Unraveling the Differences of the Hydrolytic Activity of Trypanosoma cruzi trans-Sialidase and Trypanosoma rangeli Sialidase: A Quantum Mechanics–Molecular Mechanics Modeling Study

Chagas’ disease, also known as American trypanosomiasis, is a lethal, chronic disease that currently affects more than 10 million people in Central and South America. The trans-sialidase from Trypanosoma cruzi (T. cruzi, TcTS) is a crucial enzyme for the survival of this parasite: sialic acids from the host are transferred to the cell surface glycoproteins of the trypanosome, thereby evading the host’s immune system. On the other hand, the sialidase of T. rangeli (TrSA), which shares 70% sequence identity with TcTS, is a strict hydrolase and shows no trans-sialidase activity. Therefore, TcTS and TrSA represent an excellent framework to understand how different catalytic activities can be achieved with extremely similar structures. By means of combined quantum mechanics–molecular mechanics (QM/MM, SCC-DFTB/Amberff99SB) calculations and umbrella sampling simulations, we investigated the hydrolysis mechanisms of TcTS and TrSA and computed the free energy profiles of these reactions. The results, together with our previous computational investigations, are able to explain the catalytic mechanism of sialidases and describe how subtle differences in the active site make TrSA a strict hydrolase and TcTS a more efficient trans-sialidase.Fil: Bueren Calabuig, Juan A.. University of Florida; Estados UnidosFil: Pierdominici Sottile, Gustavo. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Roitberg, Adrián. University of Florida; Estados Unido

Molecular Dynamics Simulations of Substrate Release from Trypanosoma cruzi UDP-Galactopyranose Mutase

The enzyme UDP-galactopyranose mutase (UGM) represents a promising drug target for the treatment of infections with Trypanosoma cruzi. We have computed the Potential of Mean Force for the release of UDP-galactopyranose from UGM, using Umbrella Sampling simulations. The simulations revealed the conformational changes that both substrate and enzyme undergo during the process. It was determined that the galactopyranose portion of the substrate is highly mobile and that the opening/closing of the active site occurs in stages. Previously uncharacterized interactions with highly conserved residues were also identified. These findings provide new pieces of information that contribute to the rational design of drugs against T. cruzi.Fil: Cossio Pérez, Rodrigo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pierdominici Sottile, Gustavo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sobrado, Pablo. No especifíca;Fil: Palma, Juliana Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentin

Positively charged residues in the head domain of P2X4 receptors assist the binding of ATP

P2X receptors are a family of trimeric cationic channels located in the membrane of mammalian cells. They open in response to the binding of ATP. The differences between the closed and open structures have been described in detail for some members of the family. However, the order in which the conformational changes take place as ATP enters the binding cleft, and the residues involved in the intermediate stages, are still unknown. Here, we present the results of umbrella sampling simulations aimed to elucidate the sequence of conformational changes that occur during the reversible binding of ATP to the P2X4 receptor. The simulations also provided information about the interactions that develop in the course of the process. In particular, they revealed the existence of a metastable state which assists the binding. This state is stabilized by positively charged residues located in the head domain of the receptor. Based on these findings, we propose a novel mechanism for the capture of ATP by P2X4 receptors.Fil: Racigh, Vanesa Elizabeth. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ormazábal, Agustín. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Palma, Juliana Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Pierdominici Sottile, Gustavo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Efectos cuánticos en reacciones enzimáticas de transferencia de hidrógeno

Pierdominici Sottile, G. (2017). Efectos cuánticos en reacciones enzimáticas de transferencia de hidrógeno. (Tesis de posgrado). Bernal, Argentina: Universidad Nacional de Quilmes.En el presente trabajo se utilizó un método mixto clásico-cuántico para estudiar la etapa determinante de la velocidad de oxidación de metilamina, catalizada por la enzima metilamino deshidrogenasa (MADH). En esta etapa, un protón es transferido desde un grupo metilo del sustrato hacia una base del sitio activo. Los resultados experimentales indican que el traspaso del protón ocurre por efecto túnel, un efecto cuántico que sólo recientemente fue observado en enzimas. En el capítulo 2 se exponen los principios básicos de química cuántica. En el capítulo 3 se explican las diferentes metodologías computacionales empleadas para calcular la energía electrónica de un sistema molecular. Con estas herramientas se discuten, en el capítulo 4, los fundamentos de los métodos de mecánica y dinámica molecular. Concretamente se describen los campos de fuerzas, el método Empirical Valence Bond, y los principios del método mixto clásico-cuántico implementado. El evento estudiado y su relevancia se exponen en el capítulo 5. Ya en la segunda parte del trabajo, en el capítulo 6, se presentan los resultados de cálculos cuánticos efectuados sobre modelos del sitio activo de la enzima. Luego, en el 7, se muestra un estudio de dinámica molecular clásica, realizado a fin de analizar los movimientos de la enzima en la etapa previa a la transferencia del protón. Posteriormente, en el capítulo 8 se examina la influencia del residuo Thr122 en la transferencia del protón. El detalle de la metodología mixta clásico-cuántica utilizada para simular la etapa determinante de la velocidad de la reacción y calcular el efecto isotópico cinético, junto con los resultados obtenidos se exponen en el capítulo 9. Finalmente, en el capítulo 10, se presentan las conclusiones generales del trabajo y las perspectivas para su continuación en el futuro

Fortuitous Correlations in Molecular Dynamics Simulations: Their Harmful Influence on the Probability Distributions of the Main Principal Components

Nonsense correlations frequently develop between independent random variables that evolve with time. Therefore, it is not surprising that they appear between the components of vectors carrying out multidimensional random walks, such as those describing the trajectories of biomolecules in molecular dynamics simulations. The existence of these correlations does not imply in itself a problem. Still, it can present a problem when the trajectories are analyzed with an algorithm such as the Principal Component Analysis (PCA) because it seeks to maximize correlations without discriminating whether they have physical origin or not. In this Article, we employ random walks occurring on multidimensional harmonic potentials to evaluate the influence of fortuitous correlations in PCA. We demonstrate that, because of them, this algorithm affords misleading results when applied to a single trajectory. The errors do not only affect the directions of the first eigenvectors and their eigenvalues, but the very definition of the molecule’s “essential space” may be wrong. Additionally, the main principal component’s probability distributions present artificial structures which do not correspond with the shape of the potential energy surface. Finally, we show that the PCA of two realistic protein models, human serum albumin and lysozyme, behave similarly to the simple harmonic models. In all cases, the problems can be mitigated and eventually eliminated by doing PCA on concatenated trajectories formed from a large enough number of individual simulations.Fil: Palma, Juliana Isabel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pierdominici Sottile, Gustavo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Molecular Dynamics of Biomolecules

Para cumplir con su función biológica, proteínas y ácidos nucleicos realizan precisos cambios conformacionales. Los mismos, son producidos por la coordinación de pequeñas fluctuaciones ejecutadas por sus átomos. Curiosamente, a simple vista, estos átomos parecen moverse de manera caótica. Las simulaciones de dinámica molecular (MD, por {it Molecular Dynamics}) constituyen una herramienta particularmente versátil para investigar la conexión entre los cuasi-aleatorios movimientos atómicos y los cambios conformacionales. De esta manera, permiten construir puentes que van desde la secuencia a la estructura; y desde allí a la dinámica y la función biológica.Los fundamentos de las simulaciones MD son simples e intuitivos. Sin embargo, al momento de implementar un estudio particular, es posible tomar muchas malas decisiones que no impedirán realizar el cálculo, sino que subrepticiamente harán que sus resultados sean carentes de cualquier valor.En este artículo, presentaremos los conceptos fundamentales de las simulaciones MD, resaltando aquellos aspectos que son críticos para asegurar su utilidad. Además, discutiremos sus fortalezas y limitaciones, destacando cómo las mismas fueron evolucionando con el tiempo. Asimismo, brindaremos ejemplos paradigmáticos que ilustran las capacidades actuales de esta valiosa herramienta computacional.To fulfill their biological function, proteins and nucleic acids undergo preci-se conformational changes. These changes are brought about by the coordination of small fluctuations executed by their atoms. Interestingly, at first glance, these atoms seem to move in a chaotic manner. Molecular dynamics simulations (MD) are a particularly versatile tool for investigating the connection between quasi-random atomic motions and conformational changes. In this way, they allow us to build bridges from sequence to structure, and from there to dynamics and biological function. The fundamentals of MD simulations are simple and intuitive. However, when implementing a specific study, it is possible to make many poor decisions that will not prevent the calculation from being performed but will surreptitiously render its results devoid of any value. In this article, we will present the fundamental concepts of MD simulations, highlighting those aspects that are critical to ensuring their utility. Additionally, we will discuss their strengths and limitations, emphasizing how they have evolved over time. Furthermore, we will provide paradigmatic examples that illustrate the current capabilities of this valuable computational tool.Fil: Palma, Juliana Isabel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pierdominici Sottile, Gustavo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

New insights into the meaning and usefulness of principal component analysis of concatenated trajectories

A comparison between different conformations of a given protein, relating both structure and dynamics, can be performed in terms of combined principal component analysis (combined-PCA). To that end, a trajectory is obtained by concatenating molecular dynamics trajectories of the individual conformations under comparison. Then, the principal components are calculated by diagonalizing the correlation matrix of the concatenated trajectory. Since the introduction of this approach in 1995 it has had a large number of applications. However, the interpretation of the eigenvectors and eigenvalues so obtained is based on intuitive foundations, because analytical expressions relating the concatenated correlation matrix with those of the individual trajectories under consideration have not been provided yet. In this article, we present such expressions for the cases of two, three, and an arbitrary number of concatenated trajectories. The formulas are simple and show what is to be expected and what is not to be expected from a combined-PCA. Their correctness and usefulness is demonstrated by discussing some representative examples. The results can be summarized in a simple sentence: the correlation matrix of a concatenated trajectory is given by the average of the individual correlation matrices plus the correlation matrix of the individual averages. From this it follows that the combined-PCA of trajectories belonging to different free energy basins provides information that could also be obtained by alternative and more straightforward means.Fil: Pierdominici Sottile, Gustavo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Palma, Juliana Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentin

On the Uses of PCA to Characterise Molecular Dynamics Simulations of Biological Macromolecules: Basics and Tips for an Effective Use

Principal Component Analysis (PCA) is a procedure widely used to examine data collected from molecular dynamics simulations of biological macromolecules. It allows for greatly reducing the dimensionality of their configurational space, facilitating further qualitative and quantitative analysis. Its simplicity and relatively low computational cost explain its extended use. However, a judicious implementation of PCA requires the knowledge of its theoretical grounds as well as its weaknesses and capabilities. In this article, we review these issues and discuss several strategies developed over the last years to mitigate the main PCA flaws and enhance the reproducibility of its results.Fil: Palma, Juliana Isabel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pierdominici Sottile, Gustavo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Free energy computations identify the mutations required to confer trans-sialidase activity into Trypanosoma rangeli sialidase

Trypanosoma rangeli's sialidase (TrSA) and Trypanosoma cruzi's trans-sialidase (TcTS) are members of the glycoside hydrolase family 33 (GH-33). They share 70% of sequence identity and their crystallographic Cα RMSD is 0.59 Å. Despite these similarities they catalyze different reactions. TcTS transfers sialic acid between glycoconjugates while TrSA can only cleave sialic acid from sialyl-glyconjugates. Significant effort has been invested into unraveling the differences between TrSA and TcTS, and into conferring TrSA with trans-sialidase activity through appropriate point mutations. Recently, we calculated the free-energy change for the formation of the covalent intermediate (CI) in TcTS and performed an energy decomposition analysis of that process. In this article we present a similar study for the formation of the CI in TrSA, as well as in a quintuple mutant (TrSA5mut), which has faint trans-sialidase activity. The comparison of these new results with those previously obtained for TcTS allowed identifying five extra mutations to be introduced in TrSA5mut that should create a mutant (TrSA10mut) with high trans-sialidase activity.Fil: Pierdominici Sottile, Gustavo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Palma, Juliana Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Roitberg, Adrián. University of Florida. Departament of Chemistry; Estados Unido