis and mass spectrometric analysis. Complex 3 was found to be redox active by cyclic voltammetry (CV) studies. Theoretical calculations were carried out to shed light on the effects of ligand fields on the stability of complexes 1–3. Complex 3 was found to be a potential catalyst for the diastereoselective cyclopropanation of heteroarenes with good to excellent yields. The ESI mass spectrometric analysis revealed the existence of solution dynamics and oligomerization of 3 in solution. Mechanistic investigation of the catalytic cycle revealed that complex 3 and its various oligomers bind to the diazoester employed, followed by dissociative insertion of the respective carbene moieties to the C2–C3 double bond of the involved aromatic heterocycle, leading to the diastereoselective cyclopropanation.

Amphipol-facilitated elucidation of the functional tetrameric complex of full-length cytochrome P450 CYP2B4 and NADPH-cytochrome P450 oxidoreductase

Interactions of membrane-bound mammalian cytochromes P450 (CYPs) with NADPH-cytochrome P450 oxidoreductase (POR), which are required for metabolism of xenobiotics, are facilitated by membrane lipids. A variety of membrane mimetics, such as phospholipid liposomes and nanodiscs, have been used to simulate the membrane to form catalytically active CYP:POR complexes. However, the exact mechanism(s) of these interactions are unclear because of the absence of structural information of full-length mammalian CYP:POR complexes in membranes. Herein, we report the use of amphipols (APols) to form a fully functional, soluble, homogeneous preparation of full-length CYP:POR complexes amenable to biochemical and structural study. Incorporation of CYP2B4 and POR into APols resulted in a CYP2B4:POR complex with a stoichiometry of 1:1, which was fully functional in demethylating benzphetamine at a turnover rate of 37.7 ± 2.2 min−1, with a coupling efficiency of 40%. Interestingly, the stable complex had a molecular weight (Mw) of 338 ± 22 kDa determined by multiangle light scattering, suggestive of a tetrameric complex of 2CYP2B4:2POR embedded in one APol nanoparticle. Moreover, negative stain electron microscopy (EM) validated the homogeneity of the complex and allowed us to generate a three-dimensional EM map and model consistent with the tetramer observed in solution. This first report of the full-length mammalian CYP:POR complex by transmission EM not only reveals the architecture that facilitates electron transfer but also highlights a potential use of APols in biochemical and structural studies of functional CYP complexes with redox partners.

ATR activation is regulated by dimerization of ATR activating proteins

The checkpoint kinase ATR regulates DNA repair, cell cycle progression, and other DNA damage and replication stress responses. ATR signaling is stimulated by an ATR activating protein, and in metazoan cells, there are at least two ATR activators: TOPBP1 and ETAA1. Current evidence indicates TOPBP1 and ETAA1 activate ATR via the same biochemical mechanism, but several aspects of this mechanism remain undefined. For example, ATR and its obligate binding partner ATR interacting protein (ATRIP) form a tetrameric complex consisting of two ATR and two ATRIP molecules, but whether TOPBP1 or ETAA1 dimerization is similarly required for ATR function is unclear. Here, we show that fusion of the TOPBP1 and ETAA1 ATR activation domains (AADs) to dimeric tags makes them more potent activators of ATR in vitro. Furthermore, induced dimerization of both AADs using chemical dimerization of a modified FKBP tag enhances ATR kinase activation and signaling in cells. ETAA1 forms oligomeric complexes mediated by regions of the protein that are predicted to be intrinsically disordered. Induced dimerization of a “mini-ETAA1” protein that contains the AAD and Replication Protein A (RPA) interaction motifs enhances ATR signaling, rescues cellular hypersensitivity to DNA damaging agents, and suppresses micronuclei formation in ETAA1-deficient cells. Together, our results indicate that TOPBP1 and ETAA1 dimerization is important for optimal ATR signaling and genome stability.

Dysfunction of Caveolae-Mediated Endocytic TβRI Degradation Results in Hypersensitivity of TGF-β/Smad Signaling in Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is a genetic disorder caused by mutations of type I collagen-related genes, and excessive transforming growth factor-beta (TGF-β) signaling is a common mechanism. TGF-β/Smad signaling has inhibitory effects on osteoblast differentiation and maturation and is mainly transduced and regulated by the internalization of a tetrameric receptor complex comprising types I and II TGF-β receptors (TβRI and TβRII). During internalization, clathrin-mediated endocytosis enhances TGF-β/Smad signaling via Smad2/3 phosphorylation and receptors recycling, while caveolae-mediated endocytosis turns off TGF-β/Smad signaling by promoting receptor ubiquitination and degradation. In this study, using an animal model of OI (Colla2oim , osteogenesis imperfecta murine [oim]/oim mouse), we found that osteoblastic cells of oim/oim mice were more sensitive to the inhibitory effects of TGF-β on osteoblast differentiation and maturation and had much higher cell membrane protein levels of TGF-β receptors than those of wild-type (wt)/wt mice. Further results showed that clathrin-mediated endocytosis of TβRI was enhanced, whereas caveolae-mediated TβRI endocytic degradation was reduced in oim/oim mice, combined with reduced caveolin-1 (Cav-1) phosphorylation. In addition, type I collagen downregulated TβRI via focal adhesion kinase (FAK) and Src activation-dependent Cav-1 phosphorylation. To further examine this mechanism, 4-week-old oim/oim and wt/wt mice were treated with either TβRI kinase inhibitor (SD-208) or vehicle for 8 weeks. SD-208 treatment significantly reduced the fracture incidence in oim/oim mice. Micro-computed tomography and biomechanical testing showed that femoral bone mass and strength were significantly improved with SD-208 treatment in both genotypes. Additionally, SD-208 significantly promoted osteoblast differentiation and bone formation and inhibited bone resorption. In conclusion, dysfunction of caveolae-mediated endocytic TβRI degradation is a possible mechanism for the enhanced TGF-β/Smad signaling in OI. Targeting this mechanism using a TβRI kinase inhibitor effectively reduced fractures and improved bone mass and strength in OI model and, thus, may offer a new strategy for the treatment of OI. © 2022 American Society for Bone and Mineral Research (ASBMR).

Phylogenetics-based identification and characterization of a superior 2,3-butanediol dehydrogenase for Zymomonas mobilis expression

BackgroundZymomonas mobilis has recently been shown to be capable of producing the valuable platform biochemical, 2,3-butanediol (2,3-BDO). Despite this capability, the production of high titers of 2,3-BDO is restricted by several physiological parameters. One such bottleneck involves the conversion of acetoin to 2,3-BDO, a step catalyzed by 2,3-butanediol dehydrogenase (Bdh). Several Bdh enzymes have been successfully expressed in Z. mobilis, although a highly active enzyme is yet to be identified for expression in this host. Here, we report the application of a phylogenetic approach to identify and characterize a superior Bdh, followed by validation of its structural attributes using a mutagenesis approach.ResultsOf the 11 distinct bdh genes that were expressed in Z. mobilis, crude extracts expressing Serratia marcescens Bdh (SmBdh) were found to have the highest activity (8.89 µmol/min/mg), when compared to other Bdh enzymes (0.34–2.87 µmol/min/mg). The SmBdh crystal structure was determined through crystallization with cofactor (NAD+) and substrate (acetoin) molecules bound in the active site. Active SmBdh was shown to be a tetramer with the active site populated by a Gln247 residue contributed by the diagonally opposite subunit. SmBdh showed a more extensive supporting hydrogen-bond network in comparison to the other well-studied Bdh enzymes, which enables improved substrate positioning and substrate specificity. This protein also contains a short α6 helix, which provides more efficient entry and exit of molecules from the active site, thereby contributing to enhanced substrate turnover. Extending the α6 helix to mimic the lower activity Enterobacter cloacae (EcBdh) enzyme resulted in reduction of SmBdh function to nearly 3% of the total activity. In great contrast, reduction of the corresponding α6 helix of the EcBdh to mimic the SmBdh structure resulted in ~ 70% increase in its activity.ConclusionsThis study has demonstrated that SmBdh is superior to other Bdhs for expression in Z. mobilis for 2,3-BDO production. SmBdh possesses unique structural features that confer biochemical advantage to this protein. While coordinated active site formation is a unique structural characteristic of this tetrameric complex, the smaller α6 helix and extended hydrogen network contribute towards improved activity and substrate promiscuity of the enzyme.

Alkaline Earth Metal Imido Complexes with Doubly Deprotonated Amidine and β‐Diketimine Ligands

Double deprotonation of the amidine DIPP‐N=C(tBu)‐NH2 or β‐diketimine DIPP‐N=C(Me)‐C(H)=C(Me)‐NH2 (DIPP = 2,6‐diisopropylphenyl) with strong benzylcalcium or strontium bases gave metal imido complexes with the anions DIPP‐N=C(tBu)‐N2– (Am2–) and DIPP‐N=C(Me)‐C(H)=C(Me)‐N2– (BDI2–) which crystallized as tetrameric complexes with typical Ca4N4 or Sr4N4 cubane frameworks. Crystal structures of [(Am)Ca·THF]4, [(BDI)Ca·THF]4 and the first Sr imido complex [(Am)Sr·THF]4 are presented. Calculated geometries of [(Am)Ca·THF]4 and [(BDI)Ca·THF]4 (B3PW91/6‐311++G**) are in good agreement with the crystal structures. Also complexes with monoanionic ligands (Am‐H)– and (BDI‐H)– are reported. Charge delocalization in the ligand backbone is discussed. Ligand–metal bonds are calculated to be circa 90 % ionic; the NPA charges on Ca is circa +1.8. The negative charge on the ligand is delocalized over the ligand backbone but there is still considerable electron density on the terminal N (–1.4). Despite this high negative charge, the reactivity of these complexes is generally low due to the strongly bound cubane core. Reaction of [(Am)Ca·THF]4 with phenyl cyanide gave the [(Am)Ca·PhCN]4, clearly demonstrating its low reactivity. Attempts to break the tetrameric cluster in smaller aggregates by addition of 18‐crown‐6 led to protonation and isolation of (Am‐H)2Ca·(18‐crown‐6). Reaction of [(Am)Ca·THF]4 with iPrN=C=NiPr gave after addition a complex with a unique amido‐guanidinate ligand.

DNA binding induces a cis-to-trans switch in Cre recombinase to enable intasome assembly

Mechanistic understanding of DNA recombination in the Cre-loxP system has largely been guided by crystallographic structures of tetrameric synaptic complexes. Those studies have suggested a role for protein conformational dynamics that has not been well characterized at the atomic level. We used solution nuclear magnetic resonance (NMR) spectroscopy to discover the link between intrinsic flexibility and function in Cre recombinase. Transverse relaxation-optimized spectroscopy (TROSY) NMR spectra show the N-terminal and C-terminal catalytic domains (CreNTD and CreCat) to be structurally independent. Amide 15N relaxation measurements of the CreCat domain reveal fast-timescale dynamics in most regions that exhibit conformational differences in active and inactive Cre protomers in crystallographic tetramers. However, the C-terminal helix αN, implicated in assembly of synaptic complexes and regulation of DNA cleavage activity via trans protein-protein interactions, is unexpectedly rigid in free Cre. Chemical shift perturbations and intra- and intermolecular paramagnetic relaxation enhancement (PRE) NMR data reveal an alternative autoinhibitory conformation for the αN region of free Cre, wherein it packs in cis over the protein DNA binding surface and active site. Moreover, binding to loxP DNA induces a conformational change that dislodges the C terminus, resulting in a cis-to-trans switch that is likely to enable protein-protein interactions required for assembly of recombinogenic Cre intasomes. These findings necessitate a reexamination of the mechanisms by which this widely utilized gene-editing tool selects target sites, avoids spurious DNA cleavage activity, and controls DNA recombination efficiency.

MEAF6 is essential for cell proliferation and plays a role in the assembly of KAT7 complexes

Myst family genes encode lysine acetyltransferases that mainly mediate histone acetylation to control transcription, DNA replication and DNA damage response. They form tetrameric complexes with PHD-finger proteins (Brpfs or Jades) and small non-catalytic subunits Ing4/5 and Meaf6. Although all the components of the complex are well-conserved from yeast to mammals, the function of Meaf6 and its homologs has not been elucidated in any species. Here we revealed the role of Meaf6 utilizing inducible Meaf6 KO ES cells. By elimination of Meaf6, proliferation ceased although histone acetylations were largely unaffected. In the absence of Meaf6, one of the Myst family members Myst2/Kat7 increased the ability to interact with PHD-finger proteins. This study is the first indication of the function of Meaf6, which shows it is not essential for HAT activity but modulates the assembly of the Kat7 complex.

Genome-wide binding of SEPALLATA3 and AGAMOUS complexes determined by sequential DNA-affinity purification sequencing.

The MADS transcription factors (TF), SEPALLATA3 (SEP3) and AGAMOUS (AG) are required for floral organ identity and floral meristem determinacy. While dimerization is obligatory for DNA binding, SEP3 and SEP3–AG also form tetrameric complexes. How homo and hetero-dimerization and tetramerization of MADS TFs affect genome-wide DNA-binding and gene regulation is not known. Using sequential DNA affinity purification sequencing (seq-DAP-seq), we determined genome-wide binding of SEP3 homomeric and SEP3–AG heteromeric complexes, including SEP3Δtet-AG, a complex with a SEP3 splice variant, SEP3Δtet, which is largely dimeric and SEP3–AG tetramer. SEP3 and SEP3–AG share numerous bound regions, however each complex bound unique sites, demonstrating that protein identity plays a role in DNA-binding. SEP3–AG and SEP3Δtet-AG share a similar genome-wide binding pattern; however the tetrameric form could access new sites and demonstrated a global increase in DNA-binding affinity. Tetramerization exhibited significant cooperative binding with preferential distances between two sites, allowing efficient binding to regions that are poorly recognized by dimeric SEP3Δtet-AG. By intersecting seq-DAP-seq with ChIP-seq and expression data, we identified unique target genes bound either in SEP3–AG seq-DAP-seq or in SEP3/AG ChIP-seq. Seq-DAP-seq is a versatile genome-wide technique and complements in vivo methods to identify putative direct regulatory targets.

Identification of Functional HLA-A*01:01-Restricted Epstein-Barr Latent Membrane Protein 2-Specific T-Cell Receptors.

BackgroundAdoptive transfer of genetically engineered T cells expressing antigen-specific T-cell receptors (TCRs) is an appealing therapeutic approach for Epstein-Barr virus (EBV)–associated malignancies of latency type II/III that express EBV antigens (LMP1/2). Patients who are HLA-A*01:01 positive could benefit from such products, since no T cells recognizing any EBV-derived peptide in this common HLA allele have been found thus far.MethodsHLA-A*01:01–restricted EBV-LMP2–specific T cells were isolated using peptide major histocompatibility complex (pMHC) tetramers. Functionality was assessed by production of interferon gamma (IFN-γ) and cytotoxicity when stimulated with EBV-LMP2–expressing cell lines. Functionality of primary T cells transduced with HLA-A*01:01–restricted EBV-LMP2–specific TCRs was optimized by knocking out the endogenous TCRs of primary T cells (∆TCR) using CRISPR-Cas9 technology.ResultsEBV-LMP2–specific T cells were successfully isolated and their TCRs were characterized. TCR gene transfer in primary T cells resulted in specific pMHC tetramer binding and reactivity against EBV-LMP2–expressing cell lines. The mean fluorescence intensity of pMHC-tetramer binding was increased 1.5–2 fold when the endogenous TCRs of CD8+ T cells was knocked out. CD8+/∆TCR T cells modified to express EBV-LMP2–specific TCRs showed IFN-γ secretion and cytotoxicity toward EBV-LMP2–expressing malignant cell lines.ConclusionsWe isolated the first functional HLA-A*01:01–restricted EBV-LMP2–specific T-cell populations and TCRs, which can potentially be used in future TCR gene therapy to treat EBV-associated latency type II/III malignancies.