Enzymatic Activity of Soybean Lipoxygenase-1 on Linoleoyl-Derivatized Agarose

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of linoleic acid to form 13(S) and 9(R) hydroperoxides. The manner in which substrates bind to the lipoxygenase family of enzymes is not known. It is believed fatty acid substrates may bind either with the aliphatic end first or with the carboxylate group facing the interior of the protein. This thesis tested a potential methyl-end first substrate binding mechanism by studying the activity of SBLO-1 to oxygenate immobilized linoleoyl residues attached to an insoluble polymer. Linoleic acid was attached to aminohexyl agarose in the presence of N-(3- dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and Nhydroxysuccinimide (NHS). The concentration of the covalently attached residues was facilitated by enriching linoleic acid with a small amount of the radioactive 14C-isotope. Functionalization yields of 3% available primary amines on the resin were obtained. Enzymatic oxygenation of the linoleoyl-residues was verified using the ferrous oxidation in xylenol orange (FOX) assay. Approximately 30% of the attached linoleoyl moieties were converted to hydroperoxides in the presence of SBLO-1. A disulfide-containing cleavable linker, cystamine, was used as part of an improved method to isolate the product in a facile manner. Cystamine was attached to NHS-activated agarose with approximately 5% overall functionalization yield of available functional groups. 14C-linoleic acid was successfully covalently linked to the cystamine moieties in the presence of EDC and NHS. The FOX assay verified the enzymatic oxygenation of the linoleoyl residues attached to cystamine-derivatized agarose. The isolation of the peroxide product was attempted in a series of extractions in organic solvents. The product was analyzed using GC/MS which did not show a new peak indicative of product. Further work is needed to successfully analyze the stereoand regiochemistry of the oxygenated product. The presence of the peroxides in this study indicated the linoleoyl residues behave as substrates of SBLO-1. It is unknown how bulky substrates bind to the active site; however, it is difficult to rationalize a carboxylate group-first binding mode. Discovery of the 13(S)-hydroperoxide product on the linoleoyl-agarose would support the claim of a potential methyl-end first binding mechanism.

Role of the N- and C-lobes of calmodulin in the activation of Ca(2+)/calmodulin-dependent protein kinase II.

Understanding the principles of calmodulin (CaM) activation of target enzymes will help delineate how this seemingly simple molecule can play such a complex role in transducing Ca (2+)-signals to a variety of downstream pathways. In the work reported here, we use biochemical and biophysical tools and a panel of CaM constructs to examine the lobe specific interactions between CaM and CaMKII necessary for the activation and autophosphorylation of the enzyme. Interestingly, the N-terminal lobe of CaM by itself was able to partially activate and allow autophosphorylation of CaMKII while the C-terminal lobe was inactive. When used together, CaMN and CaMC produced maximal CaMKII activation and autophosphorylation. Moreover, CaMNN and CaMCC (chimeras of the two N- or C-terminal lobes) both activated the kinase but with greater K act than for wtCaM. Isothermal titration calorimetry experiments showed the same rank order of affinities of wtCaM > CaMNN > CaMCC as those determined in the activity assay and that the CaM to CaMKII subunit binding ratio was 1:1. Together, our results lead to a proposed sequential mechanism to describe the activation pathway of CaMKII led by binding of the N-lobe followed by the C-lobe. This mechanism contrasts the typical sequential binding mode of CaM with other CaM-dependent enzymes, where the C-lobe of CaM binds first. The consequence of such lobe specific binding mechanisms is discussed in relation to the differential rates of Ca (2+)-binding to each lobe of CaM during intracellular Ca (2+) oscillations.

Recognition principle of the TAP transporter disclosed by combinatorial peptide libraries

Transport of peptides across the membrane of the endoplasmic reticulum for assembly with MHC class I molecules is an essential step in antigen presentation to cytotoxic T cells. This task is performed by the major histocompatibility complex-encoded transporter associated with antigen processing (TAP). Using a combinatorial approach we have analyzed the substrate specificity of human TAP at high resolution and in the absence of any given sequence context, revealing the contribution of each peptide residue in stabilizing binding to TAP. Human TAP was found to be highly selective with peptide affinities covering at least three orders of magnitude. Interestingly, the selectivity is not equally distributed over the substrate. Only the N-terminal three positions and the C-terminal residue are critical, whereas effects from other peptide positions are negligible. A major influence from the peptide backbone was uncovered by peptide scans and libraries containing d amino acids. Again, independent of peptide length, critical positions were clustered near the peptide termini. These approaches demonstrate that human TAP is selective, with residues determining the affinity located in distinct regions, and point to the role of the peptide backbone in binding to TAP. This binding mode of TAP has implications in an optimized repertoire selection and in a coevolution with the major histocompatibility complex/T cell receptor complex.

The Aer protein and the serine chemoreceptor Tsr independently sense intracellular energy levels and transduce oxygen, redox, and energy signals for Escherichia coli behavior

We identified a protein, Aer, as a signal transducer that senses intracellular energy levels rather than the external environment and that transduces signals for aerotaxis (taxis to oxygen) and other energy-dependent behavioral responses in Escherichia coli. Domains in Aer are similar to the signaling domain in chemotaxis receptors and the putative oxygen-sensing domain of some transcriptional activators. A putative FAD-binding site in the N-terminal domain of Aer shares a consensus sequence with the NifL, Bat, and Wc-1 signal-transducing proteins that regulate gene expression in response to redox changes, oxygen, and blue light, respectively. A double mutant deficient in aer and tsr, which codes for the serine chemoreceptor, was negative for aerotaxis, redox taxis, and glycerol taxis, each of which requires the proton motive force and/or electron transport system for signaling. We propose that Aer and Tsr sense the proton motive force or cellular redox state and thereby integrate diverse signals that guide E. coli to environments where maximal energy is available for growth.

(4-Aminomethyl)phenylguanidine derivatives as nonpeptidic highly selective inhibitors of human urokinase

Increased expression of the serine protease urokinase-type plasminogen activator (uPA) in tumor tissues is highly correlated with tumor cell migration, invasion, proliferation, progression, and metastasis. Thus inhibition of uPA activity represents a promising target for antimetastatic therapy. So far, only the x-ray crystal structure of uPA inactivated by H-Glu-Gly-Arg-chloromethylketone has been reported, thus limited data are available for a rational structure-based design of uPA inhibitors. Taking into account the trypsin-like arginine specificity of uPA, (4-aminomethyl)phenylguanidine was selected as a potential P1 residue and iterative derivatization of its amino group with various hydrophobic residues, and structure–activity relationship-based optimization of the spacer in terms of hydrogen bond acceptor/donor properties led to N-(1-adamantyl)-N′-(4-guanidinobenzyl)urea as a highly selective nonpeptidic uPA inhibitor. The x-ray crystal structure of the uPA B-chain complexed with this inhibitor revealed a surprising binding mode consisting of the expected insertion of the phenylguanidine moiety into the S1 pocket, but with the adamantyl residue protruding toward the hydrophobic S1′ enzyme subsite, thus exposing the ureido group to hydrogen-bonding interactions. Although in this enzyme-bound state the inhibitor is crossing the active site, interactions with the catalytic residues Ser-195 and His-57 are not observed, but their side chains are spatially displaced for steric reasons. Compared with other trypsin-like serine proteases, the S2 and S3/S4 pockets of uPA are reduced in size because of the 99-insertion loop. Therefore, the peculiar binding mode of the new type of uPA inhibitors offers the possibility of exploiting optimized interactions at the S1′/S2′ subsites to further enhance selectivity and potency. Because crystals of the uPA/benzamidine complex allow inhibitor exchange by soaking procedures, the structure-based design of new generations of uPA inhibitors can rely on the assistance of x-ray analysis.

Three-dimensional structure of human electron transfer flavoprotein to 2.1-Å resolution

Mammalian electron transfer flavoproteins (ETF) are heterodimers containing a single equivalent of flavin adenine dinucleotide (FAD). They function as electron shuttles between primary flavoprotein dehydrogenases involved in mitochondrial fatty acid and amino acid catabolism and the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase. The structure of human ETF solved to 2.1-Å resolution reveals that the ETF molecule is comprised of three distinct domains: two domains are contributed by the α subunit and the third domain is made up entirely by the β subunit. The N-terminal portion of the α subunit and the majority of the β subunit have identical polypeptide folds, in the absence of any sequence homology. FAD lies in a cleft between the two subunits, with most of the FAD molecule residing in the C-terminal portion of the α subunit. Alignment of all the known sequences for the ETF α subunits together with the putative FixB gene product shows that the residues directly involved in FAD binding are conserved. A hydrogen bond is formed between the N5 of the FAD isoalloxazine ring and the hydroxyl side chain of αT266, suggesting why the pathogenic mutation, αT266M, affects ETF activity in patients with glutaric acidemia type II. Hydrogen bonds between the 4′-hydroxyl of the ribityl chain of FAD and N1 of the isoalloxazine ring, and between αH286 and the C2-carbonyl oxygen of the isoalloxazine ring, may play a role in the stabilization of the anionic semiquinone. With the known structure of medium chain acyl-CoA dehydrogenase, we hypothesize a possible structure for docking the two proteins.

A β-hairpin structure in a 13-mer peptide that binds α-bungarotoxin with high affinity and neutralizes its toxicity

Snake-venom α-bungarotoxin is a member of the α-neurotoxin family that binds with very high affinity to the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. The structure of the complex between α-bungarotoxin and a 13-mer peptide (WRYYESSLEPYPD) that binds the toxin with high affinity, thus inhibiting its interactions with AChR with an IC50 of 2 nM, has been solved by 1H-NMR spectroscopy. The bound peptide folds into a β-hairpin structure created by two antiparallel β-strands, which combine with the already existing triple-stranded β-sheet of the toxin to form a five-stranded intermolecular, antiparallel β-sheet. Peptide residues Y3P, E5P, and L8P have the highest intermolecular contact area, indicating their importance in the binding of α-bungarotoxin; W1P, R2P, and Y4P also contribute significantly to the binding. A large number of characteristic hydrogen bonds and electrostatic and hydrophobic interactions are observed in the complex. The high-affinity peptide exhibits inhibitory potency that is better than any known peptide derived from AChR, and is equal to that of the whole α-subunit of AChR. The high degree of sequence similarity between the peptide and various types of AChRs implies that the binding mode found within the complex might possibly mimic the receptor binding to the toxin. The design of the high-affinity peptide was based on our previous findings: (i) the detection of a lead peptide (MRYYESSLKSYPD) that binds α-bungarotoxin, using a phage-display peptide library, (ii) the information about the three-dimensional structure of α-bungarotoxin/lead-peptide complex, and (iii) the amino acid sequence analysis of different AChRs.

Unraveling principles of lead discovery: from unfrustrated energy landscapes to novel molecular anchors.

The search for novel leads is a critical step in the drug discovery process. Computational approaches to identify new lead molecules have focused on discovering complete ligands by evaluating the binding affinity of a large number of candidates, a task of considerable complexity. A new computational method is introduced in this work based on the premise that the primary molecular recognition event in the protein binding site may be accomplished by small core fragments that serve as molecular anchors, providing a structurally stable platform that can be subsequently tailored into complete ligands. To fulfill its role, we show that an effective molecular anchor must meet both the thermodynamic requirement of relative energetic stability of a single binding mode and its consistent kinetic accessibility, which may be measured by the structural consensus of multiple docking simulations. From a large number of candidates, this technique is able to identify known core fragments responsible for primary recognition by the FK506 binding protein (FKBP-12), along with a diverse repertoire of novel molecular cores. By contrast, absolute energetic criteria for selecting molecular anchors are found to be promiscuous. A relationship between a minimum frustration principle of binding energy landscapes and receptor-specific molecular anchors in their role as "recognition nuclei" is established, thereby unraveling a mechanism of lead discovery and providing a practical route to receptor-biased computational combinatorial chemistry.

Mutational analysis of NM23-H2/NDP kinase identifies the structural domains critical to recognition of a c-myc regulatory element.

NM23-H2, a presumed regulator of tumor metastasis in humans, is a hexameric protein with both enzymatic (NDP kinase) and regulatory (transcriptional activation) activity. While the structure and catalytic mechanisms have been well characterized, the mode of DNA binding is not known. We examined this latter function in a site-directed mutational study and identified residues and domains essential for the recognition of a c-myc regulatory sequence. Three amino acids, Arg-34, Asn-69, and Lys-135, were found among 30 possibilities to be critical for DNA binding. Two of these, Asn-69 and Lys-135, are not conserved between NM23 variants differing in DNA-binding potential, suggesting that DNA recognition resides partly in nonconserved amino acids. All three DNA-binding defective mutant proteins are active enzymatically and appear to be stable hexamers, suggesting that they perform at the level of DNA recognition and that separate functional domains exist for enzyme catalysis and DNA binding. In the context of the known crystal structure of NM23-H2, the DNA-binding residues are located within distinct structural motifs in the monomer, which are exposed to the surface near the 2-fold axis of adjacent subunits in the hexamer. These findings are explained by a model in which NM23-H2 binds DNA with a combinatorial surface consisting of the "outer" face of the dimer. Chemical crosslinking data support a dimeric DNA-binding mode by NM23-H2.

Selenocysteine, identified as the penultimate C-terminal residue in human T-cell thioredoxin reductase, corresponds to TGA in the human placental gene.

The possible relationship of selenium to immunological function which has been suggested for decades was investigated in studies on selenium metabolism in human T cells. One of the major 75Se-labeled selenoproteins detected was purified to homogeneity and shown to be a homodimer of 55-kDa subunits. Each subunit contained about 1 FAD and at least 0.74 Se. This protein proved to be thioredoxin reductase (TR) on the basis of its catalytic activities, cross-reactivity with anti-rat liver TR antibodies, and sequence identities of several tryptic peptides with the published deduced sequence of human placental TR. Physicochemical characteristics of T-cell TR were similar to those of a selenocysteine (Secys)-containing TR recently isolated from human lung adenocarcinoma cells. The sequence of a 12-residue 75Se-labeled tryptic peptide from T-cell TR was identical with a C-terminal-deduced sequence of human placental TR except that Secys was present in the position corresponding to TGA, previously thought to be the termination codon, and this was followed by Gly-499, the actual C-terminal amino acid. The presence of the unusual conserved Cys-Secys-Gly sequence at the C terminus of TR in addition to the redox active cysteines of the Cys-Val-Asn-Val-Gly-Cys motif in the FAD-binding region may account for the peroxidase activity and the relatively low substrate specificity of mammalian TRs. The finding that T-cell TR is a selenoenzyme that contains Se in a conserved C-terminal region provides another example of the role of selenium in a major antioxidant enzyme system (i.e., thioredoxin-thioredoxin reductase), in addition to the well-known glutathione peroxidase enzyme system.

Sequence similarity analysis of Escherichia coli proteins: functional and evolutionary implications.

A computer analysis of 2328 protein sequences comprising about 60% of the Escherichia coli gene products was performed using methods for database screening with individual sequences and alignment blocks. A high fraction of E. coli proteins--86%--shows significant sequence similarity to other proteins in current databases; about 70% show conservation at least at the level of distantly related bacteria, and about 40% contain ancient conserved regions (ACRs) shared with eukaryotic or Archaeal proteins. For > 90% of the E. coli proteins, either functional information or sequence similarity, or both, are available. Forty-six percent of the E. coli proteins belong to 299 clusters of paralogs (intraspecies homologs) defined on the basis of pairwise similarity. Another 10% could be included in 70 superclusters using motif detection methods. The majority of the clusters contain only two to four members. In contrast, nearly 25% of all E. coli proteins belong to the four largest superclusters--namely, permeases, ATPases and GTPases with the conserved "Walker-type" motif, helix-turn-helix regulatory proteins, and NAD(FAD)-binding proteins. We conclude that bacterial protein sequences generally are highly conserved in evolution, with about 50% of all ACR-containing protein families represented among the E. coli gene products. With the current sequence databases and methods of their screening, computer analysis yields useful information on the functions and evolutionary relationships of the vast majority of genes in a bacterial genome. Sequence similarity with E. coli proteins allows the prediction of functions for a number of important eukaryotic genes, including several whose products are implicated in human diseases.

Protein structure-based design of potent orally bioavailable, nonpeptide inhibitors of human immunodeficiency virus protease.

A class of potent nonpeptidic inhibitors of human immunodeficiency virus protease has been designed by using the three-dimensional structure of the enzyme as a guide. By employing iterative protein cocrystal structure analysis, design, and synthesis the binding affinity of the lead compound was incrementally improved by over four orders of magnitude. An inversion in inhibitor binding mode was observed crystallographically, providing information critical for subsequent design and highlighting the utility of structural feedback in inhibitor optimization. These inhibitors are selective for the viral protease enzyme, possess good antiviral activity, and are orally available in three species.

Dalla catalisi all’inibizione della crescita da contatto: le molteplici funzioni di O-acetilserina sulfidrilasi batterica

CysK, uno degli isoenzimi di O-acetilserina sulfidrilasi (OASS) presenti in piante e batteri, è un enzima studiato da molto tempo ed il suo ruolo fisiologico nella sintesi della cisteina è stato ben definito. Recentemente sono state scoperte altre funzioni apparentemente non collegate alla sua funzione enzimatica (moonlighting). Una di queste è l’attivazione di una tossina ad attività tRNAsica, CdiA-CT, coinvolta nel sistema di inibizione della crescita da contatto (CDI) di ceppi patogeni di E. coli. In questo progetto abbiamo studiato il ruolo di CysK nel sistema CDI e la formazione di complessi con due differenti partner proteici: CdiA-CT e CysE (serina acetiltransferasi, l’enzima che catalizza la reazione precedente nella biosintesi della cisteina). I due complessi hanno le stesse caratteristiche spettrofluorimetriche e affinità molto simili, ma la cinetica di raggiungimento dell’equilibrio per il complesso tossina:CysK è più lenta che per il complesso CysE:CysK (cisteina sintasi). In entrambi i casi la formazione veloce di un complesso d’incontro è seguita da un riarrangiamento conformazionale che porta alla formazione di un complesso ad alta affinità. L’efficienza di formazione del complesso cisteina sintasi è circa 200 volte maggiore rispetto al complesso CysK:tossina. Una differenza importante, oltre alla cinetica di formazione dei complessi, è la stechiometria di legame. Infatti mentre CysE riesce a legare solo uno dei due siti attivi del dimero di CysK, nel complesso con CdiA-CT entrambi i siti attivi dell’enzima risultano essere occupati. Le cellule isogeniche esprimono un peptide inibitore della tossina (CdiI), e sono quindi resistenti all’azione tRNAsica. Tuttavia, siccome CdiI non altera la formazione del complesso CdiA-CT:CysK, CdiA-CT può esercitare comunque un ruolo nel metabolismo della cisteina e quindi nella fitness dei batteri isogenici, attraverso il legame e l'inibizione di CysK e la competizione con CysE. La via biosintetica della cisteina, un precursore di molecole riducenti, risulta essere molto importante per i batteri soprattutto in condizioni avverse come all’interno dei macrofagi nelle infezioni persistenti. Perciò questa via metabolica è di interesse per lo sviluppo di nuovi antibiotici, e in particolare le due isoforme dell’OASS negli enterobatteri, CysK e CysM, sono potenziali target per lo sviluppo di nuove molecole ad azione antibatterica. Partendo dall’analisi delle modalità di interazione con CysK del suo partner ed inibitore fisiologico, CysE, si è studiato dapprima l’interazione di pentapeptidi che mimassero la regione C-terminale di quest'ultimo, e in base ai dati ottenuti sono stati sviluppati piccoli ligandi sintetici. La struttura generale di questi composti è costituita da un gruppo acido ed un gruppo lipofilo, separati da un linker ciclopropanico che mantiene questi due gruppi in conformazione trans, ottimale per l’interazione col sito attivo dell’enzima. Sulla base di queste considerazioni, di docking in silico e di dati sperimentali ottenuti con la tecnica dell’STD-NMR e con saggi di binding spettrofluorimetrici, si è potuta realizzare una analisi di relazione struttura-attività che ha portato via via all’ottimizzazione dei ligandi. Il composto più affine che è stato finora ottenuto ha una costante di dissociazione nel range del nanomolare per entrambe le isoforme, ed è un ottimo punto di partenza per lo sviluppo di nuovi farmaci.

La tagatose-1,6-bisphosphate aldolase et la fructose-1,6-bisphosphate aldolase de classe I : mécanisme et stéréospécificité

La tagatose-1,6-biphosphate aldolase de Streptococcus pyogenes est une aldolase qui fait preuve d'un remarquable manque de spécificité vis à vis de ses substrats. En effet, elle catalyse le clivage réversible du tagatose-1,6-bisphosphate (TBP), mais également du fructose-1,6-bisphosphate (FBP), du sorbose-1,6-bisphosphate et du psicose-1,6-bisphosphate, quatre stéréoisomères, en dihydroxyacétone phosphate (DHAP) et en glycéraldéhyde-3-phosphate (G3P). Aldolase de classe I, qui donc catalyse sa réaction en formant un intermédiaire covalent obligatoire, ou base de Schiff, avec son susbtrat, la TBP aldolase de S. pyogenes partage 14 % d’identité avec l’enzyme modèle de cette famille, la FBP aldolase de muscle de mammifère. Bien que le mécanime catalytique de la FBP aldolase des mammifères ait été examiné en détails et qu’il soit approprié d’en tirer des renseignements quant à celui de la TBP aldolase, le manque singulier de stéréospécificité de cette dernière tant dans le sens du clivage que celui de la condensation n’est toujours pas éclairci. Afin de mettre à jour les caractéristiques du mécanisme enzymatique, une étude structurale de la TBP aldolase de S. pyogenes, un pathogène humain extrêmement versatile, a été entreprise. Elle a permis la résolution des structures de l’enzyme native et mutée, en complexe avec des subtrats et des inhibiteurs compétitifs, à des résolutions comprises entre 1.8 Å et 2.5 Å. Le trempage des cristaux de TBP aldolase native et mutante dans une solution saturante de FBP ou TBP a en outre permis de piéger un authentique intermédiaire covalent lié à la Lys205, la lysine catalytique. La determination des profils pH de la TBP aldolase native et mutée, entreprise afin d'évaluer l’influence du pH sur la réaction de clivage du FBP et TBP et ìdentifier le(s) résidu(s) impliqué(s), en conjonction avec les données structurales apportées par la cristallographie, ont permis d’identifier sans équivoque Glu163 comme résidu responsable du clivage. En effet, le mode de liaison sensiblement différent des ligands utilisés selon la stéréochimie en leur C3 et C4 permet à Glu163, équivalent à Glu187 dans la FBP aldolase de classe I, d’abstraire le proton sur l’hydroxyle du C4 et ainsi d’amorcer le clivage du lien C3-C4. L’étude du mécanimse inverse, celui de la condensation, grâce par exemple à la structure de l’enzyme native en complexe avec ses substrats à trois carbones le DHAP et le G3P, a en outre permis d’identifier un isomérisme du substrat G3P comme possible cause de la synthèse des isomères en C4 par cette enzyme. Ce résultat, ainsi que la decouverte d’un possible isomérisme cis-trans autour du lien C2-C3 de la base de Schiff formée avec le DHAP, identifié précedemment, permet de cerner presque complètement les particularités du mécanisme de cette enzyme et d’expliquer comment elle est capable de synthétiser les quatres stéréoisomères 3(S/R), 4(S/R). De plus, la résolution de ces structures a permis de mettre en évidence trois régions très mobiles de la protéine, ce qui pourrait être relié au rôle postulé de son isozyme chez S. pyogenes dans la régulation de l’expression génétique et de la virulence de la bactérie. Enfin, la résolution de la structure du mutant Lys229→Met de la FBP aldolase de muscle en complexe avec la forme cyclique du FBP, de même que des études cristallographiques sur le mutant équivalent Lys205→Met de la TBP aldolase de S. pyogenes et des expériences de calorimétrie ont permis d’identifier deux résidus particuliers, Ala31 et Asp33 chez la FBP aldolase, comme possible cause de la discrimination de cette enzyme contre les substrats 3(R) et 4(S), et ce par encombrement stérique des substrats cycliques. La cristallographie par rayons X et la cinétique enzymatique ont ainsi permis d'avancer dans l'élucidation du mécanisme et des propriétés structurales de cette enzyme aux caractéristiques particulières.

Travels with a donkey in the Cevennes /

IUB01410: From the library of Sinclair Lewis; inscribed: "Sinclair Lewis, N.H. Apr. '08" on front fly-leaf recto.