Fumarato hidratases ou fumarases (FH) catalisam a reação estereoespecífica reversível de hidratação do fumarato em L-malato. Essas enzimas se apresentam em todas as classes de organismos, desde procariotos a eucariotos, e podem ser encontradas nas formas mitocondrial e citosólica. A enzima tem papel importante na produção de energia pois participa do ciclo do ácido cítrico, na resposta ao dano do DNA e como supressor tumoral. A fumarase humana (HsFH), que pertence à classe II, é codificada pelo gene 1q42.1, possui 467 aminoácidos em cada monômero com peso molecular de 50,2 kDa cada. Estudos associaram mutações no gene da FH com diversas doenças humanas como acidúria fumárica, leiomiomatoses de útero e pele (MCUL), que quando associadas com um agressivo carcinoma múltiplo de células é conhecido como leiomiomatose hereditária e câncer renal (HLRCC). Apesar da grande importância da fumarase humana no metabolismo energético, ainda há pouca informação em relação ao mecanismo catalítico adotado pela enzima e o efeito estrutural e cinético causado pelas mutações envolvidas com essas doenças. Diante disso, nosso trabalho utilizou uma abordagem híbrida que envolve a caracterização biofísica, bioquímica e estrutural da enzima HsFH, e seus mutantes: N107THsFH, H180RHsFH, Q185RHsFH, K230RHsFH, G282VHsFH, E362QHsFH, S365GHsFH e N373DHsFH, identificados em pacientes. Estudos cinéticos foram realizados em sete diferentes pHs e, pela primeira vez para fumarases, o ensaio foi realizado com os dois substratos presentes na mesma mistura reacional, confirmando a contribuição da reação reversa para a velocidade global da enzima. De acordo com os estudos de termoflúor a proteína é estabilizada em pHs alcalinos e através da ligação de compostos no sítio ativo. A estrutura da enzima HsFH nativa foi resolvida a 1,8 Å e identificou a presença de moléculas de HEPES complexadas na região C-terminal da enzima. Os estudos cinéticos demonstraram um aumento da eficiência catalítica na presença do HEPES, sugerindo um possível papel alostérico de seu sítio de ligação para a atividade catalítica. Foram determinadas as estruturas para os mutantes N107THsFH, H180RHsFH, Q185RHsFH, K230RHsFH, E362QHsFH, S365GHsFH e N373DHsFH. As mutações Q185R, E362Q, S365G e N373D foram identificadas no sítio ativo afetando diretamente a capacidade da proteína em ligar os substratos, enquanto que a mutação H180R foi localizada no sítio B, que conduz os substratos e produtos para dentro e fora do sítio ativo. Já a mutação K230R está localizada no domínio central, mas os resultados de termoflúor demonstram um efeito direto na capacidade da enzima em acomodar o substrato. A mutação N107T, localizada longe do sítio ativo foi a única que permaneceu ativa e teve seus parâmetros cinéticos residuais determinados. O presente trabalho contribui para o entendimento das bases estruturais que correlacionam mutações na HsFH, deficiência enzimática e patologia. / Fumarate hydratases or fumarases (FH) catalyze the reversible stereospecific hydration of fumarate to L-malate. They are present in all classes of organisms, from prokaryotes to eukaryotes, and can be found in the mitochondrial and cytosolic forms. The enzyme has an important role in energy production as part of the well-known Citric Acid Cycle, in DNA damage response and as tumor suppressor. Human fumarase (HsFH) belongs to class II and is encoded by 1q42.1 gene. HsFH is tetrameric and has 467 amino acids per monomer, with predicted molecular weight of 50.2 kDa. Several studies associated FH gene mutations with some human diseases such as fumaric aciduria, multiple cutaneous and uterine leiomyomatosis (MCUL), which when associated with an aggressive form of multiple cell carcinoma is known as hereditary leiomyomatosis and renal cancer (HLRCC) syndrome. Although the major role of HsFH in energetic metabolism, there are still little structural and kinetic information about the mutants involved in these diseases. Thus, this study aims, through a hybrid approach, composed by biophysics, biochemical and structural characterization of mutants N107THsFH, H180RHsFH, Q185RHsFH, K230RHsFH, G282VHsFH, E362QHsFH, S365GHsFH and N373DHsFH identified from patients. Steady-state kinetics studies were performed in seven different pHs and, for the first time, the contribution of both substrates was analyzed simultaneously in a single kinetic assay and allowed to quantify the contribution of the reverse reaction for kinetics. According to thermofluor studies, structural stability can be achieved at alkaline pHs and suggests that ligand binding can modulate the protein stability. HsFH crystal structure was solved at 1.8 Å resolution and identified HEPES molecules complexed with the enzyme C-terminal region. Kinetics studies with HEPES showed an increase of the catalytic efficiency and suggests that HEPES binding site might have an allosteric role. Crystal structures for the mutants N107THsFH, H180RHsFH, Q185RHsFH, K230RHsFH, E362QHsFH, S365GHsFH and N373DHsFH were determined. The mutations Q185R, E362Q, S365G and N373D were identified in the active site and affect the substrate binding capacity directly, while mutation H180R was localized in the B site, which conducts the substrates and products in and out the active site. The mutation K230R is localized in the central domain, but thermofluor results demonstrate a direct effect on the ability of the enzyme to accommodate the substrate. The N107T mutation located far from the active site was the only one that remained active and had its residual kinetic parameters determined. The present work contributes to the understanding of the structural bases that correlate mutations in HsFH, enzymatic deficiency and pathology.

<p><p>One of the most significant discoveries from mutational analysis of protein interfaces is that often a large percentage of interface residues negligibly perturb the binding energy upon mutation, while residues in a few critical "hotspots" drastically reduce affinity when mutated. The organization of protein interfaces into hotspots has a number of important implications. For example, small interfaces can have high affinity, and when multiple binding partners are generated to the same protein, they are predisposed to binding the same regions and often have the same hotspots. Even small molecules that bind to interfaces and disrupt protein-protein interactions (PPIs) tend to bind at hotspots. This suggests that some hotspot-forming sites on protein surfaces are <italic>intrinsically</italic> more apt to form protein interfaces. These observations paint a hotspot-centric picture of PPI energetics, and present a question of fundamental importance which remains largely unanswered: <italic>why are hotspots hot?</italic></p></p><p><p>In order to gain insight into the nature of hotspots I experimentally examined the small, but high-affinity interface between the synthetically evolved ankyrin repeat protein Off7 with E. coli maltose binding protein by characterization of mutant variants and redesigned interfaces. In order to characterize many mutants, I developed two high-throughput assays to measure protein-protein binding that integrate with existing technology for the high-throughput fabrication of genes. The first is an ELISA-based method using in vitro expressed protein for semi-quantitative analysis of affinity. Starting from DNA encoding protein partners, binding data is obtained in just a few hours; no exogenous purification is required. For the second assay, I develop data fitting methods and thermodynamic framework for determination of binding free energies from binding-induced shifts in protein thermal stability monitored with Sypro Orange.</p></p><p><p>Analysis of Off7/MBP variants using these methods reveals that conservative mutagenesis or local computational repacking is tolerated for many residues in the interface without drastic loss of affinity, except for a single essential hotspot. This hotspot contains a Tyr-His-Asp hydrogen bonding network reminiscent of a common catalytic motif. Substitution of the tyrosine with phenylalanine shows that a single hydrogen bond across the interface is critical for binding. Analysis of the protein database by structural bioinformatics shows that, although rare, this motif is present in other naturally evolved interfaces. Such a triad was found in the homodimeric interface of PH0642 from Pyrococcus horikoshii, and is conserved between many homologues in the nitrilase superfamily, meeting one of the key criteria by which potential hotspots can be identified. This analysis supports a number of analogies between hotspot residues and catalytic residues in enzyme active sites, and raises the intriguing possibility that hotspots may be associated with other structural motifs that could be used for identification or design of PPIs.</p></p> / Dissertation

Submitted by Ricardo Cedraz Duque Moliterno (ricardo.moliterno@uefs.br) on 2016-01-13T23:50:52Z No. of bitstreams: 1 TESE-FINAL-FRANCO-HENRIQUE-CORRIGIDO-V4.pdf: 27152448 bytes, checksum: 7d448c937bb21040514eedeaa37f689f (MD5) / Made available in DSpace on 2016-01-13T23:50:52Z (GMT). No. of bitstreams: 1 TESE-FINAL-FRANCO-HENRIQUE-CORRIGIDO-V4.pdf: 27152448 bytes, checksum: 7d448c937bb21040514eedeaa37f689f (MD5) Previous issue date: 2015-11-06 / Conselho Nacional de Pesquisa e Desenvolvimento Cient?fico e Tecnol?gico - CNPq / According to WHO, Leishmaniasis is the second most important disease caused by protozoans. However, the available therapeutic arsenal for its treatment is limited and has low efficacy and safety profile. Once Leishmania ssp. are pteridine auxotrophs key enzymes of the folate metabolism have been targeted to circumvent this dilemma. However, Dihydrofolate Reductase-Thymidylate Synthase (DHFR-TS) inhibitors are ineffective against Leishmania major due to an alternative folate pathway regulated by Pteridine Reductase 1 (PTR1). Thus, identifying molecules that act on both enzymes is crucial to develop new leishmanicidal drugs. For that reason, the main goal of this study is to identify, through in silico approaches, (pharmacophore models), putative PTR1 inhibitors that also show structural requirements for L. major DHFR-TS inhibition. The pharmacophore models 10 and 20, PTR1 (2 H-bond donors, 4 H-bond acceptors and 3 hydrophobic centers) and DHFR-TS inhibitors (2 H-bond acceptors and 2 hydrophobic centers) respectively, show high performance to differentiate true-binders from decoys (AUCPTR1=0.90; AUCDHFR-TS=0.86) and to explain the structure-activity relationships for the inhibitors under study. Thus, these models were employed sequentially to select 10 molecules whose effect over the thermal stability of LmPTR1 was investigated by ThermoFluor?. According to this assay, two molecules stabilize LmPTR1: Z80393 (?Tm = 1.02?C) and Z33165 (?Tm = 0.9?C). Binding displacement assays with biopterin or NADPH showed that Z80393 binds within the substrate binding site, whereas Z33165 binds in the cofactor binding site. Z80303 effect over the catalytic activity of PTR1 was investigated by fluorimetry. This approach allowed us to determine the inhibitor?s potency (IC50=32.31 ? 1.18 ?M). Finally, Z80303 putative binding profile was generated by molecular docking and analyzed by Molecular Dynamics (productive phase= 15 ns). The results show that during 70% of the simulation, Z80393 H-bonds to Ser-111 and Arg-17 residues. Therefore, this study not only led to identification of a new class of LmPTR1 inhibitors, but also allowed us to determine its potency, mode of inhibition and binding profile towards its therapeutic target. / A leishmaniose tem sido indicada pela OMS como a segunda protozoose mais importante em termos de mortalidade e preval?ncia. Entretanto, o repert?rio de f?rmacos dispon?veis ? limitado e apresenta, na maioria dos casos, baixos ?ndices de efic?cia e seguran?a. Embora os protozo?rios do g?nero Leishmania sejam auxotr?ficos para folatos, inibidores da Diidrofolato Redutase-Timidilato Sintase (DHFR-TS) s?o pouco eficazes contra esse parasito. A baixa suscetibilidade se explica pela presen?a da Pteridina Redutase 1 (PTR1) que atua como via alternativa para a redu??o de ?cido f?lico ou de pteridinas n?o conjugadas, quando DHFR-TS est? inibida. Diante desse cen?rio, mol?culas que atuam sobre PTR1 e DHFR-TS de Leishmania ssp. parecem ser promissoras para o desenvolvimento de f?rmacos contra a leishmaniose. Por essa raz?o, o objetivo desse trabalho foi identificar, por triagem in silico (modelo farmacof?rico), potenciais inibidores de PTR1 que apresentem os requisitos estruturais m?nimos para inibir tamb?m DHFR de L. major. Os modelos farmacof?ricos 10 e 20, baseados em inibidores de PTR1 (2 doadores de lig. H, 4 aceitadores de lig. H e 3 centros hidrof?bicos) e DHFR-TS (2 aceitadores de lig. H e 2 centros hidrof?bicos) respectivamente, mostraram desempenho satisfat?rio em discriminar inibidores verdadeiros de falsos positivos (AUCPTR1=0,90; AUCDHFR-TS=0,86), al?m de explicarem a rela??o entre a estrutura qu?mica e a atividade biol?gica. Esses modelos foram usados sequencialmente para selecionar 10 mol?culas que tiveram seu efeito sobre a estabilidade t?rmica de LmPTR1 investigado por ThermoFluor?. Nesse ensaio foram identificadas duas mol?culas que estabilizaram LmPTR1: Z80393 (?Tm = 1,02?C) e Z33165 (?Tm = 0,9?C). Ensaios de deslocamento com biopterina ou NADPH mostraram que Z80393 compete com o substrato, enquanto Z33165 interage no s?tio do cofator. O efeito de Z80393 sobre a atividade catal?tica de LmPTR1 foi investigado por fluorimetria, permitindo determinar a pot?ncia desse inibidor (IC50=32,31 ? 1,18 ?M). Por fim, um modelo de intera??o para esse inibidor foi gerado por acoplamento molecular e a pose obtida foi analisada atrav?s de uma Din?mica Molecular com fase produtiva de 15 ns. Os resultados obtidos mostram que durante 70% da simula??o, Z80393 faz liga??es de H com os res?duos Ser-111 e Arg-17. Portanto, o presente trabalho n?o s? levou a identifica??o de uma nova classe de inibidores de LmPTR1, mas tamb?m permitiu caracterizar sua pot?ncia, modalidade de inibi??o e perfil de intera??o com seu alvo terap?utico.

Lactobacillus kunkeei is one of the most abundant bacteria within the honey crop of the honey bee. Genome sequencing of L. kunkeei isolated from honey bees all over the world showed several genes unique for L. kunkeei. Among these orphan genes, an array of four to five highly conserved genes coding for giant extracellular proteins were found. Cryogenic electron microscopy imaging of a giant-protein preparation from L. kunkeei A00901 showed an overall structure similar to a long string with a knot at the end. Further analysis showed high similarity between the different giants at the N-terminus, and secondary structure predictions showed that the same region was rich in β-sheets.  These results, combined with the knowledge of other large extracellular proteins, led to the hypothesis that the “knot” domain is located at the N-terminus and that these proteins are used by the cell to latch on to the intestine lining or other cells in the honey crop. In this study, predictions were made to locate the N-terminal domains of two of these giant proteins. Four different constructs were made for each protein, where three constructs were designed for expression and purification of the N-terminal domain with different end-positions, and one construct was for a predicted β-solenoid domain located downstream from the N-terminal domain. The protein constructs were recombinantly produced in E. coli, and three of the N-terminal constructs from both proteins were purified. Thermal stability was tested using nano differential scanning fluorimetry (nanoDSF), Thermofluor, and circular dichroism (CD), which all showed characteristic melting curves at low melting temperatures, ranging from 33 °C to 44 °C, for all three constructs. During CD measurements, all three constructs showed refolding after thermal denaturation and a higher abundance of antiparallel β-sheets over α-helices. Looking at the protein structure, small angle X-ray scattering data indicated that all three proteins formed elongated structures. These results indicate that a folded domain has been found for both proteins. Although, further analysis will be required to determine the boundaries of the N-terminal domains, and to elucidate if these domains have anything to do with ligand binding and the L. kunkeei ability to latch onto the honey crop.