The organic cation transporters (OCTs) play a critical role in the absorption, distribution and elimination of many drugs, hormones, herbal medicines, and environmental toxins. Given the broad substrate specificity of OCTs, they fall victim to the high susceptibility for contributing to harmful drug-drug interactions. Further defining how human (h)OCTs mechanistically bind to its broad array of substrates will provide significant insight to the understanding and prediction of drug-drug interactions in polypharmacy patients and the advancement of future rational drug design for therapeutics targeting OCTs. The goal of the current study was to elucidate the critical amino acid residues for transporter-substrate binding interactions on human (h)OCT1 and 2 utilizing in silico molecular modeling techniques (homology modeling and automated docking), as well as in vitro mutagenesis and kinetic transport experiments. Three-dimensional homology models were generated for hOCT1 and 2 using Piriformospora indica phosphate transporter (PiPT) serving as template. A putative binding pocket was identified and used to dock the prototypical substrate MPP+. Docking studies revealed five residues for each transporter (hOCT1 and hOCT2) that may be critical for substrate-transporter interactions. The in silico data was used to guide subsequent in vitro site-directed mutagenesis and kinetic analysis. Four hOCT1 mutants (Gln241Lys, Thr245Lys, Tyr361Ala, and Glu447Lys) and three hOCT2 mutants (Gln242Lys, Tyr362Phe, and Tyr362Ala) showed complete loss of MPP+ transporter activity. Decreased affinity for MPP+ was observed for Phe244Ser and Thr245Ser in hOCT1, and Tyr245Ala in hOCT2. All amino acid residues highlighted in the in vitro experiments may be potentially critical for substrate-transporter interactions particularly Tyr361, Phe244 and Thr245 in hOCT1; and Tyr362 and Tyr245 in hOCT2. Docking of known structurally divergent hOCT1 and hOCT2 substrates revealed similar binding interactions as that identified for MPP+, albeit with some unique residues, suggesting the presence of a large central cavity within both transporters. Through the combination of in silico and in vitro experiments, a putative binding pocket was defined and several residues important for substrate-transporter interaction were identified and verified for hOCT1 and hOCT2. Further defining how OCTs biochemically interact with their broad array of substrates will provide significant insight to the understanding and prediction of drug-drug interactions in polypharmacy patients and the advancement of future rational drug design for therapeutics targeting OCT1 and OCT2.

It has been shown that the amino terminus and second extracellular loop (EC2) of CXCR2 are crucial for ligand binding and receptor activation. The lack of an ionic lock motif in the third intracellular loop of CXCR2 focuses an investigation of the mechanism by which these two extracellular regions contribute to receptor recognition and activation. The first objective of this investigation was to predict the structure of CXCR2 based on known structures of crystallized GPCRs. Rhodopsin, β2-adrenergic receptor, CXCR4 were used for homology modeling of CXCR2 structure. Highly conserved motifs found in sequence alignments of the template GPCRs were helpful to generate CXCR2 models. We also studied solvent accessibility of residues in the EC2 of CXCR2 in the inactive state. Most of the residues in the EC2 were found to be solvent accessible in the inactive state, suggesting the residues might be involved in ligand recognition. Second, we studied the role of charged residues in the EC2 of CXCR2 in ligand binding and receptor activation using constitutively active mutants (CAM) of CXCR2, D9K and D9R. Combinatorial mutations consisting of the CAM in the amino terminus and single mutations of charged residues in the EC2 were generated to study two concepts including “attraction” and “repulsion” models. The mutant receptors were used to test their effects on cell surface expression, ligand binding, receptor activation through PLC-β3, and cellular transformation. All the mutations in the repulsion model result in CXCR2 receptors that are unable to bind ligand, suggesting that each of the Arg residues in the EC2 are important for ligand recognition. Interestingly, mutations in the attraction model partially inhibited receptor activation by the CAM D9K, suggesting that Glu198 and Asp199 residues in the EC2 are associated with receptor activation. Furthermore, a novel CAM, E198A/D199A, was identified in this study. These negatively charged residues are very close to a conserved disulfide bond linking the EC2 and the third transmembrane. In this sense, these current discoveries concerning the structural basis of CXCR2 and interdisciplinary approaches would provide new insights to investigate unknown mechanisms of interaction with its cognate ligands and receptor activation.

Many pathogenic bacteria use their iron acquisition mechanisms to live inside hosts. Streptococcus pyogenes is a pathogenic bacterium that uses streptococcal iron acquisition ABC transporter to obtain heme. SiaA (HtsA, spy1795), a lipoprotein located on the cell surface, serves as a heme binding protein. To understand the iron-uptake mechanism, histidine 229, one of the two proposed axial ligands in SiaA, was mutated to alanine. SiaA H229A was expressed in E. coli, lysed by French Press, and purified by fast protein liquid chromatography (FPLC). SDS-PAGE indicated that pure protein was isolated. Nickel affinity FPLC gave purer H229A when 0.5 M imidazole was added to the binding buffer. Overall, histidine 229 is likely to be an axial ligand in wild type SiaA, as shown by the fact the mutant readily lost heme as evidenced by UV-vis spectra.

The structure of 11S cruciferin has been solved; however, how the individual subunits contribute to its physico-chemical and functional properties are not well known. The cruciferin isoforms in Arabidopsis thaliana, CRUA, CRUB, and CRUC, were investigated with respect to their molecular structures and the relationship of structural features to the physico-chemical and functional properties of cruciferin using homology modeling and various analytical techniques. Comparison of these models revealed that hydrophobicity and electrostatic potential distribution on the surface of the CRUC homotrimer had more favorable interfacial, solubility, and thermal properties than those of CRUA or CRUB. Flavor binding and pepsin digestion were associated with hypervariable regions (HVRs) and center core regions, respectively, moreso for CRUA and CRUB homotrimers than for CRUC. Chemical imaging of a single cell area in wild type (WT) and double-knockout seeds (CRUAbc, CRUaBc, and CRUabC) using synchrotron FT-IR microscopy (amide I band, 1650 cm-1, νC=O) showed that seed storage proteins were concentrated in the cell center and protein storage vacuoles, whereas lipids were closer to the cell wall. Secondary structure components of proteins of double-knockout lines did not show major differences. Changes in protein secondary structure components of pepsin-treated CRUabC (CRUC) mutant were minimal, indicating low enzyme accessibility. A three-step chromatographic procedure allowed isolation of the hexameric form of cruciferin with high purity (>95%). Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopic analysis of the secondary structure of these proteins revealed cruciferins were folded into higher order secondary structures; 44−50% β-sheets and 7−9% α-helices. The relative subunit ratio was approximately 1:3:6 (CRUA:CRUB:CRUC) in the WT cruciferin. The Tm values of purified cruciferin at pH 7.4 (μ = 0.0) were in the order of WT = CRUA = CRUB < CRUC. The order of surface hydrophobicity as determined by ANS (1-anilinonaphthalene-8-sulfonate) probe binding was CRUA > CRUB = WT >> CRUC. Intrinsic fluorescence studies revealed a compact molecular structure for the CRUC homohexamer compared to the CRUA and CRUB homohexamers. The order of emulsion forming abilities was CRUA = CRUB > WT > CRUC (no emulsion formation) and the order of heat-induced network structure strength was WT > CRUA = CRUB > CRUC (no gel formation). The inability of CRUC to form gels or emulsions may be attributed to its low surface hydrophobicity and molecular compactness. At pH 2.0, CRUC hexamers dissociated into trimers which allowed the formation of an O/W emulsion and heat-induced network structures. Solubility of cruciferin as a function of pH at low ionic strength gave two minima around pH 4 and 7.4 yielding a “W” shape solubility profile deviating from the typical “U” or “V” shape solubility profile of other 11S globulins. The high ionic strength (μ = 0.5) was not favorable for emulsification, heat-induced gel formation, or solubilization for all cruciferins. Furthermore, the CRUA and CRUB homohexamers exhibited rapid pepsinolysis, while the CRUC homohexamer and WT heterohexamer were digested more slowly. Although fairly well conserved regions were found in the primary structure of these three cruciferin subunits, differences were found in the hypervariable regions and extended loop regions resulting in slight differences in 3D structures and interactions that occur during association to form superstructures, such as hexamers. These differences were reflected in the physico-chemical and techno-functional properties of hexamers and trimers composed of each subunit. In silico predictions for certain functionalities were highly correlated with empirical data from laboratory experiments.

Les canaux Ca2+ activés par le voltage (CaV) sont des protéines membranaires qui génèrent des courants Ca2+ dans les cellules excitables suite à une dépolarisation membranaire. Ces complexes oligomériques sont classifiés selon les propriétés structurelles de la sous-unité principale qui forme le pore du canal, soit la sous-unité CaVα1. La sous-unité auxiliaire CaVβ module l’expression membranaire et la dépendance au voltage du « gating » de la sous-unité CaVα1 des canaux HVA (« high-voltage-activated ») CaV1 et CaV2. La sous-unité CaVβ est formée par un domaine SH3 (« Src homology-3 ») connecté à un domaine GK (« guanylate kinase-like ») par le biais d’un domaine variable HOOK. Dans le but d’identifier les résidus dans la CaVβ3 qui sont responsables de la densité membranaire du CaV2.3, nous avons produit des mutants de la sous-unité auxiliaire le long de ses domaines fonctionnels. Cela dit, la délétion complète du domaine SH3 ainsi que la délétion du domaine HOOK n’ont pas modifié la densité membranaire de CaV2.3 ni ses propriétés d’activation. Cependant, la délétion de cinq résidus dans le domaine GK interrompt l’expression membranaire et l’expression fonctionnelle de CaV2.3. La mutation de résidus identifiés précédemment comme soutenant une affinité de liaison de l’ordre du nanomolaire dans le domaine GK de CaVβ n’a pas modifié de manière significative l’adressage membranaire de CaV2.3. Toutefois, les mutations de quatre résidus leucine dans les régions α3, α6, β10 et α9 du domaine GK ont grandement réduit l’adressage membranaire du canal CaV2.3. Nos résultats confirment que le domaine GK contient les déterminants moléculaires responsables de la fonction chaperone de CaVβ. Cela dit, l’adressage membranaire induit par CaVβ semble être déterminé par des éléments structuraux qui ne sont pas strictement dépendants d’une liaison à haute affinité de CaVβ sur CaVα1. / Voltage-activated Ca2+ channels (CaV) are membrane proteins that play a key role in promoting Ca2+ influx in response to membrane depolarization in excitable cells. They form oligomeric complexes that are classified according to the structural properties of the pore-forming CaVα1 subunit. Auxiliary CaVβ subunits modulate cell-surface expression and voltage-dependent gating of high-voltage-activated (HVA) CaV1 and CaV2 α1 subunits. CaVβ subunits are formed by a Src homology-3 (SH3) domain and a guanylate kinase-like (GK) domain connected through a variable HOOK-domain. In order to identify the residues responsible for the CaVβ3-induced membrane density of CaV2.3, we produced mutants along CaVβ3’s fonctionnal domains. Complete deletion of the SH3 domain as well as deletion of the HOOK domain did not alter plasma membrane targeting of CaV2.3 nor its typical activation gating. In contrast, 5-residue deletions in the GK domain disrupted cell surface trafficking and functional expression of CaV2.3. Mutations of residues known to carry nanomolar affinity binding in the GK domain of CaVβ did not significantly alter cell surface density. Mutations of a quartet of leucine residues in the α3, α6, β10, and α9 regions of the GK domain, each expected to curtail protein-protein interaction, were found to significantly impair cell surface targeting of CaV2.3 channels. Altogether, our results confirm that the GK domain includes the molecular determinants carrying the chaperone function of CaVβ. However, CaVβ-induced cell surface targeting appears to be determined by structural elements that are not strictly dominated by high-affinity binding of CaVβ onto CaVα1.

Differentes études ont montré que la sensibilité au Ca2+ du canal KCa3.1, un canal potassique indépendant du voltage, était conférée par la protéine calmoduline (CaM) liée de façon constitutive au canal. Cette liaison impliquerait la région C-lobe de la CaM et un domaine de $\ikca$ directement relié au segment transmembranaire S6 du canal. La CaM pourrait égalment se lier au canal de façon Ca2+ dépendante via une interaction entre un domaine de KCa3.1 du C-terminal (CaMBD2) et la région N-lobe de la CaM. Une étude fut entreprise afin de déterminer la nature des résidus responsables de la liaison entre le domaine CaMBD2 de KCa3.1 et la région N-lobe de la CaM et leur rôle dans le processus d'ouverture du canal par le Ca2+. Une structure 3D du complexe KCa3.1/CaM a d'abord été générée par modélisation par homologie avec le logiciel MODELLER en utilisant comme référence la structure cristalline du complexe SK2.2/CaM (PDB: 1G4Y). Le modèle ainsi obtenu de KCa3.1 plus CaM prévoit que le segment L361-S372 dans KCa3.1 devrait être responsable de la liaison dépendante du Ca2+ du canal avec la région N-lobe de la CaM via les résidus L361 et Q364 de KCa3.1 et E45, E47 et D50 de la CaM. Pour tester ce modèle, les résidus dans le segment L361-S372 ont été mutés en Cys et l'action du MTSET+ (chargé positivement) et MTSACE (neutre) a été mesurée sur l'activité du canal. Des enregistrements en patch clamp en configuration ``inside-out`` ont montré que la liaison du réactif chargé MTSET+ au le mutant Q364C entraîne une forte augmentation du courant, un effet non observé avec le MTSACE. De plus les mutations E45A et E47A dans la CaM, ont empêché l'augmentation du courant initié par MTSET+ sur le mutant Q364C. Une analyse en canal unitaire a confirmé que la liaison MTSET+ à Q364C cause une augmentation de la probabilité d'ouverture de KCa3.1 par une déstabilisation de l'état fermé du canal. Nous concluons que nos résultats sont compatibles avec la formation de liaisons ioniques entre les complexes chargés positivement Cys-MTSET+ à la position 364 de KCa3.1 et les résidus chargés négativement E45 et E47 dans la CaM. Ces données confirment qu'une stabilisation électrostatique des interactions CaM/KCa3.1 peut conduire à une augmentation de la probabilité d'ouverture du canal en conditions de concentrations saturantes de Ca2+. / The Ca2+ sensitivity of the voltage-insensitive calcium activated potassium channel of intermediate conductance KCa3.1 is conferred by calmodulin (CaM) constitutively bound to the membrane-proximal region of the channel intracellular C-terminus. A study was performed to investigate the nature of the residues involved in the CaM/KCa3.1 interactions and determine how these interactions could modulate the channel gating properties. A 3D-structure of the KCa3.1/CaM complex was first generated by homology modeling with MODELLER using as template the crystal structure of SK2.2/CaM complex (PDB: 1G4Y). The resulting structural model of KCa3.1 plus CaM predicts that the segment L361-S372 in KCa3.1 should be responsible for the Ca2+-dependent binding of the channel to the CaM-N lobe, with residues L361 and Q364 facing residues E45, E47 and D50 of CaM. To test this model residues in L361-S372 segment were substituted by Cys and the action of MTSET+ (positive charged) and MTSACE (neutral charged) measured on channel activity. Inside-out patch clamp recordings showed that the binding of the charged MTSET+ reagent to the Q364C mutant resulted in a strong current increase, an effect not seen with the neutral MTSACE. The mutations E45A and E47A in CaM prevented the current increase initiated by MTSET+ on the Q364C mutant. A single channel analysis confirmed that the binding of MTSET+ to Q364C caused an increase in the channel open probability by a destabilization of the channel closed state. Altogether, our results are compatible with the formation of ionic bonds between the positively charged Cys-MTSET+ complex at position 364 in KCa3.1 and the negatively charged E45 and E47 residues in CaM, and confirm that an electrostatic stabilization of the CaM/KCa3.1 interactions can lead to an increase in the channel open probability at saturating Ca2+.

Title: Molecular dynamics simulations of ion channel TRPA1 Author: Mgr. Vlastimil Zíma Institute: Institute of Physics of Charles University Supervisor: RNDr. Ivan Barvík, PhD., Institute of Physics of Charles Uni- versity Abstract: The ion channel TRPA1 is one of the members of the transient receptor potential channel family. These channels have recently been an im- portant objective of research, because they play important roles in various cellular processes and organismic mechanisms. Especially they are involved in most of the senses. We focused mainly on the TRPA1 ion channel due to its involvement in the pain sensation in humans. Because the molecular mechanisms behind the gating of this channel are not fully understood, their description is a key for a design of new analgesics targeting this channel. We used a homology modeling and molecular dynamics simulations in conjunc- tion with electrophysiological experiments to provide a valuable new insight into the channel mechanisms. We contributed by describing of a putative binding site for calcium ions. Further, many functionally important amino acids were found in the S1-S4 transmembrane domain. Keywords: voltage-gated ion channel, TRPA1 channel, molecular dynamics, homology modeling 1

Tracking the evolution of function in enzyme superfamilies is key in understanding how important biological functions and mechanisms have evolved. New genes are being sequenced at a rate that far surpasses the ability of characterization by wet-lab techniques. Moreover, bioinformatics allows for the use of methods not amenable to wet lab experimentation. We now face a situation in which we are aware of the existence of many gene families but are ignorant of what they do and how they function. Even for families with many structurally and functionally characterized members, the prediction of function of ancestral sequences can be used to elucidate past patterns of evolution and highlight likely future trajectories. In this thesis, we apply in silico structure and function methods to predict the functions of protein sequences from two diverse superfamily case studies. In the first, the metallo-β-lactamase superfamily, many members have been structurally and functionally characterised. In this work, we asked how many times the same function has independently evolved in the same superfamily using ancestral sequence reconstruction, homology modelling and alignment to catalytic templates. We found that in only 5% of evolutionary scenarios assessed, was there evidence of a lactam hydrolysing ancestor. This could be taken as strong evidence that metallo-β-lactamase function has evolved independently on multiple occasions. This finding has important implications for predicting the evolution of antibiotic resistance in this protein fold. However, as discussed, the interpretation of this statistic is not clear-cut. In the second case study, we analysed protein sequences of the DUF-62 superfamily. In contrast to the metallo-β-lactmase superfamily, very few members of this superfamily have been structurally and functionally characterised. We used the analysis of alignment, gene context, species tree reconciliation and comparison of the rates of evolution to ask if other functions or cellular roles might exist in this family other than the ones already established. We find that multiple lines of evidence present a compelling case for the evolution of different functions within the Archaea, and propose possible cellular interactions and roles for members of this enzyme family.

Made available in DSpace on 2014-12-17T14:03:28Z (GMT). No. of bitstreams: 1 LudovicoM.pdf: 1651172 bytes, checksum: 32d873e29ab629d06ae3a79806c6523e (MD5) Previous issue date: 2008-07-02 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Serines proteinases inhibitors (PIs) are widely distributed in nature and are able to inhibit both in vitro and in vivo enzymatic activites. Seed PIs in than leguminous are classified in seven families, Bowman-Birk and Kunitz type families that most studied representing an important role in the first line of defense toward insects pests. Some Kunitz type inhibitors possess activities serine and cysteine for proteinases named bifunctional inhibitor, as ApTKI the inhibitor isolate from seed of Adenanthera pavonina. The A. pavonina inhibitor presenting the uncommon property and was used for interaction studies between proteinases serine (trypsin) and cysteine (papain). In order to determinate the in vitro interaction of ApTKI against enzymes inhibitor purification was carried cut by using chromatographic techniques and inhibition assays. The 3D model of the bifunctional inhibitor ApTKI was constructed SWISS-MODEL program by homology modeling using soybean trypsin inhibitor (STI, pdb:1ba7), as template which presented 40% of identity to A. pavonina inhibitor. Model quality was evaluated by PROCHECK program. Moreover in silico analyzes of formed complex between the enzymes and ApTKI was evaluated by HEX 4.5 program. In vitro results confirmed the inhibitory assays, where the inhibitor presented the ability to simultaneously inhibit trypsin and papain. The residues encountered in the inhibitor model of folder structural three-dimensional that make contact to enzymes target coud explain the specificity pattern against serine and cysteine proteinases / Os inibidores de proteinases ser?nicas (IPs) est?o extensamente distribu?dos na natureza e inibem a atividade enzim?tica in vitro e in vivo. Estes IPs em sementes de leguminosas compreendem sete fam?lias, no entanto as fam?lias Bowman-Birk e do tipo Kunitz s?o as mais estudadas e representam um papel importante na primeira linha de defesa contra insetos pragas. Alguns inibidores do tipo Kunitz possuem atividades para proteinases ser?nicas e ciste?nicas sendo denominados inibidores bifuncionais, como o inibidor ApTKI da semente de Adenanthera pavonina. O inibidor de A. pavonina por apresentar essa caracter?stica incomum aos inibidores dessa fam?lia foi utilizado para o estudo da intera??o entre as proteinases ser?nica (tripsina) e ciste?nica (papa?na). Para determinar a intera??o in vitro de ApTKI e as enzimas alvo foi realizada a purifica??o do inibidor a partir de t?cnicas cromatogr?ficas e ensaios de inibi??o. O modelo 3D do inibidor bifuncional ApTKI foi constru?do pelo programa SWISS-MODEL atrav?s da metodologia de modelagem por homologia utilizando como molde o inibidor de tripsina de soja (STI, pdb:1ba7) que apresentou 40% de identidade com a prote?na alvo. A qualidade do modelo foi avaliada pelo programa PROCHECK. Para a an?lise do complexo in silico entre as enzimas alvo e o inibidor foi utilizado o programa HEX 4.5. Estes resultados confirmaram os ensaios inibit?rios in vitro, onde ApTKI apresentou a capacidade de inibir simultaneamente tripsina e papa?na. Algumas das diferen?as observadas nos res?duos do sitio reativo explicam a forte afinidade para tripsina e a fraca para papa?na

Les complexes collecteurs de lumière des bactéries photosynthétiques absorbent l'énergie solaire, et transfèrent l'énergie avec grande efficacité aux centres réactionnels, siùge où elle est captée pour l'utilisation par la cellule. Nous savons peu des détails du transfert d'énergie entre les différents complexes collecteurs de lumière. Dans cette thèse, j'ai isolé différents complexes collecteurs de lumière à partir de plusieurs souches de bactéries pourpres. J'ai construit de modèles 3D par homologie et les structures possibles de dimères ont également été examinés. Les sites de pontage dans ces protéines montrent la possibilité de construire des dimères avec des structures biologiquement pertinentes. J'ai développé un protocole pour construire de dimères de protéines collectrices de lumière fortement oligomériques. Le protocole que j'ai mis en place contient trois grandes étapes : d'abord la réaction de lysines dans les complexes à un très faible degré de réaction, et la purification des protéines marquées. Ensuite, les groupes réactifs de dibenzocyclooctyne (DBCO) ou de l'azoture sont introduits au complexe. Finalement, la réaction sans cuivre de cycloaddition azoture-alcyne promue par distorsion a pour conséquence la synthèse de dimères. / The light harvesting apparatus of photosynthetic bacteria absorb the energy from sunlight and transfer the energy with high efficiency to the reaction center, where it is captured for use by the cell. We know little about the details of energy transfer between different light-harvesting complexes. In this thesis I isolated several different types of light-harvesting complex from various stains of purple bacteria. 3D models were built, based on homology modeling, and possible dimer structures were examined. The cross linking sites in these protein shown the possiblity of forming biologically relevant dimer structures. I have developed a protocol to make dimers, from highly oligomeric light harvesting proteins. The protocol developed contains three main steps: first reaction of lysines in the complex at a very low degrees of reaction and purifying the labelled protein. Then coupling the reactive groups of dibenzylcyclootyne (DBCO) or of azide separately to the different complexes. Finally, the copper free strain promoted azide-alkyne cycloaddition reaction occurred to synthesize the dimer.