Domain Structure Of Crystals Research Articles

Crystal structure of the Arabidopsis SPIRAL2 C-terminal domain reveals a p80-Katanin-like domain.

Epidermal cells of dark-grown plant seedlings reorient their cortical microtubule arrays in response to blue light from a net lateral orientation to a net longitudinal orientation with respect to the long axis of cells. The molecular mechanism underlying this microtubule array reorientation involves katanin, a microtubule severing enzyme, and a plant-specific microtubule associated protein called SPIRAL2. Katanin preferentially severs longitudinal microtubules, generating seeds that amplify the longitudinal array. Upon severing, SPIRAL2 binds nascent microtubule minus ends and limits their dynamics, thereby stabilizing the longitudinal array while the lateral array undergoes net depolymerization. To date, no experimental structural information is available for SPIRAL2 to help inform its mechanism. To gain insight into SPIRAL2 structure and function, we determined a 1.8 Å resolution crystal structure of the Arabidopsis thaliana SPIRAL2 C-terminal domain. The domain is composed of seven core α-helices, arranged in an α-solenoid. Amino-acid sequence conservation maps primarily to one face of the domain involving helices α1, α3, α5, and an extended loop, the α6-α7 loop. The domain fold is similar to, yet structurally distinct from the C-terminal domain of Ge-1 (an mRNA decapping complex factor involved in P-body localization) and, surprisingly, the C-terminal domain of the katanin p80 regulatory subunit. The katanin p80 C-terminal domain heterodimerizes with the MIT domain of the katanin p60 catalytic subunit, and in metazoans, binds the microtubule minus-end factors CAMSAP3 and ASPM. Structural analysis predicts that SPIRAL2 does not engage katanin p60 in a mode homologous to katanin p80. The SPIRAL2 structure highlights an interesting evolutionary convergence of domain architecture and microtubule minus-end localization between SPIRAL2 and katanin complexes, and establishes a foundation upon which structure-function analysis can be conducted to elucidate the role of this domain in the regulation of plant microtubule arrays.

Molecular mechanism of human ISG20L2 for the ITS1 cleavage in the processing of 18S precursor ribosomal RNA.

The exonuclease ISG20L2 has been initially characterized for its role in the mammalian 5.8S rRNA 3' end maturation, specifically in the cleavage of ITS2 of 12S precursor ribosomal RNA (pre-rRNA). Here, we show that human ISG20L2 is also involved in 18S pre-rRNA maturation through removing the ITS1 region, and contributes to ribosomal biogenesis and cell proliferation. Furthermore, we determined the crystal structure of the ISG20L2 nuclease domain at 2.9 Å resolution. It exhibits the typical αβα fold of the DEDD 3'-5' exonuclease with a catalytic pocket located in the hollow near the center. The catalytic residues Asp183, Glu185, Asp267, His322 and Asp327 constitute the DEDDh motif in ISG20L2. The active pocket represents conformational flexibility in the absence of an RNA substrate. Using structural superposition and mutagenesis assay, we mapped RNA substrate binding residues in ISG20L2. Finally, cellular assays revealed that ISG20L2 is aberrantly up-regulated in colon adenocarcinoma and promotes colon cancer cell proliferation through regulating ribosome biogenesis. Together, these results reveal that ISG20L2 is a new enzymatic member for 18S pre-rRNA maturation, provide insights into the mechanism of ISG20L2 underlying pre-rRNA processing, and suggest that ISG20L2 is a potential therapeutic target for colon adenocarcinoma.

Kinetics of the Domain Structure in Uniform Electric Field in LiNbO3 with Surface Layer Modified by Soft Proton Exchange

The creation of periodic domain structures in lithium niobate crystals makes it possible to realize highly efficient nonlinear optical interactions in optical waveguides. The influence of near‐surface H+ ion concentration gradient on the domain kinetics in uniform electric field in lithium niobate crystals modified by soft proton exchange is studied. The spatial distribution of the H+ ions concentration in the surface layer is obtained by confocal Raman microscopy. The observed anomalous evolution of the domain structure at the surface represents the formation of stripe domains and their anisotropic growth along three directions. The domain imaging in the bulk allows revealing a comb‐like domain shape. The obtained strong influence of the proton exchange duration on the threshold field is attributed to the action of the concentration gradient in the near‐surface layer, which is considered as an analog of the internal bias electric field. It is shown that the internal bias field is proportional to the increase in the concentration gradient in the surface layer. The mechanism of the comb‐like domain formation is proposed. The obtained results can be used for development of domain engineering methods in optical waveguides.

Eriodictyol Flavanones Based Virtual Screening of Bioactive Compounds from ChEMBL 2D Database with Classic 3-point Pharmacophore Screening Method for HER2 Inhibitors for Breast Cancer

Understanding binding interactions between flavanones and human epidermal growth factor receptor 2 (HER2) is an important step in developing effective treatments for breast cancer, and this study applies computational methods to do just that. This research presents a comprehensive computational methodology for identifying potential HER2 inhibitors with a focus on breast cancer treatment. The study leverages a combination of structural and pharmacophore-based approaches, starting with bioactive compound selection from the ChEMBL 2D database. The PDB-REDO refined crystal structure of Kinase domain of Human HER2 (erbB2) was used to conduct molecular docking simulations with the identified drugs. The Kleywegt-like plot analysis demonstrates the improved structural quality of the HER2 kinase domain after refinement, showing enhanced agreement with experimental data. Molecular docking simulations, conducted using the AutoDock tool, reveal the binding affinity and interaction patterns of selected compounds with the HER2 receptor. Virtual screening results highlight compounds with high binding affinity, favorable interaction patterns, and structural compatibility as potential lead candidates. To ensure safety and efficacy, ADMETox filtering was employed, providing insights into the compound’s toxicity profile and pharmacokinetic attributes. The selected compound, Eriodictyol (C20H20O6), exhibits a generally favorable safety profile, with predicted inactivity across multiple toxicity classifications and endpoints. While immunotoxicity is predicted, the overall low probabilities suggest a relatively low risk. Physicochemical and pharmacokinetic assessments indicate Eriodictyol’s potential for drug development. With a molecular weight of 356.37 g/mol, balanced lipophilicity, and high gastrointestinal absorption, the compound aligns with drug-likeness criteria. However, careful consideration is warranted due to its inhibitory effects on certain enzymes and alerts for catechol_A and isolated_alkene. In conclusion, this integrated computational approach streamlines the identification of potential HER2 inhibitors, offering a systematic strategy for drug discovery. Eriodictyol emerges as a promising candidate, demonstrating a favorable safety profile and pharmacokinetic attributes, paving the way for further in-depth studies and development as a potential therapeutic agent for breast cancer.

Structural and enzymatic evidence for the methylation of the ACK1 tyrosine kinase by the histone lysine methyltransferase SETD2

SETD2 (SET-domain containing protein 2) is a histone methyltransferase (HMT) of the SET family responsible for the trimethylation of K36 of histone H3, thus producing the epigenetic mark H3K36me3. Recent studies have shown that certain SET family HMTs, such as SMYD2, SMYD3 or SETDB1 can also methylate protein kinases and therefore be involved in signaling pathways. Here we provide structural and enzymatic evidence showing that SETD2 methylates the protein tyrosine kinase ACK1 in vitro. ACK1 is recognized as a major integrator of signaling from various receptor tyrosine kinases. Using ACK1 peptides and recombinant proteins, we show that SETD2 methylates the K514 residue of ACK1 generating K514 mono, di or tri-methylation. Interestingly, K514 is found in a “H3K36-like” motif of ACK1 which is known to be post-translationally modified and to be involved in protein-protein interaction. The crystal structure of SETD2 catalytic domain in complex with an ACK1 peptide further provides the structural basis for the methylation of ACK1 K514 by SETD2. Our work therefore strongly suggests that ACK1 could be a novel non-histone substrate of SETD2 and further supports that SET HMTs, such as SETD2, could be involved in both epigenetic regulations and cell signaling.

Demonstrating the importance of porcine reproductive and respiratory syndrome virus papain-like protease 2 deubiquitinating activity in viral replication by structure-guided mutagenesis.

Deubiquitination of cellular substrates by viral proteases is a mechanism used to interfere with host cellular signaling processes, shared between members of the coronavirus- and arterivirus families. In the case of Arteriviruses, deubiquitinating and polyprotein processing activities are accomplished by the virus-encoded papain-like protease 2 (PLP2). Several studies have implicated the deubiquitinating activity of the porcine reproductive and respiratory syndrome virus (PRRSV) PLP2 in the downregulation of cellular interferon production, however to date, the only arterivirus PLP2 structure described is that of equine arteritis virus (EAV), a distantly related virus. Here we describe the first crystal structure of the PRRSV PLP2 domain both in the presence and absence of its ubiquitin substrate, which reveals unique structural differences in this viral domain compared to PLP2 from EAV. To probe the role of PRRSV PLP2 deubiquitinating activity in host immune evasion, we selectively removed this activity from the domain by mutagenesis and found that the viral domain could no longer downregulate cellular interferon production. Interestingly, unlike EAV, and also unlike the situation for MERS-CoV, we found that recombinant PRRSV carrying PLP2 DUB-specific mutations faces significant selective pressure to revert to wild-type virus in MARC-145 cells, suggesting that the PLP2 DUB activity, which in PRRSV is present as three different versions of viral protein nsp2 expressed during infection, is critically important for PRRSV replication.

LİGNAN SEKOİZOLARİSİRESİNOL DIGLUKOSIT ÜZERİNE İN VİTRO VE İN SİLİKO ÇALIŞMALAR

Objective: Lignans are important biologically active compounds in diphenolic structure. Secoisolariciresinol diglucoside (SDG) is a significant type of lignan known to have anti-cancer properties. This study aimed to investigate the antiproliferative activity properties of SDG on hepatocellular carcinoma cells (HepG2), colorectal cancer cells (DLD-1), lung carcinoma (A549), and prostate cancer (PC3) cell lines. Material and Method: Cell viability of cancer cells was determined by the MTT method after treatment with various concentrations of SDG at 48 or 72 hours. The DFT (Density Functional Theory) analysis of the SDG was performed using Spartan'10 and visualized. Drug-likeness and absorption, distribution, metabolism, excretion, and toxicity (ADME-Tox) properties of this compound were examined. Molecular docking was carried out to research the biological activity of SDG. Result and Discussion: Our results showed that SDG exhibited significant cytotoxicity only against DLD-1 cells with IC50 value of 37.45 µM, but inactive against other cancer cell lines as in vitro. 4UYA, which biomarker for colon cancer, is the crystal structure of the MLK4 kinase domain. The binding energy value for the SDG-MLK4 kinase domain was calculated as -6.1 kcal/mol. Anticancer potential was verified by in vitro assay and in silico molecular docking study. In conclusion, this study revealed the protective aspect of SDG against colon cancer and showed that it has promising anticancer activity.

First Validated Model of Zymogen Factor XII Provides Insights into Protein Assembly and Activation

Factor XII (FXII), the zymogen of the plasma protease FXIIa, contributes to thrombo-inflammatory processes. As part of the kallikrein-kinin system, FXIIa converts prekallikrein to the protease kallikrein (PKa), which then cleaves high-molecular-weight kininogen (HK) to release the vasoactive peptide bradykinin. In the disorder hereditary angioedema, dysregulation of FXII activation leads to excessive bradykinin production. When blood is exposed to artificial surfaces, such as during cardiopulmonary bypass or ECMO, FXII is rapidly converted to FXIIa by contact activation, and contributes to thrombus formation by activating factor XI. FXII circulates in plasma in a “closed” form that is resistant to activation by PKa and other proteases. Surface-binding “opens” the FXII structure, facilitating activation. Relatively little is known about the structural features that maintain the closed FXII conformation as the full-length protein has been refractory to crystallization. Recently, a crystal structure was reported for part of the non-catalytic portion of FXII. An intriguing feature of the structure is an interaction between the fibronectin type 2 (FN2) and kringle (KNG) domains at opposite ends of the polypeptide that forms a ring-like structure. Functional studies by our group and others indicate these domains are key to maintaining the closed conformation. Here we describe a model for FXII prepared with the program AlphaFold (AF), and its validation using site-directed mutagenesis to disrupt interactions predicted to maintain the closed conformation. The human FXII model in the AF database was modified by removing residues 300-316 from the proline-rich region and by reference to crystal structures of the FXIIa catalytic domain (CD) bound to inhibitors. Molecular graphics and interdomain interaction analyses were performed in ChimeraX. The refined model predicts 15 interdomain bonds involving residues in FN2, KNG, and the CD. Of the 22 amino acids involved in these bonds, 15 are highly conserved in FXII across 25 vertebrate species. We expressed human FXII in which these residues were individually changed to alanine (Table). Alanine replacements for amino acids not predicted to be involved in interdomain interactions were prepared as controls. Four variants (FXII-D253A, FXII-W257A, FXII-W268A and FXII-E502A) were activated in cell culture and one (FXII-R61A) did not express. FXII-R61A, FXII-W257A, and FXII-W268A were not studied further. FXII-D253A was replaced with FXII-D253K. The active site serine of FXII-E502A was replaced with alanine to prevent autocatalysis (FXII-E502A,S544A). In the absence of a surface, FXII-R36A, FXII-E225A, FXII-D253K, and FXII-K346A were activated by PKa (~15 fold) faster than wild type FXII (FXII-WT) in a chromogenic substrate assay, consistent with an open conformation. Using densitometric analyses of stained SDS- polyacrylamide gels, we noted that these four FXII variants and FXII-E502A,S544A were converted to FXIIa by PKa more rapidly than FXII-WT and other variants. FXII-R36A, FXII-E225A, FXII-D253K, and FXII-K346A bound to prekallikrein with higher affinity than FXII-WT in a plate-binding assay, consistent with a change in conformation. When the four variants were added to human plasma in the absence of a surface, they caused a rapid cleavage of HK by accelerating reciprocal activation with prekallikrein. Our functional analyses indicate that non-covalent bonds between R36 on FN2 and E502 on the CD; R36 and E225 on KNG; and D253 on KNG and K346 adjacent to the activation cleavage site are critical for maintaining FXII in a closed conformation. The instability of FXII variants with substitutions for tryptophans at positions 257 and 268 also implicate interactions between these residues and K346. In the AF model these bonds appear to maintain FXII in a conformation in which access to the activation cleavage site is limited. Our binding studies also suggest the closed conformation mask a binding site for prekallikrein and PKa. These bonds are likely disrupted when FXII binds to a surface, as part of the contact activation mechanism that enhances FXII activation.

ESRP1 controls biogenesis and function of a large abundant multiexon circRNA.

While the majority of circRNAs are formed from infrequent back-splicing of exons from protein coding genes, some can be produced at quite high level and in a regulated manner. We describe the regulation, biogenesis and function of circDOCK1(2-27), a large, abundant circular RNA that is highly regulated during epithelial-mesenchymal transition (EMT) and whose formation depends on the epithelial splicing regulator ESRP1. CircDOCK1(2-27) synthesis in epithelial cells represses cell motility both by diverting transcripts from DOCK1 mRNA production to circRNA formation and by direct inhibition of migration by the circRNA. HITS-CLIP analysis and CRISPR-mediated deletions indicate ESRP1 controls circDOCK1(2-27) biosynthesis by binding a GGU-containing repeat region in intron 1 and detaining its splicing until Pol II completes its 157 kb journey to exon 27. Proximity-dependent biotinylation (BioID) assay suggests ESRP1 may modify the RNP landscape of intron 1 in a way that disfavours communication of exon 1 with exon 2, rather than physically bridging exon 2 to exon 27. The X-ray crystal structure of RNA-bound ESRP1 qRRM2 domain reveals it binds to GGU motifs, with the guanines embedded in clamp-like aromatic pockets in the protein.

A divergent tumor overexpressed gene domain and oligomerization contribute to SPIRAL2 function in stabilizing microtubule minus ends.

The acentrosomal cortical microtubules (MTs) of higher plants dynamically assemble into specific array patterns that determine the axis of cell expansion. Recently, the Arabidopsis (Arabidopsis thaliana) SPIRAL2 (SPR2) protein was shown to regulate cortical MT length and light-induced array reorientation by stabilizing MT minus ends. SPR2 autonomously localizes to both the MT lattice and MT minus ends, where it decreases the minus end depolymerization rate. However, the structural determinants that contribute to the ability of SPR2 to target and stabilize MT minus ends remain unknown. Here, we present the crystal structure of the SPR2 N-terminal domain, which reveals a unique tumor overexpressed gene (TOG) domain architecture with 7 HEAT repeats. We demonstrate that a coiled-coil domain mediates the multimerization of SPR2, which provides avidity for MT binding, and is essential to bind soluble tubulin. In addition, we found that an SPR2 construct spanning the TOG domain, basic region, and coiled-coil domain targets and stabilizes MT minus ends similar to full-length SPR2 in plants. These results reveal how a TOG domain, which is typically found in microtubule plus-end regulators, has been appropriated in plants to regulate MT minus ends.

Identification of a Staphylococcus aureus amidase catalytic domain inhibitor to prevent biofilm formation by sequential virtual screening, molecular dynamics simulation and biological evaluation

Staphylococcus aureus (S. aureus) is one of the common causes of implant associated biofilm infections and their biofilms are resistant to antibiotics. S. aureus amidase (AM) protein, a cell wall hydrolase that cleaves the amide bond between N-acetylmuramic acid and L-alanine residue of the stem peptide, is several fold over-expressed under biofilm conditions. Previous studies demonstrated an autolysin mutant in S. aureus that lacks the AM protein, is highly impaired in biofilm development. We carried out a structure-based small molecule design using the crystal structure of AM protein catalytic domain to identify inhibitors that can block amidase activity and therefore inhibits S. aureus biofilm formation. Sequential virtual screening followed by pharmacokinetic analysis and bioassay studies filtered 25 small molecules from different databases. Two compounds from the SPECS database, SPECS-1 and SPECS-2, were selected based on the best docking score and minimum biofilm inhibitory concentration towards S. aureus biofilms. SPECS-1 and SPECS-2 were further tested for their structural/energetic stability in complex with the AM protein using molecular dynamics simulation and MM-GBSA techniques. In vitro, biofilm inhibition studies on different surfaces confirmed that treatment with SPECS-1 and SPECS-2 at a concentration of 250 μg/ml exhibited significant prevention and disruption of S. aureus biofilms. Finally, the in vitro anti-biofilm activities of these two compounds were validated against Methicillin-resistant S. aureus clinical isolates. We concluded that the discovered compounds SPECS-1 and SPECS-2 are safe and exhibit biofilm preventive and disruption activity for inhibiting the S. aureus biofilms and hence can be used to treat implant-associated biofilm infections.

Large bi-axial tensile strain effect in epitaxial BiFeO3 film grown on single crystal PrScO3

A BiFeO3 film is grown epitaxially on a PrScO3 single crystal substrate which imparts ~ 1.45% of biaxial tensile strain to BiFeO3 resulting from lattice misfit. The biaxial tensile strain effect on BiFeO3 is investigated in terms of crystal structure, Poisson ratio, and ferroelectric domain structure. Lattice resolution scanning transmission electron microscopy, precession electron diffraction, and X-ray diffraction results clearly show that in-plane interplanar distance of BiFeO3 is the same as that of PrScO3 with no sign of misfit dislocations, indicating that the biaxial tensile strain caused by lattice mismatch between BiFeO3 and PrScO3 are stored as elastic energy within BiFeO3 film. Nano-beam electron diffraction patterns compared with structure factor calculation found that the BiFeO3 maintains rhombohedral symmetry, i.e., space group of R3c. The pattern analysis also revealed two crystallographically distinguishable domains. Their relations with ferroelectric domain structures in terms of size and spontaneous polarization orientations within the domains are further understood using four-dimensional scanning transmission electron microscopy technique.

Structure-guided discovery of aminopeptidase ERAP1 variants capable of processing antigens with novel PC anchor specificities.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) belongs to the oxytocinase subfamily of M1 aminopeptidases (M1APs), which are a diverse family of metalloenzymes involved in a wide range of functions and have been implicated in various chronic and infectious diseases of humans. ERAP1 trims antigenic precursors into correct sizes (8-10 residues long) for Major Histocompatibility Complex (MHC) presentation, by a unique molecular ruler mechanism in which it makes concurrent bindings to substrate N- and C-termini. We have previously determined four crystal structures of ERAP1 C-terminal regulatory domain (termed ERAP1_C domain) in complex with peptide carboxyl (PC)-ends that carry various anchor residues, and identified a specificity subsite for recognizing the PC anchor side chain, denoted as the SC subsite to follow the conventional notations: S1 site for P1, S2 site for P2, and so forth. In this study, we report studies on structure-guided mutational and hydrolysis kinetics, and peptide trimming assays to further examine the functional roles of this SC subsite. Most strikingly, a point mutation V737R results in a change of substrate preference from a hydrophobic to a negatively charged PC anchor residue; the latter is presumed to be a poor substrate for WT ERAP1. These studies validate the crystallographic observations that this SC subsite is directly involved in binding and recognition of the substrate PC anchor and presents a potential target to modulate MHC-restricted immunopeptidomes.

Sulisobenzone is a potent inhibitor of the global transcription factor Cra

Transcription is carried out by the RNA polymerase and is regulated through a series of interactions with transcription factors. Catabolite activator repressor (Cra), a LacI family transcription factor regulates the virulence gene expression in Enterohaemorrhagic Escherichia coli (EHEC) and thus is a promising drug target for the discovery of antivirulence molecules. Here, we report the crystal structure of the effector molecule binding domain of Cra from E. coli (EcCra) in complex with HEPES molecule. Based on the EcCra-HEPES complex structure, ligand screening was performed that identified sulisobenzone as an potential inhibitor of EcCra. The electrophoretic mobility shift assay (EMSA) and in vitro transcription assay validated the sulisobenzone binding to EcCra. Moreover, the isothermal titration calorimetry (ITC) experiments demonstrated a 40-fold higher binding affinity of sulisobenzone (KD 360 nM) compared to the HEPES molecule. Finally, the sulisobenzone bound EcCra complex crystal structure was determined to elucidate the binding mechanism of sulisobenzone to the effector binding pocket of EcCra. Together, this study suggests that sulisobenzone may be a promising candidate that can be studied and developed as an effective antivirulence agent against EHEC.

The regulation of RGLG2-VWA by Ca2+ ions

RGLG2, an E3 ubiquitin ligase in Arabidopsis thaliana, affects hormone signaling and participates in drought regulation. Here, we determined two crystal structures of RGLG2 VWA domain, representing two conformations, open and closed, respectively. The two structures reveal that Ca2+ ions are allosteric regulators of RGLG2-VWA, which adopts open state when NCBS1(Novel Calcium ions Binding Site 1) binds Ca2+ ions and switches to closed state after Ca2+ ions are removed. This mechanism of allosteric regulation is identical to RGLG1-VWA, but distinct from integrin α and β VWA domains. Therefore, our data provide a backdrop for understanding the role of the Ca2+ ions in conformational change of VWA domain. In addition, we found that RGLG2closed, corresponding to low affinity, can bind pseudo-ligand, which has never been observed in other VWA domains.

The unexpected diffuse phase transition in relaxor-PbTiO3 ferroelectrics via acceptor modification

The diffuse phase transition (DPT) and domain structure are essential to the functional properties of relaxor ferroelectrics and are extremely sensitive to the ion doping. The utilization of acceptor doping has been observed to be an effective method in enhancing the high-power properties of relaxor ferroelectric materials. The present study aims to examine the acceptor-doped DPT and domain structure in single crystals of Pb(In1/2Nb1/2)O3-Pb(Mg1/3Nb2/3)O3-PbTiO3. An unexpected physical phenomenon was identified in which Mn-doping increased dielectric diffusion and lowered domain size while suppressing dielectric relaxation. Traceability investigations suggest that Mn-doping inhibited the growth of polar nanoregions by enhancing random electric fields, which increases dielectric diffusion while decreasing the domain size. Meanwhile, Mn doping redistributes the relaxation time function, which results in a reduction in dielectric relaxation. The current research deepens the understanding of the physical basis of DPT and can be applied to the development of high-performance piezoelectric materials.

Structural Modeling of Cytokine-Receptor-JAK2 Signaling Complexes Using AlphaFold Multimer.

Homodimeric class 1 cytokine receptors include the erythropoietin (EPOR), thrombopoietin (TPOR), granulocyte colony-stimulating factor 3 (CSF3R), growth hormone (GHR), and prolactin receptors (PRLR). These cell-surface single-pass transmembrane (TM) glycoproteins regulate cell growth, proliferation, and differentiation and induce oncogenesis. An active TM signaling complex consists of a receptor homodimer, one or two ligands bound to the receptor extracellular domains, and two molecules of Janus Kinase 2 (JAK2) constitutively associated with the receptor intracellular domains. Although crystal structures of soluble extracellular domains with ligands have been obtained for all of the receptors except TPOR, little is known about the structure and dynamics of the complete TM complexes that activate the downstream JAK-STAT signaling pathway. Three-dimensional models of five human receptor complexes with cytokines and JAK2 were generated here by using AlphaFold Multimer. Given the large size of the complexes (from 3220 to 4074 residues), the modeling required a stepwise assembly from smaller parts, with selection and validation of the models through comparisons with published experimental data. The modeling of active and inactive complexes supports a general activation mechanism that involves ligand binding to a monomeric receptor followed by receptor dimerization and rotational movement of the receptor TM α-helices, causing proximity, dimerization, and activation of associated JAK2 subunits. The binding mode of two eltrombopag molecules to the TM α-helices of the active TPOR dimer was proposed. The models also help elucidate the molecular basis of oncogenic mutations that may involve a noncanonical activation route. Models equilibrated in explicit lipids of the plasma membrane are publicly available.

Structural basis for evolutionarily conserved interactions between TFIIS and Paf1C

The elongation factor TFIIS interacts with Paf1C complex to facilitate processive transcription by Pol II. We here determined the crystal structure of the trypanosoma TFIIS LW domain in a complex with the LFG motif of Leo1, as well as the structures of apo-form TFIIS LW domains from trypanosoma, yeast and human. We revealed that all three TFIIS LW domains possess a conserved hydrophobic core that mediates their interactions with Leo1. Intriguingly, the structural study revealed that trypanosoma Leo1 binding induces the TFIIS LW domain to undergo a conformational change reflected in the length and orientation of α6 helix that is absent in the yeast and human counterparts. These differences explain the higher binding affinity of the TFIIS LW domain interacting with Leo1 in trypanosoma than in yeast and human, and indicate species-specific variations in the interactions. Importantly, the interactions between the TFIIS LW domain and an LFG motif of Leo1 were found to be critical for TFIIS to anchor the entire Paf1C complex. Thus, in addition to revealing a detailed structural basis for the TFIIS-Paf1C interaction, our studies also shed light on the origin and evolution of the roles of TFIIS and Paf1C complex in regulation of transcription elongation.

Structure of BTK kinase domain with the second-generation inhibitors acalabrutinib and tirabrutinib.

Bruton's tyrosine kinase (BTK) is the target of the therapeutic agent, Ibrutinib, that treats chronic lymphocyte leukemia (CLL), mantle cell lymphoma (MCL) and other B cell malignancies. Ibrutinib is a first in class, covalent BTK inhibitor that limits B-cell survival and proliferation. Designing new inhibitors of BTK has been an important objective for advancing development of improved therapeutic agents against cancer and autoimmune disorders. Based on the success of Ibrutinib, several second-generation irreversible BTK inhibitors have been developed that exhibit fewer off-target effects. However, the binding-mode and their interaction with Btk have not been experimentally determined and evaluated at atomic resolution. Here we determined the first crystal structure of the BTK kinase domain in complex with acalabrutinib. In addition, we report a structure of the BTK/tirabrutinib complex and compare these structures with previously solved structures. The structures provide insight in the superior selectivity reported for acalabrutinb and guide future BTK inhibitor development.

Redox driven B12-ligand switch drives CarH photoresponse

CarH is a coenzyme B12-dependent photoreceptor involved in regulating carotenoid biosynthesis. How light-triggered cleavage of the B12 Co-C bond culminates in CarH tetramer dissociation to initiate transcription remains unclear. Here, a series of crystal structures of the CarH B12-binding domain after illumination suggest formation of unforeseen intermediate states prior to tetramer dissociation. Unexpectedly, in the absence of oxygen, Co-C bond cleavage is followed by reorientation of the corrin ring and a switch from a lower to upper histidine-Co ligation, corresponding to a pentacoordinate state. Under aerobic conditions, rapid flash-cooling of crystals prior to deterioration upon illumination confirm a similar B12-ligand switch occurs. Removal of the upper His-ligating residue prevents monomer formation upon illumination. Combined with detailed solution spectroscopy and computational studies, these data demonstrate the CarH photoresponse integrates B12 photo- and redox-chemistry to drive large-scale conformational changes through stepwise Co-ligation changes.