Browsing by Subject "Molecular Dynamics Simulation"
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Item Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations(Springer Nature, 2016-11-22) Zuo, Zhicheng; Liu, JinThe CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg(2+) ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg(2+) bound. The simulation results showed that binding of two Mg(2+) ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.Item Interaction of Ligands for PET with the Dopamine D3 Receptor: In Silico and In Vitro Methods(MDPI, 2021-04-02) Hsieh, Chia-JI; Riad, Aladdin; Lee, Ji Youn; Sahlholm, Kristoffer; Xu, Kuiying; Luedtke, Robert R.; Mach, Robert H.[(18)F]Fallypride and [(18)F]Fluortriopride (FTP) are two different PET radiotracers that bind with sub-nanomolar affinity to the dopamine D3 receptor (D3R). In spite of their similar D3 affinities, the two PET ligands display very different properties for labeling the D3R in vivo: [(18)F]Fallypride is capable of binding to D3R under "baseline" conditions, whereas [(18)F]FTP requires the depletion of synaptic dopamine in order to image the receptor in vivo. These data suggest that [(18)F]Fallypride is able to compete with synaptic dopamine for binding to the D3R, whereas [(18)F]FTP is not. The goal of this study was to conduct a series of docking and molecular dynamic simulation studies to identify differences in the ability of each molecule to interact with the D3R that could explain these differences with respect to competition with synaptic dopamine. Competition studies measuring the ability of each ligand to compete with dopamine in the beta-arrestin assay were also conducted. The results of the in silico studies indicate that FTP has a weaker interaction with the orthosteric binding site of the D3R versus that of Fallypride. The results of the in silico studies were also consistent with the IC50 values of each compound in the dopamine beta-arrestin competition assays. The results of this study indicate that in silico methods may be able to predict the ability of a small molecule to compete with synaptic dopamine for binding to the D3R.Item Rigid Residue Scan Simulations Systematically Reveal Residue Entropic Roles in Protein Allostery(PLOS, 2016-04-26) Kalescky, Robert; Zhou, Hongyu; Liu, Jin; Tao, PengIntra-protein information is transmitted over distances via allosteric processes. This ubiquitous protein process allows for protein function changes due to ligand binding events. Understanding protein allostery is essential to understanding protein functions. In this study, allostery in the second PDZ domain (PDZ2) in the human PTP1E protein is examined as model system to advance a recently developed rigid residue scan method combining with configurational entropy calculation and principal component analysis. The contributions from individual residues to whole-protein dynamics and allostery were systematically assessed via rigid body simulations of both unbound and ligand-bound states of the protein. The entropic contributions of individual residues to whole-protein dynamics were evaluated based on covariance-based correlation analysis of all simulations. The changes of overall protein entropy when individual residues being held rigid support that the rigidity/flexibility equilibrium in protein structure is governed by the La Chatelier's principle of chemical equilibrium. Key residues of PDZ2 allostery were identified with good agreement with NMR studies of the same protein bound to the same peptide. On the other hand, the change of entropic contribution from each residue upon perturbation revealed intrinsic differences among all the residues. The quasi-harmonic and principal component analyses of simulations without rigid residue perturbation showed a coherent allosteric mode from unbound and bound states, respectively. The projection of simulations with rigid residue perturbation onto coherent allosteric modes demonstrated the intrinsic shifting of ensemble distributions supporting the population-shift theory of protein allostery. Overall, the study presented here provides a robust and systematic approach to estimate the contribution of individual residue internal motion to overall protein dynamics and allostery.Item Structure and Dynamics of Cas9 HNH Domain Catalytic State(Springer Nature, 2017-12-08) Zuo, Zhicheng; Liu, JinThe bacterial CRISPR-Cas9 immune system has been harnessed as a powerful and versatile genome-editing tool and holds immense promise for future therapeutic applications. Despite recent advances in understanding Cas9 structures and its functional mechanism, little is known about the catalytic state of the Cas9 HNH nuclease domain, and identifying how the divalent metal ions affect the HNH domain conformational transition remains elusive. A deeper understanding of Cas9 activation and its cleavage mechanism can enable further optimization of Cas9-based genome-editing specificity and efficiency. Using two distinct molecular dynamics simulation techniques, we have obtained a cross-validated catalytically active state of Cas9 HNH domain primed for cutting the target DNA strand. Moreover, herein we demonstrate the essential roles of the catalytic Mg(2+) for the active state formation and stability. Importantly, we suggest that the derived catalytic conformation of the HNH domain can be exploited for rational engineering of Cas9 variants with enhanced specificity.