determination-protein-ligand-interactions-using-differential-scanning

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

University of Exeter

Serine hydroxymethyltransferase is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the interconversion of serine and glycine using tetrahydropteroylglutamate as the one-carbon carrier. In all pyridoxal phosphate-dependent enzymes, amino acid substrates are bound and released through a transaldimination process, in which an internal aldimine and an external aldimine are interconverted via gem-diamine intermediates. Bioinformatic analyses of serine hydroxymethyltransferase sequences and structures showed the presence of two highly conserved residues, a tyrosine and an arginine, engaged in a cation-pi interaction. In Escherichia coli serine hydroxymethyltranferase, the hydroxyl group of this conserved tyrosine (Tyr55) is located in a position compatible with a role as hydrogen exchanger in the transaldimination reaction. Because of the location of Tyr55 at the active site, the enhancement of its acidic properties caused by the cation-pi interaction with Arg235, and the hydrogen bonds established by its hydroxyl group, a role of this residue as acid-base catalyst in the transaldimination process was envisaged. The role played by this cation-pi interaction in the E. coli serine hydroxymethyltransferase was investigated by crystallography and site-directed mutagenesis using Y55F and three R235 mutant forms. The crystal structure of the Y55F mutant suggests that the presence of Tyr55 is indispensable for a correct positioning of the cofactor and for the maintenance of the structure of several loops involved in substrate and cofactor binding. The kinetic properties of all mutant enzymes are profoundly altered. Substrate binding and rapid kinetic experiments showed that both Y55 and R235 are required for a correct progress of the transaldimination reaction.

Role of a conserved active site cation-pi interaction in Escherichia coli serine hydroxymethyltransferase.

Serine hydroxymethyltransferase is a ubiquitous representative of the family of fold type I, pyridoxal 5'-phosphate-dependent enzymes. The reaction catalyzed by this enzyme, the reversible transfer of the Cβ of serine to tetrahydropteroylglutamate, represents a link between amino acid and folates metabolism and operates as a major source of one-carbon units for several essential biosynthetic processes. Serine hydroxymethyltransferase has been intensively investigated because of the interest aroused by the complex mechanism of the hydroxymethyltransferase reaction and its broad substrate and reaction specificity. Although the increasing availability of crystallographic data and the characterization of several site-specific mutants helped in understanding previous functional and structural studies, they also represent the starting point of novel investigations. This review will focus on recently highlighted catalytic, structural, and evolutionary aspects of serine hydroxymethyltransferase. This article is part of a Special Issue entitled: Pyridoxal phosphate Enzymology.

Serine hydroxymethyltransferase: a model enzyme for mechanistic, structural, and evolutionary studies.

Treatment of cultured bovine adrenal chromaffin cells with the catecholamine transport blocker reserpine was shown previously to increase enkephalin levels severalfold. To explore the biochemical mechanism of this effect, we examined the effect of reserpine treatment on the activities of three different peptide precursor processing enzymes: carboxypeptidase E (CPE) and the prohormone convertases (PCs) PC1/3 and PC2. Reserpine treatment increased both CPE and PC activity in extracts of cultured chromaffin cells; total protein levels were unaltered for any enzyme. Further analysis showed that the increase in CPE activity was due to an elevated V(max), with no change in the K(m) for substrate hydrolysis or the levels of CPE mRNA. Reserpine activation of endogenous processing enzymes was also observed in extracts prepared from PC12 cells stably expressing PC1/3 or PC2. In vitro experiments using purified enzymes showed that catecholamines inhibited CPE, PC1/3, and PC2, with dopamine quinone the most potent inhibitor (IC(50) values of ∼50-500 μM); dopamine, norepinephrine, and epinephrine exhibited inhibition in the micromolar range. The inhibition of purified CPE with catecholamines was time-dependent and, for dopamine quinone, dilution-independent, suggesting covalent modification of the protein by the catecholamine. Because the catecholamine concentrations found to be inhibitory to PC1/3, PC2, and CPE are well within the physiological range found in chromaffin granules, we conclude that catecholaminergic transmitter systems have the potential to exert considerable dynamic influence over peptidergic transmitter synthesis by altering the activity of peptide processing enzymes.

Regulation of neuropeptide processing enzymes by catecholamines in endocrine cells.

The prohormone convertases PC1/3 and PC2 are eukaryotic serine proteases involved in the proteolytic maturation of peptide hormone precursors and are implicated in a variety of pathological conditions, including obesity, diabetes, and neurodegenerative diseases. In this work, we screened 45 compounds obtained by derivatization of a 2,5-dideoxystreptamine scaffold with guanidinyl and aryl substitutions for convertase inhibition. We identified four promising PC1/3 competitive inhibitors and three PC2 inhibitors that exhibited various inhibition mechanisms (competitive, noncompetitive, and mixed), with sub- and low micromolar inhibitory potency against a fluorogenic substrate. Low micromolar concentrations of certain compounds blocked the processing of the physiological substrate proglucagon. The best PC2 inhibitor effectively inhibited glucagon synthesis, a known PC2-mediated process, in a pancreatic cell line; no cytotoxicity was observed. We also identified compounds that were able to stimulate both 87 kDa PC1/3 and PC2 activity, behavior related to the presence of aryl groups on the dideoxystreptamine scaffold. By contrast, inhibitory activity was associated with the presence of guanidinyl groups. Molecular modeling revealed interactions of the PC1/3 inhibitors with the active site that suggest structural modifications to further enhance potency. In support of kinetic data suggesting that PC2 inhibition probably occurs via an allosteric mechanism, we identified several possible allosteric binding sites using computational searches. It is noteworthy that one compound was found to both inhibit PC2 and stimulate PC1/3. Because glucagon acts in functional opposition to insulin in blood glucose homeostasis, blocking glucagon formation and enhancing proinsulin cleavage with a single compound could represent an attractive therapeutic approach in diabetes.

Inhibition of prohormone convertases PC1/3 and PC2 by 2,5-dideoxystreptamine derivatives.

Cyclosporin A, a cyclic peptide produced by the fungus Tolypocladium inflatum, is a widely employed immunosuppressant drug. Its biosynthesis is strictly dependent on the action of the pyridoxal 5'-phosphate-dependent enzyme alanine racemase, which produces the d-alanine incorporated in the cyclic peptide. This enzyme has a different fold with respect to bacterial alanine racemases. The interest elicited by T. inflatum alanine racemase not only relies on its biotechnological relevance, but also on its evolutionary and structural similarity to the promiscuous enzymes serine hydroxymethyltransferase and threonine aldolase. The three enzymes represent a model of divergent evolution from an ancestral enzyme that was able to catalyse all the reactions of the modern enzymes. A protocol to express and purify with high yield recombinant T. inflatum alanine racemase was developed. The catalytic properties of the enzyme were characterized. Similarly to serine hydroxymethyltransferase and threonine aldolase, T. inflatum alanine racemase was able to catalyse retroaldol cleavage and transamination reactions. This observation corroborates the hypothesis of the common evolutionary origin of these enzymes. A three-dimensional model of T. inflatum alanine racemase was constructed on the basis of threonine aldolase crystal structure. The model helped rationalise the experimental data and explain the catalytic properties of the enzymes.

Alanine racemase from Tolypocladium inflatum: a key PLP-dependent enzyme in cyclosporin biosynthesis and a model of catalytic promiscuity.

The calcium-dependent serine endoproteases prohormone convertase 1/3 (PC1/3) and prohormone convertase 2 (PC2) play important roles in the homeostatic regulation of blood glucose levels, hence implicated in diabetes mellitus. Specifically, the absence of PC2 has been associated with chronic hypoglycemia. Since there is a reasonably good conservation of the catalytic domain between species translation of inhibitory effects is likely. In fact, similar results have been found using both mouse and human recombinant enzymes. Here, we employed computational structure-based approaches to screen 14,400 compounds from the Maybridge small molecule library towards mouse PC2. Our most remarkable finding was the identification of a potent and selective PC2 inhibitor. Kinetic data showed the compound to be an allosteric inhibitor. The compound identified is one of the few reported selective, small-molecule inhibitors of PC2. In addition, this new PC2 inhibitor is structurally different and of smaller size than those reported previously. This is advantageous for future studies where structural analogues can be built upon.

Identification of a small molecule that selectively inhibits mouse PC2 over mouse PC1/3: a computational and experimental study.

L-threonine aldolases (L-TAs) represent a family of homologous pyridoxal 5'-phosphate-dependent enzymes found in bacteria and fungi, and catalyse the reversible cleavage of several L-3-hydroxy-α-amino acids. L-TAs have great biotechnological potential, as they catalyse the formation of carbon-carbon bonds, and therefore may be exploited for the bioorganic synthesis of L-3-hydroxyamino acids that are biologically active or constitute building blocks for pharmaceutical molecules. Many L-TAs, showing different stereospecificity towards the Cβ configuration, have been isolated. Because of their potential to carry out diastereoselective syntheses, L-TAs have been subjected to structural, functional and mechanistic studies. Nevertheless, their catalytic mechanism and the structural bases of their stereospecificity have not been elucidated. In this study, we have determined the crystal structure of low-specificity L-TA from Escherichia coli at 2.2-Å resolution, in the unliganded form and cocrystallized with L-serine and L-threonine. Furthermore, several active site mutants have been functionally characterized in order to elucidate the reaction mechanism and the molecular bases of stereospecificity. No active site catalytic residue was revealed, and a structural water molecule was assumed to act as the catalytic base in the retro-aldol cleavage reaction. Interestingly, the very large active site opening of E. coli L-TA suggests that much larger molecules than L-threonine isomers may be easily accommodated, and L-TAs may actually have diverse physiological functions in different organisms. Substrate recognition and reaction specificity seem to be guided by the overall microenvironment that surrounds the substrate at the enzyme active site, rather than by one ore more specific residues.

On the catalytic mechanism and stereospecificity of Escherichia coli L-threonine aldolase.

Capsular polysaccharides (CPSs) are protective structures on the surfaces of many Gram-negative bacteria. The principal CPS of the human pathogen and Tier 1 select agent Burkholderia pseudomallei consists of a linear repeat of --3)--2-O-acetyl-6-deoxy-β-d-manno-heptopyranose-(1-. This CPS is critical to the virulence of this emerging pathogen and represents a key target for the development of novel therapeutics. wcbI is one of several genes in the CPS biosynthetic cluster whose deletion leads to significant attenuation of the pathogen; unlike most others, it has no homologues of known function and no detectable sequence similarity to any protein with an extant structure. Here, the crystal structure of WcbI bound to its proposed product, coenzyme A, is reported at 1.38 Å resolution, solved using the halide-soak method with multiple anomalous dispersion. This structure reveals that WcbI incorporates a previously described 100-amino-acid subdomain into a novel, principally helical fold (310 amino acids). This fold adopts a cradle-like structure, with a deep binding pocket for CoA in the loop-rich cradle. Structural analysis and biophysical assays suggest that WcbI functions as an acetyltransferase enzyme, whilst biochemical tests suggest that another functional module might be required to assist its activity in forming the mature B. pseudomallei capsule.

Mirella Vivoli

Department of Biosciences

University of Exeter

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry