Imidazo[1,2‐c]pyrimidin‐5(6H)‐one as a novel core of cyclin‐dependent kinase 2 inhibitors: Synthesis,activity measurement,docking, and quantum mechanical scoring

We report on the synthesis, activity testing, docking, and quantum mechanical scoring of novel imidazo[1,2‐c]pyrimidin‐5(6H)‐one scaffold for cyclin‐dependent kinase 2 (CDK2) inhibition. A series of 26 compounds substituted with aromatic moieties at position 8 has been tested in in vitro enzyme assays and shown to inhibit CDK2. 2D structure‐activity relationships have ascertained that small substituents at position 8 (up to the size of naphtyl or methoxyphenyl) generally lead to single‐digit micromolar IC₅₀ values, whereas bigger substituents (substituted biphenyls) decreased the compounds' activities. The binding modes of the compounds obtained using Glide docking have exhibited up to 2 hinge‐region hydrogen bonds to CDK2 and differed in the orientation of the inhibitor core and the placement of the 8‐substituents. Semiempirical quantum mechanics‐based scoring identified probable favourable binding modes, which will serve for future structure‐based design and synthetic optimization of substituents of the heterocyclic core. In summary, we have identified a novel core for CDK2 inhibition and will explore it further to increase the potencies of the compounds and also monitor selectivities against other protein kinases.     

Recognition in action: DNA mimicry

Proteins that mimic DNA present a surface that is similar in shape and chemical character to the DNA double helix. These DNA mimics bind to DNA-binding proteins, taking the place of DNA. Natural DNA mimics play roles in genetic regulation and defense.     

Characterization of the complex between mannose-binding lectin trimer and mannose-binding lectin-associated serine proteases

Mannose-binding lectin (MBL) is an oligomeric serum lectin involved in innate immunity. Human MBL is complexed with three types of serine proteases (MASP-1, MASP-2 and MASP-3) and two types of their truncated forms (sMAP and MAp44). When an MBL complex binds to carbohydrates of pathogens, the complement system is activated via the lectin pathway. Human MBL is a mixture of different sized oligomers that range mainly from trimers to hexamers. It has been suggested that different MBL oligomers may have distinct MASP compositions. In the present study, an MBL trimer (MBL-I) exclusive of other oligomers was isolated from human serum by chromatography. Immunoblot analysis of MBL-I revealed that it had been co-purified with MASP-1 and sMAP. This suggests that MASP-1 and sMAP are bound to each other in MBL-I. The MBL-I complex was found to activate C2, but to lack the ability to activate C4 due to the absence of MASP-2.     

Laying out ground rules for protein-aided nanofabrication: ZnO synthesis at 70°C as a case study

Designer proteins that incorporate solid-binding peptides hold promise to control the nucleation, growth, morphology, and assembly of inorganic phases under mild conditions of temperature and pressure. However, protein-aided nanofabrication remains more art than science and some materials can only be synthesized at temperatures that cause most mesophilic proteins to unfold. Using zinc oxide (ZnO) synthesis at 70°C as case study, we show here that seemingly unimportant variables, such as the carry-over concentration of Tris buffer and the "empty" host protein scaffold can exert a significant influence on materials morphology. We also show that, once well-controlled conditions are established, thermodynamic predictions and adsorption isotherms are powerful tools to understand how various ZnO-binding sequence inserted within the thermostable framework of Escherichia coli thioredoxin A (TrxA) affect inorganic morphogenesis.     

Balance between DNA‐binding affinity and specificity enables selective recognition of longer target sequences in vivo

Although genome‐editing enzymes such as TALEN and CRISPR/Cas9 are being widely used, they have an essential limitation in that their relatively high‐molecular weight makes them difficult to be delivered to cells. To develop a novel genome‐editing enzyme with a smaller molecular weight, we focused on the engrailed homeodomain (EHD). We designed and constructed proteins composed of two EHDs connected by a linker to increase sequence specificity. In bacterial one‐hybrid assays and electrophoresis mobility shift assay analyses, the created proteins exhibited good affinity for DNA sequences consisting of two tandemly aligned EHD target sequences. However, they also bound to individual EHD targets. To avoid binding to single target sites, we introduced amino acid mutations to reduce the protein–DNA affinity of each EHD monomer and successfully created a small protein with high specificity for tandem EHD target sequences.     

Insights into nonspecific binding of homeodomains from a structure of MATalpha2 bound to DNA

The 2.1-A resolution crystal structure of the MATalpha2 homeodomain bound to DNA reveals the unexpected presence of two nonspecifically bound alpha2 homeodomains, in addition to the two alpha2 homeodomains bound to canonical alpha2 binding sites. One of the extra homeodomains makes few base-specific contacts, while the other extra homeodomain binds to DNA in a previously unobserved manner. This unusually bound homeodomain is rotated on the DNA, making possible major groove contacts by side-chains that normally do not contact the DNA. This alternate docking may represent one way in which homeodomains sample nonspecific DNA sequences.     

Free energy of proton binding in proteins

In this article we use literature data on the titration of denatured ribonuclease to test the accuracy of proton-binding distributions obtained using our recent approach employing moments. We find that using only the local slope of the titration curve at a small number of points (five, for example) we can reproduce the detailed proton-binding distribution at all pH values. Our method gives the complete proton-binding polynomial for a given protein and each coefficient in this polynomial in turn yields the free energy for binding a given number of protons in all ways to the protein. Using these net free energies, we can then compute the average proton-binding free energy per proton as a function of the fraction of protons bound. We find that this function is remarkably similar for different proteins, even for proteins that exhibit quite different titration behavior. For the special case of binding to independent sites, we obtain simple relations for the first and last terms in the free energy per-proton function. For this special case we also can calculate the distribution functions giving the probability that a molecule has a given number of positive or negative charges and the joint distribution that a molecule simultaneously has a given number of positive and negative charge.     

Starch-synthase III family encodes a tandem of three starch-binding domains

The starch-synthase III (SSIII), with a total of 1025 residues, is one of the enzymes involved in plants starch synthesis. SSIII from Arabidopsis thaliana contains a putative N-terminal transit peptide followed by a 557-amino acid SSIII-specific domain (SSIII-SD) with three internal repeats and a C-terminal catalytic domain of 450 amino acids. Here, using computational characterization techniques, we show that each of the three internal repeats encodes a starch-binding domain (SBD). Although the SSIII from A. thaliana and its close homologous proteins show no detectable sequence similarity with characterized SBD sequences, the amino acid residues known to be involved in starch binding are well conserved.     

Structural and biochemical characterization of native and recombinant single insulin-like growth factor-binding domain protein (SIBD-1) from the Central American hunting spider Cupiennius salei (Ctenidae)

Cupiennius salei single insulin-like growth factor binding domain protein (SIBD-1) is an 8.6 kDa Cys-, Pro-, and Gly-rich protein, discovered in the hemocytes of the Central American hunting spider Cupiennius salei. SIBD-1 exhibits high sequence similarity to the N-terminal domain of the insulin-like growth factor-binding protein superfamily and has been reported to play an important role in the spider's immune system. Here, the recombinant expression and the elucidation of the three-dimensional structure of recombinant SIBD-1 and the characterization of the sugar moiety at Thr2 of native SIBD-1 is described in detail.     

NMR solution structures of actin depolymerizing factor homology domains

Actin is one of the most conserved proteins in nature. Its assembly and disassembly are regulated by many proteins, including the family of actin‐depolymerizing factor homology (ADF‐H) domains. ADF‐H domains can be divided into five classes: ADF/cofilin, glia maturation factor (GMF), coactosin, twinfilin, and Abp1/drebrin. The best‐characterized class is ADF/cofilin. The other four classes have drawn much less attention and very few structures have been reported. This study presents the solution NMR structure of the ADF‐H domain of human HIP‐55‐drebrin‐like protein, the first published structure of a drebrin‐like domain (mammalian), and the first published structure of GMF β (mouse). We also determined the structures of mouse GMF γ, the mouse coactosin‐like domain and the C‐terminal ADF‐H domain of mouse twinfilin 1. Although the overall fold of the five domains is similar, some significant differences provide valuable insights into filamentous actin (F‐actin) and globular actin (G‐actin) binding, including the identification of binding residues on the long central helix. This long helix is stabilized by three or four residues. Notably, the F‐actin binding sites of mouse GMF β and GMF γ contain two additional β‐strands not seen in other ADF‐H structures. The G‐actin binding site of the ADF‐H domain of human HIP‐55‐drebrin‐like protein is absent and distorted in mouse GMF β and GMF γ.     

Insight into the stability of the hydrophobic binding proteins of Escherichia coli: assessing the proteins for use as biosensors

Spectroscopic methods were used to monitor the unfolding of the leucine specific (LS) and the leucine-isoleucine-valine (LIV) binding proteins. Our studies indicate that ligand-free protein undergoes a simple two-state unfolding, whereas the protein-ligand complex undergoes a three-state unfolding model. Ligand binding causes significant stabilization of both proteins. There is correlation between ligand hydrophobicity and protein stabilization: the most hydrophobic ligand, isoleucine, causes the most significant stabilization of LIV protein. A disulfide bond present in N-domain of both proteins makes a large contribution to the protein stability of these periplasmic binding receptors.     

Mining the soluble chloroplast proteome by affinity chromatography

Chloroplasts are fundamental organelles enabling plant photoautotrophy. Besides their outstanding physiological role in fixation of atmospheric CO(2), they harbor many important metabolic processes such as biosynthesis of amino acids, vitamins or hormones. Technical advances in MS allowed the recent identification of most chloroplast proteins. However, for a deeper understanding of chloroplast function it is important to obtain a complete list of constituents, which is so far limited by the detection of low-abundant proteins. Therefore, we developed a two-step strategy for the enrichment of low-abundant soluble chloroplast proteins from Pisum sativum and their subsequent identification by MS. First, chloroplast protein extracts were depleted from the most abundant protein ribulose-1,5-bisphosphate carboxylase/oxygenase by SEC or heating. Further purification was carried out by affinity chromatography, using ligands specific for ATP- or metal-binding proteins. By these means, we were able to identify a total of 448 proteins including 43 putative novel chloroplast proteins. Additionally, the chloroplast localization of 13 selected proteins was confirmed using yellow fluorescent protein fusion analyses. The selected proteins included a phosphoglycerate mutase, a cysteine protease, a putative protein kinase and an EF-hand containing substrate carrier protein, which are expected to exhibit important metabolic or regulatory functions.     

NMR characterization of a pH-dependent equilibrium between two folded solution conformations of the pheromone-binding protein from Bombyx mori

NMR spectroscopic changes as a function of pH in solutions of the pheromone-binding protein of Bombyx mori (BmPBP) show that BmPBP undergoes a conformational transition between pH 4.9 and 6.0. At pH below 4.9 there is a single "acid form" (A), and a homogeneous "basic form" (B) exists at pH above 6.0. Between pH 5 and 6, BmPBP exists as a mixture of A and B in slow exchange on the NMR chemical shift time scale, with the transition midpoint at pH 5.4. The form B has a well-dispersed NMR spectrum, indicating that it represents a more structured, "closed" conformation than form A, which has a significantly narrower chemical shift dispersion. Conformational transitions of the kind observed here may explain heterogeneity reported for a variety of odorant-binding proteins, and it will be of interest to further investigate possible correlations with pH-dependent regulation of ligand binding and release in the biological function of this class of proteins.     

Differential expression of two C‐type lectins in grass carp Ctenopharyngodon idella and their response to grass carp reovirus

The cDNAs of two C‐type lectins in grass carp Ctenopharyngodon idella, galactose‐binding lectin (galbl) and mannose‐binding lectin (mbl), were cloned and analysed in this study. Both of them exhibited the highest expression level in liver, whereas their expression pattern differed in early phase of embryonic development. Following exposure to grass carp reovirus (GCRV), the mRNA expression level of galbl and mbl was significantly up‐regulated in liver and intestine.     

Fluorescent dyes as probes to study lipid-binding proteins

We studied the equilibrium binding of two hydrophobic fluorescent dyes, ANS and bisANS, to four members of a family of intracellular lipid-binding proteins: IFABP, CRABP I, CRABP II, and ILBP. The spectral and binding parameters for the probes bound to the proteins were determined. Typically, there was a single binding site on each protein for the ligands. However, IFABP cooperatively bound a second bisANS molecule in the binding pocket. Comparative analysis of affinities and spectral characteristics for the two probes allowed us to examine the contributions of electrostatic and hydrophobic interactions to the binding process, and to address some aspects of the internal structure of the studied proteins.