Improvement of the Activity of a Fungal Versatile-Lipase Toward Triglycerides: An in silico Mechanistic Description

Some enzymes that belong to the Candida rugosa-like lipase family (abH03. 01) combine the activities of lipases and sterol esterases. Thus, they can act on water-insoluble carboxylic esters releasing long-chain fatty acids but also on sterol esters, although with different activity and affinity. The differences in the catalytic properties among the proteins of this family are explained by small changes in the hydrophobicity of some regions. One of such versatile enzymes is the sterol esterase/lipase from Ophiostoma piceae (OPE) that acts very efficiently on the two types of substrates. Structurally, OPE is characterized by the presence of a lid formed by a α-helix and two 310-helices rich in hydrophobic amino acids. In this study, the ope gene was modified by directed mutagenesis in order to change specific amino acids in the lid region to modify its structure with the aim of increasing its hydrophobicity. Several recombinant forms of OPE were heterologously produced in Pichia pastoris. In silico molecular dynamics simulations have been used to decipher the mechanistic principles behind the improvements in substrate catalysis. The analyses suggested that the enhanced activity toward hydrophobic substrates such as triglycerides could be due to a better stabilization of the substrate in the lid region as a result of an increased hydrophobicity and an improved topology. These results indicate that in silico simulations can be useful for the optimization of the activity of lipases from the C. rugose-like family for different biotechnological applications

Desarrollo y validación experimental de un método para la obtención de estructuras de complejos ligando/receptor basado en Resonancia Magnética Nuclear

284 p.-44 fig.-13 tab.[EN]Molecular recognition plays a key role in many cellular processes. Knowledge about the structure of the molecular complexes implicated in these cellular events is therefore important to understand cellular mechanisms. For cell function, it is important that most of the processes are reversible, and thus the molecules are often weakly associated. X-ray diffraction is a widely used technique in the structural analysis of molecular complexes; however, it has the disadvantage of not being useful in solving the structure of molecular complexes with low association constants. In these complexes, symmetry in the crystal is affected by the abundance of empty binding pockets. In the case of reversible molecular complexes formed by ligands with low molecular weight with macromolecules, the crystallographic methods are not able to extract information in order to describe the entire structure. This disadvantage can be resolved using complementary Nuclear Magnetic Resonance (NMR) techniques, and integrating the structural information obtained by both techniques. NMR is an extremely versatile technique that can be used to obtain structural information from molecules or their molecular complexes. For biological systems, NMR is a very useful technique to study the structure of proteins, lipids or polysaccharides. Two-dimensional transferred nuclear Overhauser effect spectroscopy (TR-NOESY) and Saturation Transfer Difference experiments (STD-NMR) are NMR methods widely used to study biological interactions. Both techniques allow studying molecular complexes with low association constants because they can measure magnetization from molecules in solution. TR-NOESY provides information about the conformation of low molecular weight ligands when bound to macromolecules and STD-NMR contains information about the binding epitope of a small molecule bound to a protein. In this work, we have designed a hybrid methodology, able to obtain the structures of macromolecular complexes by using STD-NMR data, TR-NOESY conformational state of the ligand and the partially solved protein-ligand structure obtained by X-ray diffraction. In order to ensure that the methodology solves the molecular complexes analytically, a new auto-consistent algorithm is implemented to solve the binding pose of protein-ligand complexes based on STD-NMR data. For ensuring that the algorithm is auto-consistent, five new descriptors have been implemented to differentially evaluate the fitting of the STD-NMR maps back calculate from a binding pose with the experimental data.The algorithm uses the structure of macromolecule partially solved by X-ray diffraction, the STD-NMR data and the TR-NOESY conformation of ligand to estimate chemical-physical parameters of the system (such as the apparent binding constant and the protein apparent correlation time) and to solve the structure of molecular complex fitting the experimental STD-NMR map and the calculated one. The structure of the molecular complex with the new structural information obtained can then be iteratively refined using steps of electron density and STD-NMR maps fitting until the solved structure is found to be consistent with both techniques. Finally, the structure of dactylolide, a microtubule-stabilizing agent, was solved bound to the luminal taxane site in T2R tetramers using the hybrid methodology. Dactylolide has a fast chemical interchange between free and bound states, which leads to a reduction of crystallographic resolution in the binding site. The analysis of dactylolide-T2R structure and the crystallographic structure of zampanolide, a structural homologous of dactylolide, allows characterizing the covalent reaction mechanism of drugs in the Taxol site.[ES]El conocimiento estructural de los complejos moleculares entre proteínas y moléculas pequeñas permite obtener información útil para el estudio de la maquinaria celular. Entre los complejos moleculares, existen muchas interacciones de baja afinidad que regulan interacciones transitorias. La resolución estructural de estos complejos proteína-ligando de baja afinidad tiene vital importancia, por su relevancia en los mecanismos de regulación celular. No obstante, la resolución estructural es ardua y en muchos casos llega a ser inviable debido a sus bajas constantes de asociación. En estas condiciones, es difícil extraer información estructural suficiente por DRX o crio-ME para poder resolver analíticamente la estructura de los complejos. Sin embargo, diversas técnicas de RMN permiten extraer parte de la información estructural de este tipo de complejos. Con objeto de resolver complejos moleculares controlados por un intercambio químico rápido, se diseñó una metodología híbrida basada en datos de RMN y DRX capaz de abarcar la resolución estructural de complejos proteína-ligando con bajas constantes de asociación. La metodología combina datos estructurales cristalográficos de la proteína, datos conformacionales del ligando obtenidos por TR-NOESY y datos del contacto entre la proteína y el ligando obtenidos mediante STD-NMR, para resolver la estructura de complejos proteína-ligando de baja afinidad. Para combinar los datos de STD-NMR con el resto de las técnicas es necesario el desarrollo de una metodología fiable que determine automáticamente la orientación del ligando en el sitio de unión. Por ello se diseñaron nuevas variantes de la función de error residual entre los datos experimentales y los modelos de estructuras generadas para evaluar la calidad de los modelos. Estas variantes del factor junto con el factor R-NOE clásico se implementaron en el programa STD-MaPa. STD-MaPa es el núcleo de la metodología hibrida de RMN-DRX y fue creado para automatizar el acoplamiento guiado por datos de STD-NMR de las moléculas de ligando en el sitio activo conocido de los complejos proteína-ligando. La metodología implementada en el programa fue probada usando como referencia cinco complejos moleculares que tienen sus estructuras resueltas por DRX y cuyos datos de STD-NMR eran conocidos. El STD-MaPa resolvió correctamente las cinco estructuras cristalográficas.Por último, se estudió la estructura de la dactilolida unida al tetrámero de tubulina. La dactilolida forma parte de una nueva familia de drogas capaces de unirse covalentemente a la -tubulina evitando así la resistencia tumoral por sobreexpresión de bombas de membrana. Esta droga se une covalentemente a la proteína pero esta unión, presenta una cinética de asociación muy lenta, lo que impide su resolución estructural por DRX. En este trabajo se consiguió resolver la estructura de la dactilolida unida no covalentemente al tetrámero de tubulina con la metodología híbrida RMN-DRX. Además, se identificaron dos orientaciones en la subunidad de tubulina menos accesible del cristal del tetrámero T2R en una proporción del 83% y 17%. Analizando la orientación predominante y la estructura cristalográfica de su homólogo estructural zampanolida se pudo describir el mecanismo de reacción covalente de esta familia de drogas para la unión a la -tubulina. Este mecanismo permitirá el diseño de nuevos fármacos que presenten la unión covalente y puedan eludir los mecanismos de resistencia a fármacos de las células cancerígenas.Fundación de Investigación de la UAB (UABRF), los Institutos Nacionales para la salud (NIH) y la National Science Foundation (NSF) de EE. UU. otorgando licencia de CORCEMA-STPeer reviewe

High-throughput preparation of alkyl 4-aryl substituted-2-methyl-6-thioxo-1,4,5,6-tetrahydropyridine-3-carboxylates under microwave irradiation

17 p.-1 fig.-2 tab.An efficient high-throughput synthesis of 4-aryl substituted 1,4,5,6-tetrahydro-2-methyl-6-thioxopyridine-3-carboxylates 5a-p was developed by using Lawesson’s reagent, a very effective thionating reagent for carbonyl compounds, under conventional conditions and microwave irradiation. In order to gain a better understanding of the structure of the heterocycles obtained, theoretical calculations at the ab initio level were carried out.The part of this work carried out in Barcelona was partially supported by CICYT (CTQ2009-07758), the Generalitat de Catalunya (2009SGR 1024), the Institute for Research in Biomedicine, and the Barcelona Science Park. This work was also supported by the MICINN of Spain (project CT2008-00795/BQU and Consolider-Ingenio 2010C-07-25200).Peer reviewe

Versatile lipases from the Candida rugosa-like family: a mechanistic insight using computational approaches

26 p.-5 fig.Lipases are enzymes able to catalyze the hydrolysis or synthesis of triglycerides, depending on the reaction conditions, whereas sterol esterases show the same ability on sterol esters. Structurally, both kinds of enzymes display an α/β-hydrolase fold, with a substrate-binding pocket formed by a hydrophobic cavity covered by a mobile lid. However, it has been reported that some lipases from the Candida rugosa-like family display wide substrate specificity on both triglycerides and sterol esters. Among them, enzymes with different biotechnological applications, such as the lipase isoenzymes produced by C. rugosa and the sterol esterase from Ophiostoma piceae, have been exhaustively characterized and their crystal structures are available. Differences in substrate affinity among these proteins have been attributed to changes in their hydrophobicity. In this work, we analyzed the full catalytic mechanisms of these proteins using molecular dynamics tools, gaining insight into their mechanistic properties. In addition, we developed an in silico protocol to predict the substrate specificity using C. rugosa and O. piceae lipases as model enzymes and triglycerides and cholesterol esters with different fatty acid chain lengths as model substrates. The protocol was validated by comparing the in silico results with those described in the literature. These results would be useful to perform virtual screening of substrates for enzymes of the C. rugosa-like family with unknown catalytic properties.This work was supported by the Spanish projects BIO2015-73697-JIN from MEICOMP co-financed with FEDER funds and RETOPROSOST2 S/2018/EMT-4459 from Comunidad de Madrid. The authors would also like to thank IBISBA1.0 project (H2020 730976) and the SusPlast-CSIC Interdisciplinary Platform for their support.Peer reviewe

Taxanes with high potency inducing tubulin assembly overcome tumoural cell resistances

49 p.-7 tab.- 7 fig.We have found that four taxanes with chemical modifications at positions C10 and C13 were active against all types of taxane resistant cell lines, resistant by p-Gp overexpression, by mutations in the β-tubulin binding site or by overexpression of the highly dynamic βIII-tubulin isotype. We have characterized the interaction of taxanes with high activity on chemotherapy resistant tumoural cells with microtubules, and also studied their cellular effects. The biochemical property enhanced in comparison with other taxanes is their potency at inducing tubulin assembly, despite the fact that their interactions with the microtubule binding sites (pore and luminal) are similar as studied by NMR and SAXS. A differential interaction with the S7-S9 loop (M-loop) is responsible for their enhanced assembly induction properties. The chemical changes in the structure also induce changes in the thermodynamic properties of the interaction, indicating a higher hydrophilicity and also explaining their properties on p-Gp and βIII overexpressing cells and on mutant cells. The effect of the compounds on the microtubular network is different from those observed with the classical (docetaxel and paclitaxel) taxanes, inducing different bundling in cells with microtubules being very short, indicating a very fast nucleation effect and reflecting their high assembly induction power.This research was supported by BIPPED2 (S2010/BMD‐2457) (JFD), and MHit (CAM S2010/BMD-2353 to JJB) projects of the Comunidad de Madrid, the BIO2010‐16351 (to JFD) and CTQ2012-32025 (to JJB) projects from the Ministry of Economy and Competitiveness of Spain, a NSFC grant (No. 30930108) to (WF), the NIH/NCI CA103314 project (to IO) and a Ramon y Cajal grant to Dr. A. Canales.Peer reviewe

Insights into the interaction of discodermolide and docetaxel with tubulin. Mapping the binding sites of microtubule-stabilizing agents by using an integrated NMR and computational approach

41 p.-7 fig.-3 fig.-supl.The binding interactions of two antitumor agents that target the paclitaxel site, docetaxel and discodermolide, to unassembled α/β-tubulin heterodimers and microtubules have been studied using biochemical and NMR techniques. The use of discodermolide as a water-soluble paclitaxel biomimetic and extensive NMR experiments allowed the detection of binding of microtubule-stabilizing agents to unassembled tubulin α/β-heterodimers. The bioactive 3D structures of docetaxel and discodermolide bound to α/β-heterodimers were elucidated and compared to those bound to microtubules, where subtle changes in the conformations of docetaxel in its different bound states were evident. Moreover, the combination of experimental TR-NOE and STD NMR data with CORCEMA-ST calculations indicate that docetaxel and discodermolide target an additional binding site at the pore of the microtubules, which is different from the internal binding site at the lumen previously determined by electron crystallography. Binding to this pore site can then be considered as the first ligand-protein recognition event that takes place in advance of the drug internalization process and interaction with the lumen of the microtubulesThis work was supported in part by grant BIO2010-16351 and BQU2009-08536 from MICINN (to J.F.D. and J.J.B., respectively), BIPPED-CM from Comunidad de Madrid (J.F.D., J.J.B., and J.M.A.), and EPSRC (I.P.). We also thank CESGA and the CAI NMR of Universidad Complutense for providing access to their facilitiesPeer reviewe

Modulation of microtubule interprotofilament interactions by modified taxanes

11 páginas, 4 figuras, 2 tablas -- PAGS nros. 2970-2980Microtubules assembled with paclitaxel and docetaxel differ in their numbers of protofilaments, reflecting modification of the lateral association between αβ-tubulin molecules in the microtubule wall. These modifications of microtubule structure, through a not-yet-characterized mechanism, are most likely related to the changes in tubulin-tubulin interactions responsible for microtubule stabilization by these antitumor compounds. We have used a set of modified taxanes to study the structural mechanism of microtubule stabilization by these ligands. Using small-angle x-ray scattering, we have determined how modifications in the shape and size of the taxane substituents result in changes in the interprotofilament angles and in their number. The observed effects have been explained using NMR-aided docking and molecular dynamic simulations of taxane binding at the microtubule pore and luminal sites. Modeling results indicate that modification of the size of substituents at positions C7 and C10 of the taxane core influence the conformation of three key elements in microtubule lateral interactions (the M-loop, the S3 β-strand, and the H3 helix) that modulate the contacts between adjacent protofilaments. In addition, modifications of the substituents at position C2 slightly rearrange the ligand in the binding site, modifying the interaction of the C7 substituent with the M-loopThis work was supported in part by grants BIO2010-16351, BQU2009-08536, MAT2008-03232, from the Ministerio de Ciencia e Innovación (to J.F.D., J.J.B., and A.N., respectively) and National Natural Science Foundation of China (NSFC) grant 30930108 to W.S.F. The ESRF and the Netherlands Organization for Scientific Research (NWO) are gratefully acknowledged for beamtime on beamline BM26B (38) and the beamline staff is thanked for their supportPeer reviewe

Zampanolide binding to tubulin indicates crosstalk of taxane site with colchicine and nucleotide sites

41 p.-8 fig.-2 tab. Field, Jessica J. et al.The marine natural product zampanolide and analogues thereof constitute a new chemotype of taxoid site microtubule-stabilizing agents with a covalent mechanism of action. Zampanolide-ligated tubulin has the switch-activation loop (M-loop) in the assembly prone form and, thus, represents an assembly activated state of the protein. In this study, we have characterized the biochemical properties of the covalently modified, activated tubulin dimer, and we have determined the effect of zampanolide on tubulin association and the binding of tubulin ligands at other binding sites. Tubulin activation by zampanolide does not affect its longitudinal oligomerization but does alter its lateral association properties. The covalent binding of zampanolide to β-tubulin affects both the colchicine site, causing a change of the quantum yield of the bound ligand, and the exchangeable nucleotide binding site, reducing the affinity for the nucleotide. While these global effects do not change the binding affinity of 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC) (a reversible binder of the colchicine site), the binding affinity of a fluorescent analogue of GTP (Mant-GTP) at the nucleotide E-site is reduced from 12 ± 2 × 105 M-1 in the case of unmodified tubulin to 1.4 ± 0.3 × 105 M-1 in the case of the zampanolide tubulin adduct, indicating signal transmission between the taxane site and the colchicine and nucleotide sites of β-tubulin.This work was supported in part by grants BFU2016-75319-R (AEI/FEDER, UE) from Ministerio de Economia y Competitividad and the Cancer Society of New Zealand, and the Wellington Medical Research Foundation (JJF & JHM). The authors acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery”. The CNIC is supported by the Ministerio de Ciencia e Innovación and the Fundación Pro CNIC.Peer reviewe