Using normal mode analysis on protein structural models. How far can we go on our predictions?

International audienceNormal Mode Analysis is a fast and inexpensive approach that is largely used to gain insight into functional protein motions, and more recently to create conformations for further computational studies. However, when the protein structure is unknown, the use of computational models is necessary. Here, we analyze the capacity of normal mode analysis in internal coordinate space to predict protein motion, its intrinsic flexibility and atomic displacements, using protein models instead of native structures, and the possibility to use it for model refinement. Our results show that normal mode analysis is quite insensitive to modelling errors, but that calculations are strictly reliable only for very accurate models. Our study also suggests that internal normal mode analysis is a more suitable tool for the improvement of structural models, and for integrating them with experimental data or in other computational techniques, such as protein docking or more refined molecular dynamics simulations

Structural insights into the allosteric site of Arabidopsis NADP-malic enzyme 2: role of the second sphere residues in the regulatory signal transmission

Structure–function studies contribute to deciphering how small modifcations in the primary structure could introduce desirable characteristics into enzymes without afecting its overall functioning. Malic enzymes (ME) are ubiquitous and responsible for a wide variety of functions. The availability of a high number of ME crystal structures from diferent species facilitates comparisons between sequence and structure. Specifcally, the structural determinants necessary for fumarate allosteric regulation of ME has been of particular interest. NADP-ME2 from Arabidopsis thaliana exhibits a distinctive and complex regulation by fumarate, acting as an activator or an inhibitor according to substrate and efector concentrations. However, the 3D structure for this enzyme is not yet reported. In this work, we characterized the NADP-ME2 allosteric site by structural modeling, molecular docking, normal mode analysis and mutagenesis. The regulatory site model and its docking analysis suggested that other C4 acids including malate, NADP-ME2 substrate, could also ft into fumarate’s pocket. Besides, a non-conserved cluster of hydrophobic residues in the second sphere of the allosteric site was identifed. The substitution of one of those residues, L62, by a less fexible residue as tryptophan, resulted in a complete loss of fumarate activation and a reduction of substrate afnities for the active site. In addition, normal mode analysis indicated that conformational changes leading to the activation could originate in the region surrounding L62, extending through the allosteric site till the active site. Finally, the results in this work contribute to the understanding of structural determinants necessary for allosteric regulation providing new insights for enzyme optimization.Fil: Gerrard Wheeler, Mariel Claudia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET); Argentina.Fil: Arias, Cintia Lucía. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET); Argentina.Fil: Da Fonseca Rezende e Mello, Juliana. Universidade Federal do Rio de Janeiro. Faculdade de Farmácia. Laboratório de Modelagem Molecular & QSAR (ModMolQSAR); Brazil.Fil: Cirauqui Diaz, Nuria. Universidade Federal do Rio de Janeiro. Faculdade de Farmácia. Laboratório de Modelagem Molecular & QSAR (ModMolQSAR); Brazil.Fil: Rodrigues, Carlos Rangel. Universidade Federal do Rio de Janeiro. Faculdade de Farmácia. Laboratório de Modelagem Molecular & QSAR (ModMolQSAR); Brazil.Fil: Drincovich, María Fabiana. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET); Argentina.Fil: Mendonça Teles de Souza, Alessandra. Universidade Federal do Rio de Janeiro. Faculdade de Farmácia. Laboratório de Modelagem Molecular & QSAR (ModMolQSAR); Brazil.Fil: Alvarez, Clarisa Ester. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET); Argentina

Multiscale molecular simulations to investigate adenylyl cyclase‐based signaling in the brain

Adenylyl cyclases (ACs) play a key role in many signaling cascades. ACs catalyze the production of cyclic AMP from ATP and this function is stimulated or inhibited by the binding of their cognate stimulatory or inhibitory Gα subunits, respectively. Here we used simulation tools to uncover the molecular and subcellular mechanisms of AC function, with a focus on the AC5 isoform, extensively studied experimentally. First, quantum mechanical/molecular mechanical free energy simulations were used to investigate the enzymatic reaction and its changes upon point mutations. Next, molecular dynamics simulations were employed to assess the catalytic state in the presence or absence of Gα subunits. This led to the identification of an inactive state of the enzyme that is present whenever an inhibitory Gα is associated, independent of the presence of a stimulatory Gα. In addition, the use of coevolution-guided multiscale simulations revealed that the binding of Gα subunits reshapes the free-energy landscape of the AC5 enzyme by following the classical population-shift paradigm. Finally, Brownian dynamics simulations provided forward rate constants for the binding of Gα subunits to AC5, consistent with the ability of the protein to perform coincidence detection effectively. Our calculations also pointed to strong similarities between AC5 and other AC isoforms, including AC1 and AC6. Findings from the molecular simulations were used along with experimental data as constraints for systems biology modeling of a specific AC5-triggered neuronal cascade to investigate how the dynamics of downstream signaling depend on initial receptor activation