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Essential Dynamics on Different Biological System: Fis Protein, tvMyb1 Transcriptional Factor and BACE1 Enzyme Lucas J. Gutiérrez a, b, Ricardo D. Enriz a, b and Héctor A. Baldoni a, c a Área de Química General e Inorgánica, Universidad Nacional de San Luis (UNSL), Chacabuco 917, 5700 San Luis, Argentina. b Instituto Multidisciplinario de Investigaciones Biológicas de San Luis (UNSL, CONICET), Ejercito de Los Andes 950, 5700 San Luis, Argentina. c Instituto de Matemática Aplicada San Luis (UNSL, CONICET), Ejercito de Los Andes 950, 5700 San Luis, Argentina hbaldoni@unsl.edu.ar. In the present chapter we report molecular dynamics (MD) trajectories analyzed by essential dynamics method on three different molecular system of biological interest. We evaluated the conformational behavior of three macromolecular systems: i) DNA-bending protein Fis (Factor for Inversion Stimulation), ii) DNA-tvMyb1 (Trichomonas vaginalis transcriptional factor) and iii) the BACE1 (b-site amyloid cleaving enzyme 1). Methods Molecular dynamics simulation: All molecular dynamics simulations (MD) were carried out by standard procedure using an all atom force field in explicit solvent at the NPT ensemble. The temperature was coupled to Berendsen thermostat. During the MD simulations, the periodic boundary conditions were applied. The particle mesh Ewald summation was used to treat the long-range electrostatics. Essential dynamics: The essential dynamics method is based on the diagonalization of the covariance matrix built from atomic fluctuations in a trajectory from which overall translation and rotations have been removed: in which X are the separate x, y, z coordinates of the atoms, fluctuating around their average positions X0, á...ñ denotes an average over time. Here, to construct the protein covariance matrices we have used Ca atom trajectories. Indeed, it has been shown that the Ca atom contains all the information for a reasonable description of the protein large concerted motions [1]. Upon diagonalization of the covariance matrix, a set of eigenvalues and eigenvectors is obtained. The eigenvectors of the covariance matrix correspond to directions in a 3N dimensional space (where N is the number of Ca atoms), and motions along single eigenvectors correspond to concerted fluctuations of atoms. The eigenvalues represent the total mean square fluctuation of the system along the corresponding eigenvectors. To examine domain motions in a protein, we calculated the cross-correlation (normalized covariance) matrix, Cij, of the fluctuations of each of the x, y and z coordinates of the Ca atoms from their average during the last nanosecond of the simulation for all models. For the displacement vectors Dri and Drj of atoms i and j, this matrix, Cij is give by [2] where Dri is the displacement from the mean position of the ith atom and the angle brackets represent the time average over the entire trajectory. The elements of the cross-correlation matrix take values from -1 to 1. Positive values of Cij represent a motion in the same direction between atoms i and j, and negative Cij values represent a motion in the opposite direction. When Cij is close to zero, the atomic motions are uncorrelated, and their movements are random compared to each other. i) DNA-bending protein Fis Fis is a nucleoid-associated protein found in the γ and β subdivisions of proteobacteria, which include the Enterobacterieae, Pasteurellaceae, Pseudomonaceae, Vibrionaceae, Xanthomonadaceae, Burkholderiaceae, and Neisseriaceae [3?6]. Fis participates in a wide array of cellular activities such as modulation of DNA topology during growth, [7, 8] regulation of certain site-specific DNA recombination events, [9] and regulation of the transcription of a large number of genes during different stages of growth, [10, 11] including ribosomal RNA and tRNA genes and genes involved in virulence and biofilm formation [12, 13]. Most of these functions depend on the ability of Fis to interact with DNA at specific sites. The residues of the Fis protein involved at the DNA flexion and conformational changes over the DNA molecule have been previously reported. However, it is not clear yet how the movements involved in the flexion mechanism are. Our MD simulations performed on both apo-Fis and DNA-Fis displayed a hinge movement which produces the DNA bending. Also, our simulations indicated that this movement is clearly more stressed for the apo-Fis than those observed for the DNA-Fis due to the absence of the DNA. Our theoretical results are in agreement with the experimental data. ii) DNA- tvMyb1 transcriptional factor Not only does DNA contain a big amount of information, it is also consulted by the cell for that information in a myriad ways. Between others, the Myb proteins regulate gene-specific transcription in a wide range of eukaryotic system likes vertebrates, plants and protozoan parasites. Transcription factors regulate the expression of genes at the level of transcription and control many critical biological processes. These factors typically recognize DNA sequences in the promoter regions of the target genes and regulate the frequency of transcription initiation of the genes. Transcription factors contain DNA-binding domains which bind to DNA with high sequence specificity and are classified based on the sequence similarity in the DNA-binding domain. Myb is one of the largest transcription factor families in plants [14], which contains DNA-binding domains composed of one, two or three repeated motifs of approximately 50 amino acids surrounded by three conserved tryptophan residues [15]. These repeated motifs adopt a helix-turn-helix conformation to recognize the major groove of target DNA sequences. Vertebrate c-Myb protein contains three tandem repeated motifs, designated as R1, R2 and R3 motifs [16]. Other Myb repeated motifs are categorized according to their similarity to the R1, R2 or R3 motifs. The transcription factor, tvMyb1 protein, was the first Myb family protein identified in the protozoan parasite Trichomonas vaginalis [17] the causative parasite responsible for the disease trichomoniasis which is one of the most common sexually transmitted diseases in humans [18]. Recently, Lou et al. reported the structural basis for the tvMyb135?141/DNA interaction investigated using chemical shift perturbations, residual dipolar couplings, and DNA specificity [19] The published experimental data indicates that the orientation between R2 and R3 motifs dramatically changes upon binding to DNA so as to recognize the DNA major groove through a number of key contacts involving residues in helices 3 and 6. In this section of our report the simulated dynamics of the tvMyb135?141/DNA complex was carried out in order to shed light about the collective movements and thermodynamic of the complex at the atomic level. The simulated dynamics of the tvMyb135?141/DNA complex furthers the understanding of DNA recognition by Myb proteins and these theoretical calculations could be applied in determining the complex structures involving proteins with multiple domains. iii) BACE1 enzyme BACE1 also called b-secretase is a key protease involved in the proteolysis of amyloid precursor protein (APP) that generates a toxic peptide amyloid beta (Ab), a pathological feature of Alzheimer disease (AD) [20]. BACE1 like other proteolytic enzymes possess an additional binding pocket so-called exosite. Because the exosite are structurally distinct from the active site of this enzyme, the nature of the molecules that bind to the exosite may be different from that of active-site-directed inhibitors. Therefore, it is possible that in some cases the exosite could provide a more pharmacologically tractable target for small molecule interactions than does the catalytic site of the enzyme. Recently we reported where is located the exosite of BACE1 [21]. In the present chapter we report how is modified the conformational behavior of this enzyme by the presence of an inhibitor in the exosite. In this study we performed MD simulations on the BACE1 with and without the inhibitor located at the exosite. In summary, our results indicate that the essential dynamics employed in the three different systems give an accurate description at molecular level about the conformational changes involved in these macromolecular systems. Our theoretical results are in complete agreement with the available experimental data giving an additional support to the molecular behaviour of such systems. References [1] Amadei, A., Linssen, A. B. M., Berendsen, H. J. C. (1993) Essential dynamics of proteins. Proteins: Struct. Funct. Genet., 17, 412-425. [2] Ichiye, T., and Karplus, M. (1991) Collective motions in proteins: a covariance analysis of atomic fluctuations in molecular dynamics and normal mode simulations. Proteins: Struct. Funct. Genet., 11, 205-217. [3] Azam, T. A. & Ishihama, A. (1999). Twelve species of the nucleoid-associated protein from Escherichia coli. Sequence recognition specificity and DNA binding affinity. J. Biol. Chem. 274, 33105?33113. [4] Boswell, S., Mathew, J., Beach, M., Osuna, R. & Colon, W. (2004). Variable contributions of tyrosine residues to the structural and spectroscopic properties of the factor for inversion stimulation. Biochemistry, 43, 2964?2977. [5] Beach, M. B. & Osuna, R. (1998). Identification and characterization of the fis operon in enteric bacteria. J. Bacteriol. 180, 5932?5946. [6] Osuna, R., Lienau, D., Hughes, K. T. & Johnson, R. C. (1995). Sequence, regulation, and functions of Fis in Salmonella typhimurium. J. Bacteriol. 177, 2021?2032. [7] Schneider, R., Travers, A. & Muskhelishvili, G. (1997). Fis modulates growth phase-dependent topological transitions of DNA in Escherichia coli. Mol. Microbiol. 26, 519?530. [8] Schneider, R., Travers, A., Kutateladze, T. & Muskhelishvili, G. (1999).A DNA architectural protein couples cellular physiology and DNA topology in Escherichia coli. Mol. Microbiol. 34, 953?964. [9] Finkel, S. E. & Johnson, R. C. (1992). The Fis protein: it´s not just for DNA inversion anymore. Mol. Microbiol. 6, 3257?3265. [10] Bradley, M. D., Beach, M. B., Jason de Koning, A. P.,Pratt, T. S. & Osuna, R. (2007). Global effects of Fis on Escherichia coli gene expression during different stages of growth. Microbiology, 153, 2922?2940. [11] Kelly, A., Goldberg, M. D., Carroll, R. K., Danino, V., Hinton, J. C. & Dorman, C. J. (2004). A global role for Fis in the transcriptional control of metabolism and type III secretion in Salmonella enterica serovar Typhimurium. Microbiology, 150, 2037?2053. [12] Bosch, L., Nilsson, L., Vijgenboom, E. & Verbeek, H. (1990). FIS-dependent trans-activation of tRNA and rRNA operons of Escherichia coli. Biochim. Biophys. Acta, 1050, 293?301. [13] Ross, W., Thompson, J. F., Newlands, J. T. & Gourse,R. L. (1990). E. coli Fis protein activates ribosomal RNA transcription in vitro and in vivo. EMBO J. 9, 3733?3742. [14] Stracke,R., Werber,M. and Weisshaar,B. (2001) The R2R3-MYB gene family in Arabidopsis thaliana. Curr. Opin. Plant Biol., 4, 447?456. [15] Lipsick, J. S. (1996) One billion years of Myb. Oncogene, 13, 223?235. [16] Ogata, K., Morikawa, S., Nakamura, H., Sekikawa, A., Inoue, T., Kanai, H., Sarai, A., Ishii, S., Nishimura, Y. (1994) Solution structure of a specific DNA complex of the Myb DNA-binding domain with cooperative recognition helices. Cell, 79, 639?648. [17] Ong, S. J., Hsu, H. M., Liu, H. W., Chu, C. H., Tai, J. H. (2006) Multifarious transcriptional regulation of adhesion protein gene ap65-1 by a novel Myb1 protein in the protozoan parasite Trichomonas vaginalis. Eukaryot. Cell, 5, 391?399. [18] Honigberg, B. M. (1978) In Parasitic Protozoa. Academic Press, Inc., New York. [19] Lou, Y.C., Wei, S. Y., Rajasekaran, M., Chou, C. C., Hsu, H. M., Tai, J. H., Chen, C. (2009) NMR structural analysis of DNA recognition by a novel Myb1 DNA-binding domain in the protozoan parasite Trichomonas vaginalis Nucleic Acids Research 37 (7), 2381-2394. [20] Sandipan, C., Sanjay, K., Soumalee, B. (2011) Conformational transition in the substrate binding domain of b-secretase exploited by NMA and its implication in inhibitor recognition: BACE1-myricetin a case study. Neurochemistry International. [21] Gutiérrez, L. J., Enriz, R. D., Baldón, H. A. (2010) Structural and Thermodynamic Characteristics of the Exosite Binding Pocket on the Human BACE1: A Molecular Modeling Approach. J. Phys. Chem. A, 114, 10261?10269