Computational protocol: The first crystal structure of human RNase 6 reveals a novel substrate-binding and cleavage site arrangement

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Protocol publication

[…] Data were collected at the XALOC BL13 beamline station of ALBA synchrotron (Spain) using a wavelength of 0.9795 Å. Data collection was performed at 100 K using a Pilatus 6M detector (Dectris®), 800 images were taken at texp=0.2 s, Δφ=0.2°. The data obtained were processed with XDS (MPI for Medical Research) []. The structure was solved by molecular replacement with Phenix Phaser-MR program using an RNase 6 model constructed upon NMR structure of RNase 7 (PDB ID: 2HKY) []. Iterative cycles of refinement and manual building were applied using PHENIX [] and Coot [] respectively until no further improvement of Rfree could be achieved. Finally, the stereochemistry of the structure was validated with SFCHECK [] and WHAT_CHECK []. shows the data collection and structure refinement statistics. [...] Molecular modelling predictions were carried out using protein–nucleotide docking and molecular dynamics (MD) simulations. Docking simulations were conducted with AutoDock 4.2.6 (Scripps Research Institute) and MD simulations were performed with GROMACS 4.5.5 []. RNase A and RNase 6 complexes with dinucleotides (CpA, UpA and UpG) were predicted. The initial RNase A–dinucleotides’ positions were determined on the basis of crystallographic data of RNase A bound to d(CpA) []. For RNase 6–dinucleotide complexes, the position of the S1 sulfate was taken as reference. Due to the inactive position of His122 in the RNase 6 crystal, the position of the histidine was adjusted to the ‘active’ conformation taking RNase A as a reference (PDB ID: 1RPG).For MD simulations the force field AMBER99SB-ILDN [] was used both for protein and RNA components. All of the complexes were centred in a cubic cell with a minimum distance of box to solute of 1.0 nm. The unit cell was filled with transferable intermolecular potential 3P (TIP3P) water [] in neutral conditions with 150 mM NaCl. Neighbour search was performed using a group cut-off scheme with a cut-off of 1.4 nm for van der Waals interactions and 0.9 nm for the other short-range Lennard–Jones interactions. For long range interactions, smooth particle mesh of Ewald (PME) [,] was used with a fourth-order interpolation scheme and 0.16-nm grid spacing for FFT. The bonds were constrained with the P-LINCS algorithm [], with an integration time step of 2 fs. The energy of the system was minimized using the steepest descendant algorithm and equilibrated in two steps. First, an initial constant volume equilibration (NVT) of 100 ps was performed with a temperature of 300 K using a modified Berendsen thermostat. Then, 100 ps of constant pressure equilibration (NPT) was run at 1 bar (100 kPa) with a Parrinello–Rahman barostat [,] at 300 K and the same thermostat. Finally, 20 ns production runs were performed under an NPT ensemble without applying restraints. Three independent simulations in periodic boundary conditions were conducted for each complex. The evolution of the average RMSD for all non-hydrogen ligand atoms after least-squares fitting to the original position was calculated.For prediction of the RNase 6–heptanucleotide complex, the RNase A–d(ATAA) crystal structure was taken as a reference (PDB ID: 1RCN []). First, the d(ATAA) co-ordinates were used to build an AUAA ribonucleotide. His122 of RNase 6 was fixed in the corresponding active conformation. Local search docking with 2000 cycles and 2000 iterations was performed with AutoDock 4.2.6 [] to adjust the AUAA position to RNase 6 active site. Then, three cytidines were added to the 5′ end of the tetranucleotide. The sulfate positions of the RNase 6 structures were taken as a reference to place the phosphates corresponding to the extended nucleotide. Then, a steepest descent energy minimization of the complex was performed with GROMACS 4.5.5. MD simulations were also applied using the same protocol described for dinucleotides. […]

Pipeline specifications

Software tools AutoDock, GROMACS, P-LINCS
Application Protein interaction analysis
Organisms Homo sapiens
Diseases Bacterial Infections
Chemicals Nucleotides, Phosphates