Small-molecule and mutational analysis of allosteric Eg5 inhibition by monastrol

Background  

A recent crystal structure of monastrol in a ternary complex with the kinesin Eg5 motor domain highlights a novel, induced-fit drug binding site at atomic resolution. Mutational obliteration of the monastrol binding site results in a monastrol-resistant, but otherwise catalytically active Eg5 motor domain. However, considering the conformational changes at this site, it is unclear what specific interactions stabilize the interaction between monastrol and the Eg5 motor domain.     

Biochemical evidence for the tyrosine involvement in cationic intermediate stabilization in mouse β-carotene 15, 15'-monooxygenase

Background  

β-carotene 15,15'-monooxygenase (BCMO1) catalyzes the crucial first step in vitamin A biosynthesis in animals. We wished to explore the possibility that a carbocation intermediate is formed during the cleavage reaction of BCMO1, as is seen for many isoprenoid biosynthesis enzymes, and to determine which residues in the substrate binding cleft are necessary for catalytic and substrate binding activity. To test this hypothesis, we replaced substrate cleft aromatic and acidic residues by site-directed mutagenesis. Enzymatic activity was measured in vitro using His-tag purified proteins and in vivo in a β-carotene-accumulating E. coli system.     

Nonnatural amino acid incorporation into the methionine 214 position of the metzincin <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> alkaline protease

Background  

The alkaline protease from Pseudomonas aeruginosa(AprA) is a member of the metzincin superfamily of metalloendoproteases. A key feature of these proteases is a conserved methionine-containing 1,4-tight β turn at the base of the active site zinc binding region.     

Orthophosphate binding at the dimer interface of <Emphasis Type="Italic">Corynebacterium callunae</Emphasis> starch phosphorylase: mutational analysis of its role for activity and stability of the enzyme

Background  

Orthophosphate recognition at allosteric binding sites is a key feature for the regulation of enzyme activity in mammalian glycogen phosphorylases. Protein residues co-ordinating orthophosphate in three binding sites distributed across the dimer interface of a non-regulated bacterial starch phosphorylase (from Corynebacterium callunae) were individually replaced by Ala to interrogate their unknown function for activity and stability of this enzyme.     

Effects of synthetic food colorants on the interaction between norfloxacin and bovine serum albumin by fluorescence spectroscopy

Abstract  

The effects of synthetic food colorants like tartrazine, sunset yellow, and erythrosine on the binding reaction between norfloxacin and bovine serum albumin (BSA) were investigated by fluorescence spectroscopy. Results showed that food colorants bound to BSA by van der Waals force and hydrogen bonding formation and norfloxacin by electrostatic interaction. In addition, marker competitive experiments suggested that the primary binding site for both norfloxacin and food colorants was located at subdomain IIA of BSA (site I). The presence of food colorants could alter the binding constant and distance between BSA and norfloxacin. The effects of colorants were dependent on their concentrations and binding affinity to BSA. The interaction could result in the change of the free, biologically active fraction of norfloxacin in blood.     

Systematic substitutions at BLIP position 50 result in changes in binding specificity for class A β-lactamases

Background

The production of β-lactamases by bacteria is the most common mechanism of resistance to the widely prescribed β-lactam antibiotics. β-lactamase inhibitory protein (BLIP) competitively inhibits class A β-lactamases via two binding loops that occlude the active site. It has been shown that BLIP Tyr50 is a specificity determinant in that substitutions at this position result in large differential changes in the relative affinity of BLIP for class A β-lactamases.

Results

In this study, the effect of systematic substitutions at BLIP position 50 on binding to class A β-lactamases was examined to further explore the role of BLIP Tyr50 in modulating specificity. The results indicate the sequence requirements at position 50 are widely different depending on the target β-lactamase. Stringent sequence requirements were observed at Tyr50 for binding Bacillus anthracis Bla1 while moderate requirements for binding TEM-1 and relaxed requirements for binding KPC-2 β-lactamase were seen. These findings cannot be easily rationalized based on the β-lactamase residues in direct contact with BLIP Tyr50 since they are identical for Bla1 and KPC-2 suggesting that differences in the BLIP-β-lactamase interface outside the local environment of Tyr50 influence the effect of substitutions.

Conclusions

Results from this study and previous studies suggest that substitutions at BLIP Tyr50 may induce changes at the interface outside its local environment and point to the complexity of predicting the impact of substitutions at a protein-protein interaction interface.

     

Low affinity PEGylated hemoglobin from Trematomus bernacchii, a model for hemoglobin-based blood substitutes

Background

The majority of peroxisomal matrix proteins destined for translocation into the peroxisomal lumen are recognised via a C-terminal Peroxisomal Target Signal type 1 by the cycling receptor Pex5p. The only structure to date of Pex5p in complex with a cargo protein is that of the C-terminal cargo-binding domain of the receptor with sterol carrier protein 2, a small, model peroxisomal protein. In this study, we have tested the contribution of a second, ancillary receptor-cargo binding site, which was found in addition to the characterised Peroxisomal Target Signal type 1.

Results

To investigate the function of this secondary interface we have mutated two key residues from the ancillary binding site and analyzed the level of binding first by a yeast-two-hybrid assay, followed by quantitative measurement of the binding affinity and kinetics of purified protein components and finally, by in vivo measurements, to determine translocation capability. While a moderate but significant reduction of the interaction was found in binding assays, we were not able to measure any significant defects in vivo.

Conclusions

Our data therefore suggest that at least in the case of sterol carrier protein 2 the contribution of the second binding site is not essential for peroxisomal import. At this stage, however, we cannot rule out that other cargo proteins may require this ancillary binding site.     

Structural explanation for the tunable substrate specificity of an <Emphasis Type="Italic">E. coli</Emphasis> nucleoside hydrolase: insights from molecular dynamics simulations

Parasitic protozoa rely on nucleoside hydrolases that play key roles in the purine salvage pathway by catalyzing the hydrolytic cleavage of the N-glycosidic bond that connects nucleobases to ribose sugars. Cytidine–uridine nucleoside hydrolase (CU–NH) is generally specific toward pyrimidine nucleosides; however, previous work has shown that replacing two active site residues with Tyr, specifically the Thr223Tyr and Gln227Tyr mutations, allows CU–NH to process inosine. The current study uses molecular dynamics (MD) simulations to gain atomic-level insight into the activity of wild-type and mutant E. coli CU–NH toward inosine. By examining systems that differ in the identity and protonation states of active site catalytic residues, key enzyme-substrate interactions that dictate the substrate specificity of CU–NH are identified. Regardless of the wild-type or mutant CU–NH considered, our calculations suggest that inosine binding is facilitated by interactions of the ribose moiety with active site residues and Ca²⁺, and π-interactions between two His residues (His82 and His239) and the nucleobase. However, the lack of observed activity toward inosine for wild-type CU–NH is explained by no residue being correctly aligned to stabilize the departing nucleobase. In contrast, a hydrogen-bonding network between hypoxanthine and a newly identified general acid (Asp15) is present when the two Tyr mutations are engineered into the active site. Investigation of the single CU–NH mutants reveals that this hydrogen-bonding network is only maintained when both Tyr mutations are present due to a π-interaction between the residues. These results rationalize previous experiments that show the single Tyr mutants are unable to efficiently hydrolyze inosine and explain how the Tyr residues work synergistically in the double mutant to stabilize the nucleobase leaving group during hydrolysis. Overall, our simulations provide a structural explanation for the substrate specificity of nucleoside hydrolases, which may be used to rationally develop new treatments for kinetoplastid diseases.

Graphical Abstract

Open image in new window

     

Displacement affinity chromatography of protein phosphatase one (PP1) complexes

Background  

Protein phosphatase one (PP1) is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PP1 is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif.     

PocketPicker: analysis of ligand binding-sites with shape descriptors

Background  

Identification and evaluation of surface binding-pockets and occluded cavities are initial steps in protein structure-based drug design. Characterizing the active site's shape as well as the distribution of surrounding residues plays an important role for a variety of applications such as automated ligand docking or in situ modeling. Comparing the shape similarity of binding site geometries of related proteins provides further insights into the mechanisms of ligand binding.     

Synthesis and binding properties of guanidinium biscarboxylates

Abstract  

The ammonium ion binding site of the enzyme glutaminase HisF inspired us to design guanidinium biscarboxylates as potential self-organized ionophores in molecular recognition. The syntheses of the title compounds based on aliphatic and aromatic building blocks, along with a general method for the preparation of δ-aminoethoxyacetic acids, are presented in this work. Investigation of the binding properties of the title compounds in dimethyl sulfoxide (DMSO) and methanol solution revealed no ammonium ion affinity, but interaction of the guanidinium moiety with acetate ions.     

Computer-assisted combinatorial design of bicyclic thymidine analogs as inhibitors of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> thymidine monophosphate kinase

Abstract  

Thymidine monophosphate kinase (TMPK_mt) is an essential enzyme for nucleotide metabolism in Mycobacterium tuberculosis, and thus an attractive target for novel antituberculosis agents. In this work, we have explored the chemical space around the 2′,3′-bicyclic thymidine nucleus by designing and in silico screening of a virtual focused library selected via structure based methods to identify more potent analogs endowed with favorable ADME-related properties. In all the library members we have exchanged the ribose ring of the template with a cyclopentane moiety that is less prone to enzymatic degradation. In addition, we have replaced the six-membered 2′,3′-ring by a number of five-membered and six-membered heterocyclic rings containing alternative proton donor and acceptor groups, to exploit the interaction with the carboxylate groups of Asp9 and Asp163 as well as with several cationic residues present in the vicinity of the TMPK_mt binding site. The three-dimensional structure of the TMPK_mt complexed with 5-hydroxymethyl-dUMP, an analog of dTMP, was employed to develop a QSAR model, to parameterize a scoring function specific for the TMPK_mt target and to select analogues which display the highest predicted binding to the target. As a result, we identified a small highly focused combinatorial subset of bicyclic thymidine analogues as virtual hits that are predicted to inhibit the mycobacterial TMPK in the submicromolar concentration range and to display favorable ADME-related properties.     

Iron-sulfur cluster biosynthesis: characterization of iron nucleation sites for assembly of the [2Fe-2S]2+ cluster core in IscU proteins

ISU (eukaryotes) and IscU (prokaryotes) are a homologous family of proteins that appear to provide a platform for assembly of [2Fe-2S] centers prior to delivery to a target apoprotein. The intermediate [2Fe-2S] IscU-bound cluster is formed by delivery of iron and sulfur to the apo-IscU, with the latter delivered through an IscS-mediated reaction. The identity of the iron donor is not yet established. In this report we characterize iron-binding sites on IscU that appear to nucleate [2Fe-2S] cluster assembly. This iron-bound form of IscU is shown to be viable for subsequent IscS-mediated assembly of holo-IscU. Following on recent reports, we demonstrate the persulfide form of IscU to be a dead-end complex that is incapable of forming holoprotein after addition of ferrous or ferric ion. The latter observation reflects the low binding affinity of persulfido IscU for iron ion.     

Biosynthesis of the proteasome inhibitor syringolin A: the ureido group joining two amino acids originates from bicarbonate

Background  

Syringolin A, an important virulence factor in the interaction of the phytopathogenic bacterium Pseudomonas syringaepv. syringaeB728a with its host plant Phaseolus vulgaris(bean), was recently shown to irreversibly inhibit eukaryotic proteasomes by a novel mechanism. Syringolin A is synthesized by a mixed non-ribosomal peptide synthetase/polyketide synthetase and consists of a tripeptide part including a twelve-membered ring with an N-terminal valine that is joined to a second valine via a very unusual ureido group. Analysis of sequence and architecture of the syringolin A synthetase gene cluster with the five open reading frames sylA-sylEallowed to formulate a biosynthesis model that explained all structural features of the tripeptide part of syringolin A but left the biosynthesis of the unusual ureido group unaccounted for.     

Insertion of a small peptide of six amino acids into the β7–β8 loop of the p51 subunit of HIV-1 reverse transcriptase perturbs the heterodimer and affects its activities

Background  

HIV-1 RT is a heterodimeric enzyme, comprising of the p66 and p51 subunits. Earlier, we have shown that the β7-β8 loop of p51 is a key structural element for RT dimerization (Pandey et al., Biochemistry 40: 9505, 2001). Deletion or alanine substitution of four amino acid residues of this loop in the p51 subunit severely impaired DNA binding and catalytic activities of the enzyme. To further examine the role of this loop in HIV-1 RT, we have increased its size such that the six amino acids loop sequences are repeated in tandem and examined its impact on the dimerization process and catalytic function of the enzyme.     

Role of the linker region in the expression of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> glucoamylase

Background  

Rhizopus oryzaeglucoamylase (RoGA) consists of three domains: an amino (N)-terminal raw starch-binding domain (SBD), a glycosylated linker domain, and a carboxy (C)-terminal catalytic domain. The 36-amino-acid linker region (residues 132–167) connects the two functional domains, but its structural and functional roles are unclear.     

A molecular modelling study of the interaction of noradrenaline with theβ 2-adrenergic receptor

Summary A model of the ₂-adrenergic receptor binding site is built from the primary structure of the receptor, experimental evidence for key binding residues and analogy with a homologous protein of partially determined structure. It is suggested that residues Trp-109, Thr-110 and Asp-113 are involved in ligand binding. Noradrenaline is successfully docked into this model, and the results of an INDO molecular orbital calculation on the complex indicate that a charge transfer interaction between Trp-109 and noradrenaline is possible.     

Bcr is a substrate for Transglutaminase 2 cross-linking activity

Background  

Breakpoint cluster region (Bcr) is a multi-domain protein that contains a C-terminal GTPase activating protein (GAP) domain for Rac. Transglutaminase 2 (TG2) regulates Bcr by direct binding to its GAP domain. Since TG2 has transglutaminase activity that has been implicated in the response to extreme stress, we investigated if Bcr can also act as a substrate for TG2.     

Small molecules targeting the NEDD8·NAE protein–protein interaction

Ubiquitination is a major controller of protein homeostasis in cells. Some ubiquitination pathways are modulated by a NEDDylation cascade, that also features E1 – 3 enzymes. The E1 enzyme in the NEDDylation cascade involves a protein–protein interaction (PPI) between NEDD8 (similar to ubiquitin) and NAE (NEDD8 Activating Enzyme). A small molecule inhibitor of the ATP binding site in NAE is in clinical trials. We hypothesized a similar effect could be induced by disrupting the NEDD8·NAE PPI, though, to the best of our knowledge, no small molecules have been reported to disrupt this to date. In the research described here, Exploring Key Orientations (EKO) was used to evaluate several chemotype designs for their potential to disrupt NEDD8·NAE; specifically, for their biases towards orientation of side-chains in similar ways to protein segments at the interface. One chemotype design was selected, and a targeted library of 24 compounds was made around this theme via solid phase synthesis. An entry level hit for disrupting NEDD8·NAE was identified from this library on the basis of its ability to bind NAE (K_i of 6.4 ± 0.3 μM from fluorescence polarization), inhibit NEDDylation, suppress formation of the corresponding E1 – 3 complexes as monitored by cell-based immunoblotting, and cytotoxicity to K562 leukemia cells via early stage apoptosis. The cell-based immunoblot assay also showed the compound caused NEDD8 to accumulate in cells, presumably due to inhibition of the downstream pathways involving the E1 enzyme. The affinity and cellular activities of the hit compound are modest, but is interesting as first in class for this mode of inhibition of NEDDylation, and as another illustration of the way EKO can be used to evaluate user-defined chemotypes as potential inhibitors of PPIs.

Discovery of the first NEDDylation inhibitor, that targets the NEDD8·NAE protein–protein interaction, was acheived using the Exploring Key Orientations (EKO) approach.