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Steroid bile acid fused tetrazoles were screened for ability to inhibit aldo–keto reductase 1C3 (AKR1C3), a target for cancer treatment. The X-ray structure of AKR1C3 in complex with a bile acid tetrazole inhibitor is presented.

Human PAICS is a bifunctional enzyme that is involved in the

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 bothin vitroandin 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.

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.

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.

3,5,7-Trisubstituted pyrazolo[4,3

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.

While protein function is largely encoded in DNA, it is also modulated by a complementary and often invisible layer of information encoded by glycans attached to the protein surface. The CD44-hyaluronan complex involved in inflammatory responses and cell migration is a prime example of the significance of glycans, as the ability of the cell surface receptor CD44 to bind its ligand, hyaluronan, is modulated by N-glycosylation. Intriguingly, how glycans can regulate this binding process, and the activation of CD44, has remained unclear. In this work, 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. While non-glycosylated CD44 is found to favor the canonical sub-micromolar binding site, 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 constitutes a challenge for protein structure determination where glycosylation is typically not observed.

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 Å resolution 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.

© International Union of Crystallography, 2018. 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 His6tag 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.In situ specific proteolytic removal of a His tag by thrombin improves the morphology and diffraction quality of crystals of human aldo-keto reductase 1C3.

Botulinum neurotoxins (BoNTs) are the most potent toxins known to man and are also used to treat an increasing number of medical disorders. They target the neuromuscular junction and inhibit synapt ...

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.

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.

The hyaluronate receptor CD44 plays role in cell adhesion and migration and is involved in tumor metastasis. The extracellular domain of CD44 comprises the hyaluronate-binding domain (HABD) and the membrane-proximal stem region; the short intracellular portion interacts with adaptor proteins and triggers signaling pathways. Binding of hyaluronate to CD44 HABD induces an allosteric conformational change, which results in CD44 shedding. A poorly characterized epitope in human CD44 HABD is recognized by the murine monoclonal antibody MEM-85, which cross-blocks hyaluronate binding to CD44 and also induces CD44 shedding. MEM-85 is of therapeutic interest, as it inhibits growth of lung cancer cells in murine models. In this work, we employed a combination of biophysical methods to determine the MEM-85 binding epitope in CD44 HABD and to provide detailed insight into the mechanism of MEM-85 action. In particular, we constructed a single-chain variable fragment (scFv) of MEM-85 as a tool for detailed characterization of the CD44 HABD–antibody complex and identified residues within CD44 HABD involved in the interaction with scFv MEM-85 by NMR spectroscopy and mutational analysis. In addition, we built a rigid body model of the CD44 HABD–scFv MEM-85 complex using a low-resolution structure obtained by small-angle X-ray scattering. The MEM-85 epitope is situated in the C-terminal part of CD44 HABD, rather than the hyaluronate-binding groove, and the binding of MEM-85 induces a structural reorganization similar to that induced by hyaluronate. Therefore, the mechanism of MEM-85 cross-blocking of hyaluronate binding is likely of an allosteric, relay-like nature.

Single-chain variable antibody fragments (scFvs) are molecules with immense therapeutic and diagnostic potential. Knowledge of their three-dimensional structure is important for understanding their antigen-binding mode as well as for protein-engineering approaches such as antibody humanization. A major obstacle to the crystallization of single-chain variable antibody fragments is their relatively poor homogeneity caused by spontaneous oligomerization. A new approach to optimization of the crystallizability of single-chain variable antibody fragments is demonstrated using a representative single-chain variable fragment derived from the anti-CD3 antibody MEM-57. A Thermofluor-based assay was utilized to screen for optimal conditions for antibody-fragment stability and homogeneity. Such an optimization of the protein storage buffer led to a significantly improved ability of the scFv MEM-57 to yield crystals.

β-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 β-N-acetylhexosaminidase isolated from its natural source. The three-dimensional structure determined and refined to a resolution of 2.3 A 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 β-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 β-N-acetylhexosaminidase (EC 3.2.1.52).

Background Relapsed acute lymphoblastic leukemia (ALL) is one of the main causes of mortality in childhood malignancies. Previous genetic studies demonstrated that chemoresistant ALL is driven by activating mutations in NT5C2, the gene encoding cytosolic 5´-nucleotidase (cN-II). However, molecular mechanisms underlying this hyperactivation are still unknown. Here, we present kinetic and structural properties of cN-II variants that represent 75 % of mutated alleles in patients who experience relapsed ALL (R367Q, R238W and L375F). Results Enzyme kinetics measurements revealed that the mutants are consitutively active without need for allosteric activators. This shows that hyperactivity is not caused by a direct catalytic effect but rather by misregulation of cN-II. X-ray crystallography combined with mass spectrometry-based techniques demonstrated that this misregulation is driven by structural modulation of the oligomeric interface within the cN-II homotetrameric assembly. These specific conformational changes are shared between the studied variants, despite the relatively random spatial distribution of the mutations. Conclusions These findings define a common molecular mechanism for cN-II hyperactivity, which provides a solid basis for targeted therapy of leukemia. Our study highlights the cN-II oligomerization interface as an attractive pharmacological target. Electronic supplementary material The online version of this article (doi:10.1186/s12915-016-0313-y) contains supplementary material, which is available to authorized users.

Deoxyribonucleoside regulator (DeoR) from Bacillus subtilis negatively regulates expression of enzymes involved in the catabolism of deoxyribonucleosides and deoxyribose. The DeoR protein is homologous to the sorbitol operon regulator family of metabolic regulators and comprises an N-terminal DNA-binding domain and a C-terminal effector-binding domain. We have determined the crystal structure of the effector-binding domain of DeoR (C-DeoR) in free form and in covalent complex with its effector deoxyribose-5-phosphate (dR5P). This is the first case of a covalently attached effector molecule captured in the structure of a bacterial transcriptional regulator. The dR5P molecule is attached through a Schiff base linkage to residue Lys141. The crucial role of Lys141 in effector binding was confirmed by mutational analysis and mass spectrometry of Schiff base adducts formed in solution. Structural analyses of the free and effector-bound C-DeoR structures provided a structural explanation for the mechanism of DeoR function as a molecular switch. Databases Atomic coordinates and structure factors for crystal structures of free C-DeoR and the covalent Schiff base complex of C-DeoR with dR5P have been deposited in the Protein Data Bank with accession codes 4OQQ and 4OQP, respectively. Structured digital abstract C-DeoR and C-DeoR bind by x-ray crystallography (View interaction) DeoR and DeoR bind by molecular sieving (1, 2)