Structural characterization of two prototypical repressors of SorC family reveals tetrameric assemblies on DNA and mechanism of function.

The SorC family of transcriptional regulators plays a crucial role in controlling the carbohydrate metabolism and quorum sensing. We employed an integrative approach combining X-ray crystallography and cryo-electron microscopy to investigate architecture and functional mechanism of two prototypical representatives of two sub-classes of the SorC family: DeoR and CggR from Bacillus subtilis. Despite possessing distinct DNA-binding domains, both proteins form similar tetrameric assemblies when bound to their respective DNA operators. Structural analysis elucidates the process by which the CggR-regulated gapA operon is derepressed through the action of two effectors: fructose-1,6-bisphosphate and newly confirmed dihydroxyacetone phosphate. Our findings provide the first comprehensive understanding of the DNA binding mechanism of the SorC-family proteins, shedding new light on their functional characteristics.

The cryo-EM structure of the BoNT/Wo-NTNH complex reveals two immunoglobulin-like domains.

The botulinum neurotoxin-like toxin from Weissella oryzae (BoNT/Wo) is one of the BoNT-like toxins recently identified outside of the Clostridium genus. We show that, like the canonical BoNTs, BoNT/Wo forms a complex with its non-toxic non-hemagglutinin (NTNH) partner, which in traditional BoNT serotypes protects the toxin from proteases and the acidic environment of the hosts' guts. We here report the cryo-EM structure of the 300 kDa BoNT/Wo-NTNH/Wo complex together with pH stability studies of the complex. The structure reveals molecular details of the toxin's interactions with its protective partner. The overall structural arrangement is similar to other reported BoNT-NTNH complexes, but NTNH/Wo uniquely contains two extra bacterial immunoglobulin-like (Big) domains on the C-terminus. Although the function of these Big domains is unknown, they are structurally most similar to bacterial proteins involved in adhesion to host cells. In addition, the BoNT/Wo protease domain contains an internal disulfide bond not seen in other BoNTs. Mass photometry analysis revealed that the BoNT/Wo-NTNH/Wo complex is stable under acidic conditions and may dissociate at neutral to basic pH. These findings established that BoNT/Wo-NTNH/Wo shares the general fold of canonical BoNT-NTNH complexes. The presence of unique structural features suggests that it may have an alternative mode of activation, translocation and recognition of host cells, raising interesting questions about the activity and the mechanism of action of BoNT/Wo as well as about its target environment, receptors and substrates.

Multivalency of nucleosome recognition by LEDGF.

Eukaryotic transcription is dependent on specific histone modifications. Their recognition by chromatin readers triggers complex processes relying on the coordinated association of transcription regulatory factors. Although various modification states of a particular histone residue often lead to differential outcomes, it is not entirely clear how they are discriminated. Moreover, the contribution of intrinsically disordered regions outside of the specialized reader domains to nucleosome binding remains unexplored. Here, we report the structures of a PWWP domain from transcriptional coactivator LEDGF in complex with the H3K36 di- and trimethylated nucleosome, indicating that both methylation marks are recognized by PWWP in a highly conserved manner. We identify a unique secondary interaction site for the PWWP domain at the interface between the acidic patch and nucleosomal DNA that might contribute to an H3K36-methylation independent role of LEDGF. We reveal DNA interacting motifs in the intrinsically disordered region of LEDGF that discriminate between the intra- or extranucleosomal DNA but remain dynamic in the context of dinucleosomes. The interplay between the LEDGF H3K36-methylation reader and protein binding module mediated by multivalent interactions of the intrinsically disordered linker with chromatin might help direct the elongation machinery to the vicinity of RNA polymerase II, thereby facilitating productive elongation.

Characterization of new highly selective pyrazolo[4,3-d]pyrimidine inhibitor of CDK7.

Targeting cyclin-dependent kinase 7 (CDK7) provides an interesting therapeutic option in cancer therapy because this kinase participates in regulating the cell cycle and transcription. Here, we describe a new trisubstituted pyrazolo[4,3-d]pyrimidine derivative, LGR6768, that inhibits CDK7 in the nanomolar range and displays favourable selectivity across the CDK family. We determined the structure of fully active CDK2/cyclin A2 in complex with LGR6768 at 2.6 Å resolution using X-ray crystallography, revealing conserved interactions within the active site. Structural analysis and comparison with LGR6768 docked to CDK7 provides an explanation of the observed biochemical selectivity, which is linked to a conformational difference in the biphenyl moiety. In cellular experiments, LGR6768 affected regulation of the cell cycle and transcription by inhibiting the phosphorylation of cell cycle CDKs and the carboxy-terminal domain of RNA polymerase II, respectively. LGR6768 limited the proliferation of several leukaemia cell lines, triggered significant changes in protein and mRNA levels related to CDK7 inhibition and induced apoptosis in dose- and time-dependent experiments. Our work supports previous findings and provides further information for the development of selective CDK7 inhibitors.

X-ray structure of human aldo-keto reductase 1C3 in complex with a bile acid fused tetrazole inhibitor: experimental validation, molecular docking and structural analysis.

Aldo-keto reductase 1C3 (AKR1C3) catalyzes the reduction of androstenedione to testosterone and reduces the effectiveness of chemotherapeutics. AKR1C3 is a target for treatment of breast and prostate cancer and AKR1C3 inhibition could be an effective adjuvant therapy in the context of leukemia and other cancers. In the present study, steroidal bile acid fused tetrazoles were screened for their ability to inhibit AKR1C3. Four C24 bile acids with C-ring fused tetrazoles were moderate to strong AKR1C3 inhibitors (37-88% inhibition), while B-ring fused tetrazoles had no effect on AKR1C3 activity. Based on a fluorescence assay in yeast cells, these four compounds displayed no affinity for estrogen receptor-α, or the androgen receptor, suggesting a lack of estrogenic or androgenic effects. A top inhibitor showed specificity for AKR1C3 over AKR1C2, and inhibited AKR1C3 with an IC50 of ∼7 µM. The structure of AKR1C3·NADP+ in complex with this C-ring fused bile acid tetrazole was determined by X-ray crystallography at 1.4 Å resolution, revealing that the C24 carboxylate is anchored to the catalytic oxyanion site (H117, Y55); meanwhile the tetrazole interacts with a tryptophan (W227) important for steroid recognition. Molecular docking predicts that all four top AKR1C3 inhibitors bind with nearly identical geometry, suggesting that C-ring bile acid fused tetrazoles represent a new class of AKR1C3 inhibitors.

Structure and activity of botulinum neurotoxin X.

Botulinum neurotoxins (BoNTs) are the most potent toxins known and are used to treat an increasing number of medical disorders. All BoNTs are naturally co-expressed with a protective partner protein (NTNH) with which they form a 300 kDa complex, to resist acidic and proteolytic attack from the digestive tract. We have previously identified a new botulinum neurotoxin serotype, BoNT/X, that has unique and therapeutically attractive properties. We present the cryo-EM structure of the BoNT/X-NTNH/X complex at 3.1 Å resolution. Unexpectedly, the BoNT/X complex is stable and protease resistant at both neutral and acidic pH and disassembles only in alkaline conditions. Using the stabilizing effect of NTNH, we isolated BoNT/X and showed that it has very low potency both in vitro and in vivo . Given the high catalytic activity and translocation efficacy of BoNT/X, low activity of the full toxin is likely due to the receptor-binding domain, which presents weak ganglioside binding and exposed hydrophobic surfaces.

Reaction Mechanism of Human PAICS Elucidated by Quantum Chemical Calculations.

Human PAICS is a bifunctional enzyme that is involved in the de novo purine biosynthesis, catalyzing the conversion of aminoimidazole ribonucleotide (AIR) into N-succinylcarboxamide-5-aminoimidazole ribonucleotide (SAICAR). It comprises two distinct active sites, AIR carboxylase (AIRc) where the AIR is initially converted to carboxyaminoimidazole ribonucleotide (CAIR) by reaction with CO2 and SAICAR synthetase (SAICARs) in which CAIR then reacts with an aspartate to form SAICAR, in an ATP-dependent reaction. Human PAICS is a promising target for the treatment of various types of cancer, and it is therefore of high interest to develop a detailed understanding of its reaction mechanism. In the present work, density functional theory calculations are employed to investigate the PAICS reaction mechanism. Starting from the available crystal structures, two large models of the AIRc and SAICARs active sites are built and different mechanistic proposals for the carboxylation and phosphorylation-condensation mechanisms are examined. For the carboxylation reaction, it is demonstrated that it takes place in a two-step mechanism, involving a C-C bond formation followed by a deprotonation of the formed tetrahedral intermediate (known as isoCAIR) assisted by an active site histidine residue. For the phosphorylation-condensation reaction, it is shown that the phosphorylation of CAIR takes place before the condensation reaction with the aspartate. It is further demonstrated that the three active site magnesium ions are involved in binding the substrates and stabilizing the transition states and intermediates of the reaction. The calculated barriers are in good agreement with available experimental data.

3,5,7-Substituted Pyrazolo[4,3-d]Pyrimidine Inhibitors of Cyclin-Dependent Kinases and Cyclin K Degraders.

3,5,7-Trisubstituted pyrazolo[4,3-d]pyrimidines have been identified as potent inhibitors of cyclin-dependent kinases (CDKs), which are established drug targets. Herein, we describe their further structural modifications leading to novel nanomolar inhibitors with strong antiproliferative activity. We determined the crystal structure of fully active CDK2/A2 with 5-(2-amino-1-ethyl)thio-3-cyclobutyl-7-[4-(pyrazol-1-yl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidine (24) at 1.7 Å resolution, confirming the competitive mode of inhibition. Biochemical and cellular assays in lymphoma cell lines confirmed the expected mechanism of action through dephosphorylation of retinoblastoma protein and RNA polymerase II, leading to induction of apoptosis. Importantly, we also revealed an interesting ability of compound 24 to induce proteasome-dependent degradation of cyclin K both in vitro and in a patient-derived xenograft in vivo. We propose that 24 has a dual mechanism of action, acting as a kinase inhibitor and as a molecular glue inducing an interaction between CDK12 and DDB1 that leads to polyubiquitination of cyclin K and its subsequent degradation.

Structural insight into DNA recognition by bacterial transcriptional regulators of the SorC/DeoR family.

The SorC/DeoR family is a large family of bacterial transcription regulators that are involved in the control of carbohydrate metabolism and quorum sensing. To understand the structural basis of DNA recognition, structural studies of two functionally characterized SorC/DeoR family members from Bacillus subtilis were performed: the deoxyribonucleoside regulator bsDeoR and the central glycolytic genes regulator bsCggR. Each selected protein represents one of the subgroups that are recognized within the family. Crystal structures were determined of the N-terminal DNA-binding domains of bsDeoR and bsCggR in complex with DNA duplexes representing the minimal operator sequence at resolutions of 2.3 and 2.1 Å, respectively. While bsDeoRDBD contains a homeodomain-like HTH-type domain, bsCggRDBD contains a winged helix-turn-helix-type motif. Both proteins form C2-symmetric dimers that recognize two consecutive major grooves, and the protein-DNA interactions have been analyzed in detail. The crystal structures were used to model the interactions of the proteins with the full DNA operators, and a common mode of DNA recognition is proposed that is most likely to be shared by other members of the SorC/DeoR family.

Structure of the native pyruvate dehydrogenase complex reveals the mechanism of substrate insertion.

The pyruvate dehydrogenase complex (PDHc) links glycolysis to the citric acid cycle by converting pyruvate into acetyl-coenzyme A. PDHc encompasses three enzymatically active subunits, namely pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. Dihydrolipoyl transacetylase is a multidomain protein comprising a varying number of lipoyl domains, a peripheral subunit-binding domain, and a catalytic domain. It forms the structural core of the complex, provides binding sites for the other enzymes, and shuffles reaction intermediates between the active sites through covalently bound lipoyl domains. The molecular mechanism by which this shuttling occurs has remained elusive. Here, we report a cryo-EM reconstruction of the native E. coli dihydrolipoyl transacetylase core in a resting state. This structure provides molecular details of the assembly of the core and reveals how the lipoyl domains interact with the core at the active site.

3H-Pyrazolo[4,3-f]quinoline-Based Kinase Inhibitors Inhibit the Proliferation of Acute Myeloid Leukemia Cells In Vivo.

The 3H-pyrazolo[4,3-f]quinoline moiety has been recently shown to be a privileged kinase inhibitor core with potent activities against acute myeloid leukemia (AML) cell lines in vitro. Herein, various 3H-pyrazolo[4,3-f]quinoline-containing compounds were rapidly assembled via the Doebner-Povarov multicomponent reaction from the readily available 5-aminoindazole, ketones, and heteroaromatic aldehydes in good yields. The most active compounds potently inhibit the recombinant FLT3 kinase and its mutant forms with nanomolar IC50 values. Docking studies with the FLT3 kinase showed a type I binding mode, where the 3H-pyrazolo group interacts with Cys694 in the hinge region. The compounds blocked the proliferation of AML cell lines harboring oncogenic FLT3-ITD mutations with remarkable IC50 values, which were comparable to the approved FLT3 inhibitor quizartinib. The compounds also inhibited the growth of leukemia in a mouse-disseminated AML model, and hence, the novel 3H-pyrazolo[4,3-f]quinoline-containing kinase inhibitors are potential lead compounds to develop into anticancer agents, especially for kinase-driven cancers.

N-Glycosylation can selectively block or foster different receptor-ligand binding modes.

While DNA encodes protein structure, glycans provide a complementary layer of information to protein function. As a prime example of the significance of glycans, the ability of the cell surface receptor CD44 to bind its ligand, hyaluronan, is modulated by N-glycosylation. However, the details of this modulation remain unclear. Based on atomistic simulations and NMR, we provide evidence that CD44 has multiple distinct binding sites for hyaluronan, and that N-glycosylation modulates their respective roles. We find that non-glycosylated CD44 favors the canonical sub-micromolar binding site, while glycosylated CD44 binds hyaluronan with an entirely different micromolar binding site. Our findings show (for the first time) how glycosylation can alter receptor affinity by shielding specific regions of the host protein, thereby promoting weaker binding modes. The mechanism revealed in this work emphasizes the importance of glycosylation in protein function and poses a challenge for protein structure determination where glycosylation is usually neglected.

Imidazo[1,2-c]pyrimidin-5(6H)-one inhibitors of CDK2: Synthesis, kinase inhibition and co-crystal structure.

Pharmacological inhibition of cyclin-dependent kinases has emerged as a possible treatment option for various cancer types. We recently identified substituted imidazo[1,2-c]pyrimidin-5(6H)-ones as inhibitors of cyclin-dependent kinase 2 (CDK2). Here, we report the synthesis of derivatives modified at positions 2, 3, 6 or 8 prepared using Suzuki-Miyaura cross-coupling, halogenation, Dimroth-type rearrangement and alkylation as the main synthetic methods. The compounds displayed micro- to submicromolar inhibition of CDK2/cyclin E activity. Binding of the most potent compound 3b to CDK2 was determined using isothermal titration calorimetry. The co-crystal structure of 3b in complex with fully active CDK2 was solved, revealing the binding mode of 3b in the ATP pocket and a hydrogen bonding interaction with hinge region residue Leu83. Evaluation against leukaemia cell lines revealed low cytotoxicity, which is in line with the high selectivity towards CDK2. This study demonstrates that substituted imidazo[1,2-c]pyrimidines can be exploited for future kinase inhibitor development.

Structural and functional analysis of the inhibition of equine glutathione transferase A3-3 by organotin endocrine disrupting pollutants.

Organotin compounds are highly toxic environmental pollutants with neurotoxic and endocrine-disrupting effects. They are potent inhibitors of glutathione transferases (GSTs), thus impeding their detoxication and antioxidant functions. Several GSTs, including equine GST A3-3 (EcaGST A3-3), exhibit steroid double-bond isomerase activity and are involved in the biosynthesis of testosterone and progesterone. We have performed enzyme kinetics analyses of the inhibition of EcaGST A3-3 by organotin compounds. We have also solved crystal structures of EcaGST A3-3 in complexes with glutathione, and with glutathione together with covalently bound triethyltin. Our structural data indicate that the tin atom forms strong bonds with a covalent character not only with the glutathione, but also with a tyrosyl residue of the enzyme itself, thereby preventing the release of the glutathione-organotin adduct and completely blocking the enzyme function. This work presents a structural basis for the general mechanism of GST inhibition by organotin compounds and contributes to the understanding of their neurotoxic and endocrine disrupting effects.

Crystal structures of human PAICS reveal substrate and product binding of an emerging cancer target.

The bifunctional human enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (PAICS) catalyzes two essential steps in the de novo purine biosynthesis pathway. PAICS is overexpressed in many cancers and could be a promising target for the development of cancer therapeutics. Here, using gene knockdowns and clonogenic survival and cell viability assays, we demonstrate that PAICS is required for growth and survival of prostate cancer cells. PAICS catalyzes the carboxylation of aminoimidazole ribonucleotide (AIR) and the subsequent conversion of carboxyaminoimidazole ribonucleotide (CAIR) and l-aspartate to N-succinylcarboxamide-5-aminoimidazole ribonucleotide (SAICAR). Of note, we present the first structures of human octameric PAICS in complexes with native ligands. In particular, we report the structure of PAICS with CAIR bound in the active sites of both domains and SAICAR bound in one of the SAICAR synthetase domains. Moreover, we report the PAICS structure with SAICAR and an ATP analog occupying the SAICAR synthetase active site. These structures provide insight into substrate and product binding and the architecture of the active sites, disclosing important structural information for rational design of PAICS inhibitors as potential anticancer drugs.

Structure and steroid isomerase activity of Drosophila glutathione transferase E14 essential for ecdysteroid biosynthesis.

Ecdysteroids are critically important for the formation of the insect exoskeleton. Cholesterol is a precursor of ecdysone and its active form 20-hydroxyecdysone, but some steps in the ecdysteroid biosynthesis pathway remain unknown. An essential requirement of glutathione (GSH) transferase GSTE14 in ecdysteroid biosynthesis has been established in Drosophila melanogaster, but its function is entirely unknown. Here, we have determined the crystal structure of GSTE14 in complex with GSH and investigated the kinetic properties of GSTE14 with alternative substrates. GSTE14 has high-ranking steroid double-bond isomerase activity, albeit 50-fold lower than the most efficient mammalian GSTs. Corresponding steroid isomerizations are unknown in insects, and their exact physiological role remains to be shown. Nonetheless, the essential enzyme GSTE14 is here demonstrated to be catalytically competent and have a steroid-binding site.

3,5,7-Substituted Pyrazolo[4,3- d]pyrimidine Inhibitors of Cyclin-Dependent Kinases and Their Evaluation in Lymphoma Models.

Cyclin-dependent kinases are therapeutic targets frequently deregulated in various cancers. By convenient alkylation of the 5-sulfanyl group, we synthesized 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2) H-pyrazolo[4,3- d]pyrimidines with various substitutions at position 5 with potent antiproliferative activity in non-Hodgkin lymphoma cell lines. The most potent derivative 4.35 also displayed activities across more than 60 cancer cell lines. The kinase profiling confirmed high selectivity of 4.35 toward cyclin-dependent kinases (CDKs) 2, 5, and 9, and the cocrystal with CDK2/cyclin A2 revealed its binding in the active site. Cultured lymphoma cell lines treated with 4.35 showed dephosphorylation of CDK substrates, cleavage of PARP-1, downregulation of XIAP and MCL-1, and activation of caspases, which collectively confirmed ongoing apoptosis. Moreover, 4.35 demonstrated significant activity in various cell line xenograft and patient-derived xenograft mouse models in vivo both as a monotherapy and as a combination therapy with the BCL2-targeting venetoclax. These findings support further studies of combinatorial treatment based on CDK inhibitors.

Crystal structure of native α-L-rhamnosidase from Aspergillus terreus.

α-L-Rhamnosidases cleave terminal nonreducing α-L-rhamnosyl residues from many natural rhamnoglycosides. This makes them catalysts of interest for various biotechnological applications. The X-ray structure of the GH78 family α-L-rhamnosidase from Aspergillus terreus has been determined at 1.38 Šresolution using the sulfur single-wavelength anomalous dispersion phasing method. The protein was isolated from its natural source in the native glycosylated form, and the active site contained a glucose molecule, probably from the growth medium. In addition to its catalytic domain, the α-L-rhamnosidase from A. terreus contains four accessory domains of unknown function. The structural data suggest that two of these accessory domains, E and F, might play a role in stabilizing the aglycon portion of the bound substrate.

In situ proteolysis of an N-terminal His tag with thrombin improves the diffraction quality of human aldo-keto reductase 1C3 crystals.

Human aldo-keto reductase 1C3 (AKR1C3) stereospecifically reduces steroids and prostaglandins and is involved in the biotransformation of xenobiotics. Its role in various cancers makes it a potential therapeutic target for the development of inhibitors. Recombinant AKR1C3 with a thrombin-cleavable N-terminal His6 tag was expressed from a pET-28(+) vector for structural studies of enzyme-inhibitor complexes. A modified in situ proteolysis approach was applied to specifically remove the His tag by thrombin cleavage during crystallization screening trials. This improved the morphology and diffraction quality of the crystals and allowed the acquisition of high-resolution diffraction data and structure solution. This approach may be generally applicable to other proteins expressed using the pET-28(+) vector.

Crystal structure of native ß-N-acetylhexosaminidase isolated from Aspergillus oryzae sheds light onto its substrate specificity, high stability, and regulation by propeptide.

ß-N-acetylhexosaminidase from the fungus Aspergillus oryzae is a secreted extracellular enzyme that cleaves chitobiose into constituent monosaccharides. It belongs to the GH 20 glycoside hydrolase family and consists of two N-glycosylated catalytic cores noncovalently associated with two 10-kDa O-glycosylated propeptides. We used X-ray diffraction and mass spectrometry to determine the structure of A. oryzae ß-N-acetylhexosaminidase isolated from its natural source. The three-dimensional structure determined and refined to a resolution of 2.3 Å revealed that this enzyme is active as a uniquely tight dimeric assembly further stabilized by N- and O-glycosylation. The propeptide from one subunit forms extensive noncovalent interactions with the catalytic core of the second subunit in the dimer, and this chain swap suggests the distinctive structural mechanism of the enzyme's activation. Unique structural features of ß-N-acetylhexosaminidase from A. oryzae define a very stable and robust framework suitable for biotechnological applications. The crystal structure reported here provides structural insights into the enzyme architecture as well as the detailed configuration of the active site. These insights can be applied to rational enzyme engineering. DATABASE: Structural data are available in the PDB database under the accession number 5OAR. ENZYME: ß-N-acetylhexosaminidase (EC 3.2.1.52).