title: Molecular dynamic simulations of protein/RNA complexes: CRISPR/Csy4 endoribonuclease.
abstract: BACKGROUND
  Many prokaryotic genomes comprise Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) offering defense against foreign nucleic acids. These immune systems are conditioned by the production of small CRISPR-derived RNAs matured from long RNA precursors. This often requires a Csy4 endoribonuclease cleaving the RNA 3'-end.
  
  
  METHODS
  We report extended explicit solvent molecular dynamic (MD) simulations of Csy4/RNA complex in precursor and product states, based on X-ray structures of product and inactivated precursor (55 simulations; ~3.7μs in total).
  
  
  RESULTS
  The simulations identify double-protonated His29 and deprotonated terminal phosphate as the likely dominant protonation states consistent with the product structure. We revealed potential substates consistent with Ser148 and His29 acting as the general base and acid, respectively. The Ser148 could be straightforwardly deprotonated through solvent and could without further structural rearrangements deprotonate the nucleophile, contrasting similar studies investigating the general base role of nucleobases in ribozymes. We could not locate geometries consistent with His29 acting as general base. However, we caution that the X-ray structures do not always capture the catalytically active geometries and then the reactive structures may be unreachable by the simulation technique.
  
  
  CONCLUSIONS
  We identified potential catalytic arrangement of the Csy4/RNA complex but we also report limitations of the simulation technique. Even for the dominant protonation state we could not achieve full agreement between the simulations and the structural data.
  
  
  CONCLUSIONS
  Potential catalytic arrangement of the Csy4/RNA complex is found. Further, we provide unique insights into limitations of simulations of protein/RNA complexes, namely, the influence of the starting experimental structures and force field limitations. This article is part of a Special Issue entitled Recent developments of molecular dynamics.
authors.affiliation: CEITEC - Central European Institute of Technology, Masaryk University, Campus Bohunice, Kamenice 5, 625 00 Brno, Czech Republic.
authors.forename: Carolina
authors.initials: C
authors.lastname: Estarellas
authors.affiliation: Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacky University Olomouc, tr. 17 listopadu 12, 771 46 Olomouc, Czech Republic.
authors.forename: Michal
authors.initials: M
authors.lastname: Otyepka
authors.affiliation: CEITEC - Central European Institute of Technology, Masaryk University, Campus Bohunice, Kamenice 5, 625 00 Brno, Czech Republic.
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Koča
authors.affiliation: Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacky University Olomouc, tr. 17 listopadu 12, 771 46 Olomouc, Czech Republic.
authors.forename: Pavel
authors.initials: P
authors.lastname: Banáš
authors.affiliation: Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno, Czech Republic.
authors.forename: Miroslav
authors.initials: M
authors.lastname: Krepl
authors.affiliation: CEITEC - Central European Institute of Technology, Masaryk University, Campus Bohunice, Kamenice 5, 625 00 Brno, Czech Republic; Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno, Czech Republic. Electronic address: sponer@ncbr.muni.cz.
authors.forename: Jiří
authors.initials: J
authors.lastname: Šponer
mesh_terms: D001665:Binding Sites; D064130:CRISPR-Associated Proteins / Q000737:chemistry* / Q000378:metabolism; D064113:CRISPR-Cas Systems; D020134:Catalytic Domain; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D018360:Crystallography, X-Ray; D004722:Endoribonucleases / Q000737:chemistry* / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D013997:Time Factors
chemical_list: D064130:CRISPR-Associated Proteins; D004722:Endoribonucleases
keywords: Cas6 superfamily; Endoribonuclease; Force field; Molecular dynamic simulation; Protein/RNA complex; RNA cleavage