Microseeding – A Powerful Tool for Crystallizing Proteins Complexed with Hydrolyzable Substrates

Hydrolysis is an often-encountered obstacle in the crystallization of proteins complexed with their substrates. As the duration of the crystallization process, from nucleation to the growth of the crystal to its final size, commonly requires several weeks, non-enzymatic hydrolysis of an “unstable” ligand occurs frequently. In cases where the crystallization conditions exhibit non neutral pH values this hydrolysis phenomenon may be even more pronounced. ChoX, the substrate binding protein of a choline ABC-importer, produced crystals with its substrate acetylcholine after one month. However, these crystals exhibited only choline, an acetylcholine hydrolysis product, in the binding site. To overcome this obstacle we devised a microseeding protocol leading to crystals of ChoX with bound acetylcholine within 24 hours. One drawback we encountered was the high twinning fraction of the crystals, possibly was due to the rapid crystal growth

Crystal Structure Determination Of Two New Red Fluorescent Proteins By X-Ray Crystallography

Ph.D. Thesis. University of Hawaiʻi at Mānoa 2017

Studying the Phosphorylation of Isocitrate Dehydrogenase in Humans

Isocitrate dehydrogenase is an important enzyme in the citric acid cycle where it catalyzes the oxidative decarboxylation of isocitrate to alpha-ketoglutarate. While there are three isoforms of isocitrate dehydrogenase (IDH1, IDH2, and IDH3), this research will focus on IDH1. The phosphorylation of isocitrate dehydrogenase is a process that has been linked to the formation of both luminal-like and basal-like breast cancer. Despite these correlations, the mechanisms that cause breast cancer development are unknown. To examine this, an enzyme activity assay for each phosphorylation variant and crystallization were conducted. The results of these indicate that phosphorylation at each site (IDH1-T77, IDH1-S188, and IDH1-S237) leads to a decrease in isocitrate dehydrogenase activity when compared to the wild type

Characterization of different crystal forms of the α-glucosidase MalA from \u3ci\u3eSulfolobus solfataricus\u3c/i\u3e

MalA is an _-glucosidase from the hyperthermophilic archaeon Sulfolobus solfataricus. It belongs to glycoside hydrolase family 31, which includes several medically interesting α-glucosidases. MalA and its selenomethionine derivative have been overproduced in Escherichia coli and crystallized in four different crystal forms. Microseeding was essential for the formation of good-quality crystals of forms 2 and 4. For three of the crystal forms (2, 3 and 4) full data sets could be collected. The most suitable crystals for structure determination are the monoclinic form 4 crystals, belonging to space group P21, from which data sets extending to 2.5 Å resolution have been collected. Self-rotation functions calculated for this form and for the orthorhombic (P212121) form 2 indicate the presence of six molecules in the asymmetric unit related by 32 symmetry

Studying the Phosphorylation of Isocitrate Dehydrogenase in Humans

Isocitrate dehydrogenase is an important enzyme in the citric acid cycle where it catalyzes the oxidative decarboxylation of isocitrate to alpha-ketoglutarate. While there are three isoforms of isocitrate dehydrogenase (IDH1, IDH2, and IDH3), this research will focus on IDH1. The phosphorylation of isocitrate dehydrogenase is a process that has been linked to the formation of both luminal-like and basal-like breast cancer. Despite these correlations, the mechanisms that cause breast cancer development are unknown. To examine this, an enzyme activity assay for each phosphorylation variant and crystallization were conducted. The results of these indicate that phosphorylation at each site (IDH1-T77, IDH1-S188, and IDH1-S237) leads to a decrease in isocitrate dehydrogenase activity when compared to the wild type

Characterization of different crystal forms of the α-glucosidase MalA from \u3ci\u3eSulfolobus solfataricus\u3c/i\u3e

MalA is an _-glucosidase from the hyperthermophilic archaeon Sulfolobus solfataricus. It belongs to glycoside hydrolase family 31, which includes several medically interesting α-glucosidases. MalA and its selenomethionine derivative have been overproduced in Escherichia coli and crystallized in four different crystal forms. Microseeding was essential for the formation of good-quality crystals of forms 2 and 4. For three of the crystal forms (2, 3 and 4) full data sets could be collected. The most suitable crystals for structure determination are the monoclinic form 4 crystals, belonging to space group P21, from which data sets extending to 2.5 Å resolution have been collected. Self-rotation functions calculated for this form and for the orthorhombic (P212121) form 2 indicate the presence of six molecules in the asymmetric unit related by 32 symmetry

Structural studies of protein from Leptospira interrogans sorovar Copenhageni potentially located at the cell envelope

Orientadores: Beatriz Gomes Guimarães, Nilson Ivo Tonin ZanchinTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Leptospira interrogans é uma bactéria espiroqueta que causa a leptospirose, uma zoonose de distribuição mundial que afeta mais de 500.000 pessoas anualmente. Pouco se sabe sobre a biologia de leptospiras, o que dificulta a elaboração de novas estratégias de prevenção e de tratamento contra a doença. Cerca de 60 % dos genes de L. interrogans codifica proteínas que não apresentam similaridade de sequência significativa com proteínas de função conhecida. Como a estrutura cristalográfica de uma proteína pode revelar vários indícios funcionais, este trabalho visou à determinação da estrutura cristalográfica das proteínas LIC10793, LIC12922 e LIC10494 de L. interrogans, que são potencialmente localizadas no envelope celular e não são funcionalmente caracterizadas. A estrutura do antígeno LIC10793 (Lp49) foi resolvida a 2 Å de resolução e revelou que essa provável lipoproteína apresenta dois domínios. O domínio N-terminal de Lp49 possui um enovelamento do tipo Imunoglobulina e sua topologia diverge das formas padrão do motivo estrutural "chave grega" (Greek key motif). O domínio C-terminal consiste em um ?- propeller formado por sete folhas ?. Comparações locais não identificaram nenhum sítio catalítico conhecido em Lp49, mas análises de sua superfície revelaram a presença deprováveis sítios de ligação a proteínas. Com base nesses indícios, na provável localização de Lp49 na membrana externa e em sua antigenicidade, postula-se que Lp49 tenha uma função de interação com outras proteínas podendo desempenhar um papel na interação entre leptospiras e seus hospedeiros. A estrutura cristalográfica de LIC12922, determinada a 3,1 Å de resolução, revelou a presença de dois domínios. O domínio NC é estruturalmente relacionado a domínios que apresentam atividade chaperona, encontrados nas proteínas SurA e trigger factor de E. coli. O domínio parvulina de LIC12922 não apresenta atividade de peptidil prolil isomerase, mas possui um provável sítio de interação a proteína que inclui o sítio de reconhecimento ao substrato proposto para o domínio parvulina P1 de SurA. Análises filogenéticas sugerem que LIC12922 e as chaperonas extracitoplasmáticas SurA, PpiD e PrsA apresentam um ancestral comum. Com base nesses indícios e na provável localização de LIC12922 no periplasma, propõe-se que LIC12922 seja uma chaperona periplasmática envolvida na biogênese de proteínas da membrana externa. LIC10494 foi expressa, purificada e cristalizada. Refinamentos das condições de cristalização não foram suficientes para se obter cristais adequados ao experimento de difração. Análises da sua sequência evidenciaram que LIC10494 apresenta uma extensa região central intrinsecamente desordenada rica em resíduos de treonina. Assim como proteínas que possuem domínios intrinsecamente desestruturados, LIC10494 apresenta mobilidade mais lenta do que o esperado em SDS-PAGE, um volume de eluição menor do que o esperado em ensaios de gel filtração e uma considerável contribuição de configurações randômicas em seu espectro de dicroísmo circularAbstract: Leptospira interrogans is a spirochaetal bacterium which causes leptospirosis, a worldwide spread zoonosis that affects more than 500,000 people annually. Little is known about the biology of leptospires, which difficults the development of new preventive and treatment strategies for the disease. About 60 % of the genes from L. interrogans encode for proteins that did not show significative sequence similarity with proteins of known function. Since the tridimensional structure of a protein can contribute to the understanding of its function, this work aimed at the crystallographic structure determination of the proteins LIC10793, LIC12922 and LIC10494 from L. interrogans, which are potentially situated at the cell envelope and are not functionally characterized. The structure of the antigen LIC10793 (Lp49) was determined at 2 Å resolution and revealed that this probable lipoprotein possesses two domains. The Lp49 N-terminal domain presents an Immunoglobulin-like fold and its topology diverges from the standard patterns of the Greek key motif. The C-terminal domain is a 7-bladed ?-propeller. Local structural comparisons did not identify known catalytic sites at Lp49, but surface analyses evidenced potential protein binding sites. Based on these results, the putative localization of Lp49 at the outer membrane and its role as an antigen, we postulate that Lp49 has a protein binding function involved in Leptospira-host interaction. The LIC12922 crystal structure, determined at 3.1 Å resolution, revealed two domains. The NC domain is structurally related to the chaperone domains of E. coli SurA and trigger factor proteins. The LIC12922 parvulin domain is devoid of peptidyl prolyl isomerase activity, but presents a putative protein binding site which includes the substrate recognition site proposed to the first parvulin domain of SurA. Phylogenetic analyses suggest that LIC12922 and the extracytoplasmic chaperones SurA, PpiD and PrsA have a common ancestor. Based on the structural and phylogenetic analyses and taking into account its probable periplasmic localization we postulate that LIC12922 is a periplasmic chaperone involved at the biogenesis of outer membrane proteins. The protein LIC10494 was expressed, purified and crystallized. In spite of extensive refinement of crystallization conditions the crystals were not adequate for diffraction experiments. Sequence analyses evidenced that LIC10494 has an extensive central region intrinsically disordered which is rich in threonine residues. Similarly to proteins that possess intrinsically disordered domains, LIC10494 presents mobility slower than expected at SDSPAGE, elution volume smaller than expected in gel filtration assays and a considerable contribution of random coil structures in circular dichroism spectrumDoutoradoGenetica de MicroorganismosDoutor em Genetica e Biologia Molecula

The p110 delta structure: mechanisms for selectivity and potency of new PI(3)K inhibitors.

Deregulation of the phosphoinositide-3-OH kinase (PI(3)K) pathway has been implicated in numerous pathologies including cancer, diabetes, thrombosis, rheumatoid arthritis and asthma. Recently, small-molecule and ATP-competitive PI(3)K inhibitors with a wide range of selectivities have entered clinical development. In order to understand the mechanisms underlying the isoform selectivity of these inhibitors, we developed a new expression strategy that enabled us to determine to our knowledge the first crystal structure of the catalytic subunit of the class IA PI(3)K p110 delta. Structures of this enzyme in complex with a broad panel of isoform- and pan-selective class I PI(3)K inhibitors reveal that selectivity toward p110 delta can be achieved by exploiting its conformational flexibility and the sequence diversity of active site residues that do not contact ATP. We have used these observations to rationalize and synthesize highly selective inhibitors for p110 delta with greatly improved potencies

Crystallization and preliminary x-ray diffraction studies of a novel bacterial esterase.

Journal ArticleResearch Support, Non-U.S. Gov'tA novel bacterial esterase has been crystallized in two forms suitable for X-ray diffraction studies. Crystals have been obtained by vapour-phase diffusion at 290 K using ammonium sulfate as precipitant. The first crystals grew in space group C2 with unit-cell parameters a = 134.7, b = 55.8, c = 110.3 A, beta = 125.1 degrees. A monoclinic data set has been collected to 2.0 A resolution. Microseeding yielded a second crystal form which grew in space group P212121 with unit-cell parameters a = 57.1, b = 115.4, c = 130.4 A. Native data from these crystals have been collected to 1.6 A resolution. A molecular envelope has been determined using an uranyl acetate derivative for phase calculation.BBSRCSmithKline Beecham PharmaceuticalsEuropean Unio

Structural Characterization of Serine Protease Complexes with Novel Inhibitors

Human Neutrophil Elastase (HNE) is a serine protease responsible for cleavage of peptide bonds conferring elasticity to the connecting tissues. For this reason, this enzyme is mainly found in the lungs, arteries and ligaments [1-2]. In case of over-expression, HNE enables the appearance of some diseases, such as Chronic Obstructive Pulmonary Disease (COPD), Rheumatoid Arthritis, Psoriasis and Arteriosclerosis [3-4]. Currently, diseases affecting the respiratory tract are one of the major causes of death in the world, so HNE is a potential drug target of considerable interest [4]. Porcine Pancreatic Elastase (PPE) is commonly used as a model for HNE, sharing 37% of amino acid sequence identity [5]. According to previous studies, the catalytic serine performs a nucleophilic attack on a carbonyl group present in the inhibitors [6]. The focus of this work was the three-dimensional structure determination of elastases (PPE and HNE) in complex with inhibitors by X-ray crystallography to characterize their interactions at atomic level. The rational is to correlate structure and function and contribute to the design of more potent and specific inhibitors. These newly synthetic compounds were provided by the group of Prof. Rui Moreira, Instituto de Investigação do Medicamento, Faculdade de Farmácia, Universidade de Lisboa. X-ray diffraction data of PPE crystals were collected at a synchrotron source and three 3D-structures of PPE in complex with inhibitors were determined at resolutions around 1.4 Ǻ. Analysis of the electron density maps revealed that the nucleophilic attack occurred at the sulfonyl group of the inhibitors, contrary to what was initially expected (which would be in the carbonyl group). In silico energy minimization studies of the docked ligand structure into the active site of HNE, show no relevant structural modifications of the protein structure upon ligand binding. Finally, crystals of HNE have already been obtained and experiments are ongoing to grow complexes of HNE with various inhibitors. References: [1] A. Thomson and S. B. Kapadia, “The specificity of the S1 and S2 subsites of elastase,” Eur. J. Biochem., vol. 102, pp. 111–116, 1979. [2] Z. Werb, M. J. Banda, J. H. McKerrow, and R. A. Sandhaus, “Elastases and elastin degradation.,” J. Invest. Dermatol., vol. 79 Suppl 1, p. 154s–159s, Jul. 1982. [3] E. F. P. Ruivo, L. M. Gonåalves, L. A. R. Carvalho, R. C. Guedes, S. Hofbauer, J. A. Brito, M. Archer, R. Moreira, and S. D. Lucas, “Clickable 4-Oxo- b -lactam-Based Selective Probing for Human Neutrophil Elastase Related Proteomes,” pp. 1–7, 2016. [4] L. R. P. Areias, E. F. P. Ruivo, M. T. Duarte, R. Moreira, S. D. Lucas, and R. C. Guedes, “RSC Advances PAPER A uni fi ed approach toward the rational design of selective low nanomolar human neutrophil elastase,” pp. 51717–51721, 2015. [5] “Uniprot.” [Online]. Available: http://www.uniprot.org/. [Accessed: 16-Aug-2017]. [6] W. Huang, Y. Yamamoto, Y. Li, D. Dou, K. R. Alliston, R. P. Hanzlik, T. D. Williams, and W. C. Groutas, “X-ray Snapshot of the Mechanism of Inactivation of Human Neutrophil Elastase by 1,2,5-Thiadiazolidin-3-one 1,1-Dioxide Derivatives.,” Society, pp. 2003–2008, 2008.Elastase Neutrófila Humana (HNE) é uma protease de serina responsável pela clivagem das ligações peptídicas que conferem elasticidade aos tecidos de conexão. Por esta razão, esta enzima é encontrada principalmente nos pulmões, artérias e ligamentos [1-2]. Em casos de sobre-expressão, esta permite o aparecimento de algumas doenças, como Doença Pulmonar Obstrutiva Crónica (DPOC), Artrite Reumatóide, Psoríase e Arteriosclerose [3-4]. Atualmente, as doenças que afetam o trato respiratório são uma das principais causas de morte no mundo, sendo então a HNE um potencial alvo terapêutico de considerável interesse [4]. A Elastase Pancreática Suína (PPE) é normalmente usada como modelo para HNE, compartilhando 37% de identidade de sequência primária [5]. De acordo com estudos anteriores, a serina catalítica realiza um ataque nucleofílico ao grupo carbonilo presente nos inibidores [6]. O foco deste trabalho foi a determinação por cristalografia de raios-X da estrutura tridimensional de elastases (HNE e PPE) complexadas com inibidores, de modo a caracterizar as respetivas interações a nível atómico. O racional é correlacionar a estrutura com a função e contribuir para o desenho de inibidores mais fortes e mais específicos. Estes novos compostos sintéticos foram fornecidos pelo grupo do Prof. Rui Moreira, Instituto de Investigação do Medicamento, Faculdade de Farmácia, Universidade de Lisboa. Os dados de difração de raios-X dos cristais de PPE foram recolhidos numa fonte de sincrotrão e três estruturas 3D de três complexos da PPE com inibidores foram determinadas com resoluções em torno dos 1,4 Å. A análise dos mapas de densidade eletrónica revelaram que o ataque nucleofílico ocorreu no grupo sulfonilo dos inibidores ao contrário do que era inicialmente esperado (que seria no grupo carbonilo). A minimização de energia in silico da estrutura do ligando acoplado no centro ativo da HNE não mostra modificações relevantes na estrutura da proteína após a ligação do ligando. Finalmente, já foram obtidos cristais de HNE, estando já em curso experiencias para o crescimento de cristais de complexos de HNE com vários inibidores. Referências: [1] A. Thomson and S. B. Kapadia, “The specificity of the S1 and S2 subsites of elastase,” Eur. J. Biochem., vol. 102, pp. 111–116, 1979. [2] Z. Werb, M. J. Banda, J. H. McKerrow, and R. A. Sandhaus, “Elastases and elastin degradation.,” J. Invest. Dermatol., vol. 79 Suppl 1, p. 154s–159s, Jul. 1982. [3] E. F. P. Ruivo, L. M. Gonåalves, L. A. R. Carvalho, R. C. Guedes, S. Hofbauer, J. A. Brito, M. Archer, R. Moreira, and S. D. Lucas, “Clickable 4-Oxo- b -lactam-Based Selective Probing for Human Neutrophil Elastase Related Proteomes,” pp. 1–7, 2016. [4] L. R. P. Areias, E. F. P. Ruivo, M. T. Duarte, R. Moreira, S. D. Lucas, and R. C. Guedes, “RSC Advances PAPER A uni fi ed approach toward the rational design of selective low nanomolar human neutrophil elastase,” pp. 51717–51721, 2015. [5] “Uniprot.” [Online]. Available: http://www.uniprot.org/. [Accessed: 16-Aug-2017]. [6] W. Huang, Y. Yamamoto, Y. Li, D. Dou, K. R. Alliston, R. P. Hanzlik, T. D. Williams, and W. C. Groutas, “X-ray Snapshot of the Mechanism of Inactivation of Human Neutrophil Elastase by 1,2,5-Thiadiazolidin-3-one 1,1-Dioxide Derivatives.,” Society, pp. 2003–2008, 2008