Mechanism of Coupled Folding and Binding in the siRNA-PAZ Complex.

The PAZ domain plays a key role in gene silencing pathway. The PAZ domain binds with siRNAs to form the multimeric RNA-induced silencing complex (RISC). RISC identifies mRNAs homologous to the siRNAs and promotes their degradation. It was found that binding with siRNA significantly enhances apo-PAZ folding. However, the mechanism by which folding is coupled to binding is poorly understood. Thus, the coupling relationship between binding and folding is very important for understanding the function of gene silencing. We have performed molecular dynamics (MD) of both bound and apo-PAZ to study the coupling mechanism between binding and folding in the siRNA-PAZ complex. Room-temperature MD simulations suggest that both PAZ and siRNA become more rigid and stable upon siRNA binding. Kinetic analysis of high-temperature MD simulations shows that both bound and apo-PAZ unfold via a two-state process. The unfolding pathways are different between bound and apo-PAZ: the order of helix III and helices I & II unfolding is switched. Furthermore, transition probability was used to determine the transition state ensemble for both bound and apo-PAZ. It was found that the transition state of bound PAZ is more compact than that of apo-PAZ. The predicted Φ-values suggest that the Φ-values of helix III and sheets of β3-β7 for bound PAZ are more native-like than those of apo-PAZ upon the binding of siRNA. The results can help us to understand the mechanism of gene silencing.

[1]  H. Berendsen,et al.  Molecular dynamics with coupling to an external bath , 1984 .

[2]  T. Oas,et al.  Linked folding and anion binding of the Bacillus subtilis ribonuclease P protein. , 2001, Biochemistry.

[3]  R. Luo,et al.  Force field influences in β‐hairpin folding simulations , 2006 .

[4]  T. Darden,et al.  Particle mesh Ewald: An N⋅log(N) method for Ewald sums in large systems , 1993 .

[5]  T. Kiefhaber,et al.  Protein folding kinetics. , 1995, Methods in molecular biology.

[6]  Ira M. Hall,et al.  Establishment and Maintenance of a Heterochromatin Domain , 2002, Science.

[7]  A Caflisch,et al.  Acid and thermal denaturation of barnase investigated by molecular dynamics simulations. , 1995, Journal of molecular biology.

[8]  P. Zamore,et al.  ATP Requirements and Small Interfering RNA Structure in the RNA Interference Pathway , 2001, Cell.

[9]  D. Patel,et al.  Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain , 2004, Nature.

[10]  Ryan Day,et al.  Direct observation of microscopic reversibility in single-molecule protein folding. , 2007, Journal of molecular biology.

[11]  A. Caudy,et al.  Role for a bidentate ribonuclease in the initiation step of RNA interference , 2001 .

[12]  Ira M. Hall,et al.  Regulation of Heterochromatic Silencing and Histone H3 Lysine-9 Methylation by RNAi , 2002, Science.

[13]  R. Bhatnagar,et al.  RNA Interference: Biology, Mechanism, and Applications , 2003, Microbiology and Molecular Biology Reviews.

[14]  A. Fersht,et al.  Protein folding and unfolding in microseconds to nanoseconds by experiment and simulation. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Bengt Nölting,et al.  Protein Folding Kinetics: Biophysical Methods , 1999 .

[16]  G. Hannon,et al.  RNAi: an ever-growing puzzle. , 2003, Trends in biochemical sciences.

[17]  T. Oas,et al.  Ligation-state hydrogen exchange: coupled binding and folding equilibria in ribonuclease P protein. , 2006, Journal of the American Chemical Society.

[18]  T. Tuschl,et al.  RNA interference is mediated by 21- and 22-nucleotide RNAs. , 2001, Genes & development.

[19]  Valerie Daggett,et al.  The complete folding pathway of a protein from nanoseconds to microseconds , 2003, Nature.

[20]  B. Simon,et al.  Structure and nucleic-acid binding of the Drosophila Argonaute 2 PAZ domain , 2003, Nature.

[21]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[22]  M Levitt,et al.  Hierarchy of structure loss in MD simulations of src SH3 domain unfolding. , 1999, Journal of molecular biology.

[23]  Stephen J. Moran,et al.  The folding pathway of spectrin R17 from experiment and simulation: using experimentally validated MD simulations to characterize States hinted at by experiment. , 2006, Journal of molecular biology.

[24]  Ray Luo,et al.  Binding induced folding in p53-MDM2 complex. , 2007, Journal of the American Chemical Society.

[25]  J M Thornton,et al.  LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. , 1995, Protein engineering.

[26]  W. L. Jorgensen,et al.  Comparison of simple potential functions for simulating liquid water , 1983 .

[27]  G. Ciccotti,et al.  Numerical Integration of the Cartesian Equations of Motion of a System with Constraints: Molecular Dynamics of n-Alkanes , 1977 .

[28]  W. Kabsch,et al.  Dictionary of protein secondary structure: Pattern recognition of hydrogen‐bonded and geometrical features , 1983, Biopolymers.

[29]  O. Voinnet.  RNA silencing as a plant immune system against viruses. , 2001, Trends in genetics : TIG.

[30]  V. Daggett,et al.  Sensitivity of the folding/unfolding transition state ensemble of chymotrypsin inhibitor 2 to changes in temperature and solvent , 2005, Protein science : a publication of the Protein Society.

[31]  S. Akanuma,et al.  A detailed unfolding pathway of a (β/α)8‐barrel protein as studied by molecular dynamics simulations , 2004 .

[32]  P. Sharp,et al.  RNAi Double-Stranded RNA Directs the ATP-Dependent Cleavage of mRNA at 21 to 23 Nucleotide Intervals , 2000, Cell.

[33]  David Baker.  Metastable states and folding free energy barriers , 1998, Nature Structural Biology.

[34]  Junmei Wang,et al.  How well does a restrained electrostatic potential (RESP) model perform in calculating conformational energies of organic and biological molecules? , 2000, J. Comput. Chem..

[35]  Gunter Meister,et al.  Argonaute proteins: mediators of RNA silencing. , 2007, Molecular cell.

[36]  S. Hammond,et al.  An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells , 2000, Nature.

[37]  A. Caudy,et al.  Argonaute2, a Link Between Genetic and Biochemical Analyses of RNAi , 2001, Science.

[38]  A. Fersht,et al.  Structure of the transition state for folding of a protein derived from experiment and simulation. , 1996, Journal of molecular biology.

[39]  A Caflisch,et al.  Role of native topology investigated by multiple unfolding simulations of four SH3 domains. , 2001, Journal of molecular biology.

[40]  V S Pande,et al.  Molecular dynamics simulations of unfolding and refolding of a beta-hairpin fragment of protein G. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[41]  B. Bass,et al.  A Role for the RNase III Enzyme DCR-1 in RNA Interference and Germ Line Development in Caenorhabditis elegans , 2001, Science.

[42]  A. Fersht,et al.  Protein Folding and Unfolding at Atomic Resolution , 2002, Cell.

[43]  B. Karki,et al.  Mechanism of β-purothionin antimicrobial peptide inhibition by metal ions: Molecular dynamics simulation study , 2006 .

[44]  Vijay S Pande,et al.  Dimerization of the p53 oligomerization domain: identification of a folding nucleus by molecular dynamics simulations. , 2005, Journal of molecular biology.

[45]  Ji-Joon Song,et al.  The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes , 2003, Nature Structural Biology.

[46]  A Caflisch,et al.  Molecular dynamics simulation of protein denaturation: solvation of the hydrophobic cores and secondary structure of barnase. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[47]  M. Karplus,et al.  Three key residues form a critical contact network in a protein folding transition state , 2001, Nature.

[48]  Jörg Gsponer,et al.  Molecular dynamics simulations of protein folding from the transition state , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[49]  A. Fersht,et al.  Synergy between simulation and experiment in describing the energy landscape of protein folding. , 1998, Proceedings of the National Academy of Sciences of the United States of America.