Computational protocol: Structural basis of actin monomer re-charging by cyclase-associated protein

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[…] For complex formation with mouse CAP1242–474, actin was thawed and prepared by first exchanging Ca2+ metal to Mg2+ during O/N dialysis (5 mM HEPES, 0.2 mM MgCl2, 0.2 mM EGTA, 0.2 mM ADP, 0.2 mM DTT, pH 8.0) at 4 °C. The actin was then treated with 20 U/ml hexokinase (Sigma-Aldrich) and 0.3 mM glucose for 1 h and mixed with CAP1242–474 in ~1:1 molar ratio. Complex was further purified by gel filtration (SD200 Increase 10/300, GE Healthcare), pre-equilibrated with 5 mM HEPES, 50 mM NaCl, 0.2 mM ADP, 0.2 mM MgCl2, 0.2 mM DTT, pH 8.0. Major peak was collected and concentrated to 6 mg/ml as above. The sample was immediately set up for crystallization with sitting drop method (1:1 ratio in 200 nL drop, Mosquito, TTP) at 20 °C, at the Crystallization Facility (Institute of Biotechnology, Helsinki). After 24 h, a single crystal appeared in Helsinki Complex screen in a well with 0.1 M Tris-HCl, 0.2 M LiCl, 20% (w/v) PEG8000, pH 8.0 of mother liquid. Crystallization conditions were further optimized; however, only a few crystals could be obtained after numerous attempts at 0.1 M Tris-HCl, 0.2 M LiCl, 20–23% (w/v) PEG8000, pH 7.9–8.5. For data collection, crystals were snap frozen in liquid N2 by fishing directly from 96-well plates and soaking for cryo-protection in 25% glycerol (v/v) containing mother liquid. Diffraction data were collected at Diamond Light Source synchrotron (Didcot, UK) at 100 K on I04 beamline with Pilatus3 6M detector. A complete data set was obtained at 0.97 Å wavelength with 0.1° oscillation angle per diffraction image with a total of 1400 images collected. Data were integrated using X-ray Detector Software, merged and scaled with AIMLESS (CCP4). The initial molecular replacement solution of 1:1 complex CARP-domain and actin was obtained with BALBES program, part of CCP4 suit, giving best result (Q factor = 0.6809) using 1k4z model for CARP domain and 3tpq for the actin with Rwork/Rfree = 0.3470/0.4470, after single REFMAC refinement round. Rounds of manual model building in COOT, introduction of translation-libration-screw grouping and refinement with BUSTER lead to final model with Rwork = 0.186 and Rfree = 0.234. It is important to note that we used a CAP1 construct (CAP1242–474) composed of the WH2 domain and the CARP domain in the crystallization trials, but electron density was observed only for the CARP domain (residues 317–473) and actin, whereas the first 74 residues of the CAP1242–474 were absent from the structure. Mass spectrometry analysis of the crystallization drops confirmed that the flexible N-terminal region of CAP1242–474 construct, containing the WH2 domain, had a tendency to degrade thus resulting in a product of a size of the CARP domain that was seen in the final electron density maps. The diffraction data was strongly anisotropic (CC1/2 > 0.3 at 2.3 Å along l-axis, 3.2 Å along hk plane) with ΔB of 56.16 Å2 between the axes which might explain a high overall B-factor (Supplementary Table ) when using individual B-factor refinement in the final model. Despite high average B, distribution of B-factors along the structure was normal. We considered anisotropic treatment of the merged data using ellipsoidal cut (STARANISO) which in our model lowers the average B-factor from ~129 to ~75 Å2 (Wilson B from 86 to 55 Å2), normal for the resolution range. However, ellipsoidal cut decreased slightly the completeness of our data in the 3.0–3.6 Å resolution range. This caused the electron density maps to be slightly discontinuous in some parts of CARP, but allowed us to observe more high-resolution details overall in the electron density maps. For these reasons, anisotropically treated data was only used to polish and finalize the final model, especially in the placement of waters and some side chains. Finally, we refined the model against either of these data sets yielding nearly identical models (RMSD ~0.5). Before deposition to PDB, the model was refined to Rwork/Rfree 18.6%/23.4% using non-treated data at 2.8 Å with I/σI = 1.3 as resolution cutoff criteria. [...] The WH2 domain of the CAP1 was obtained by homology modeling based on three structures (PDB entries 1sqk, 2a3z, and 4pl7) using the comparative modeling protocol of Rosetta (RosettaCM). This initial model of WH2 was used for MD simulations of ATP-actin-WH2 (System 3, Supplementary Table ). The model was further extended by adding PP2 and connecting it to the CARP domain from our structure. There were two possible ways of connecting WH2 to CARP domain: in cis (Supplementary Fig. ) or in trans (Supplementary Fig. ). The cis configuration requires PP2 to stretch across a distance that is at least 11 Å longer than in the trans configuration, making it both sterically and entropically unfavorable. The trans configuration has no steric clashes and in addition allows the regulatory region PP2 to be accessible, and thus represents more plausible model (Supplementary Fig. ). One thousand decoys of the ADP–actin–CAP1248–474 dimeric complex were generated. The models were ranked based on their total score, and the top ranking five models were selected to initiate atomistic MD simulations.The structures of protein molecules (actin, CARP domain, WH2, and CAP1248–474) were prepared using Propka (pKa estimation based on the crystallized complex and determination of protonation states at pH = 6.8), Chimera (placing hydrogens), VMD (protein structure building and visualization), and PyTopol (protein topology conversion from CHARMM to the GROMACS format). In all systems, E454 and H416 of the CARP domain were protonated; N-terminus of actin was acetylated; the His73 residue of actin was methylated (parameters were obtained by analogy); and the crystal water molecules and the bound Mg ion were kept. In each system containing ATP-actin, ATP was docked into the binding pocket based on the ADP position. Each system containing the WH2 domain was initiated from the top scoring five Rosetta models as described above.All simulations were carried out using GROMACS 5.1 employing the Charmm36 force field for the proteins and the TIP3P model for water. The equations of motion were integrated using a leap-frog algorithm with a 2 fs time step. All bonds involving hydrogens were constrained using the LINCS algorithm. Long-range electrostatic interactions were treated by the smooth particle mesh Ewald scheme with a real-space cutoff of 1.2 nm, a Fourier spacing of 0.12 nm, and a fourth-order interpolation. A Lennard–Jones potential with a force-switch between 1.0 and 1.2 nm was used for the van der Waals interactions.Each protein complex was placed in a rhombic dodecahedral simulation box maintaining at least a distance of 15 Å to the box sides and solvated in 0.15 M NaCl solution, ensuring neutrality of the system. Before production runs, steepest descent minimization and successive equilibration simulations (in total ~500 ps) in the NVT and NPT ensembles using the Berendsen thermostat and barostat were performed. In these simulations, initially all protein heavy atoms, and later only the Cα atoms were restrained with a force constant of 1000 kJ/mol/Å2; the time step was then ramped up from 1 to 2 fs. The number of water molecules and the average box volume at the start of the production runs are given in Supplementary Table .For production runs, each system was simulated in the NPT ensemble for ~1.2 µs. Five independent repeats were performed for each system (Supplementary Table ). The total time scale covered in the simulations was >24 μs. Protein-ADP/ATP-Mg complex, and solvent (water and NaCl) were coupled to separate temperature baths at 310 °K using the Nosé-Hoover thermostat, with a time constant of 1.0 ps. Isotropic pressure coupling was performed using the Parrinello–Rahman barostat with a reference pressure of 1 atm, a time constant of 5 ps, and a compressibility of 4.5 × 10−5 bar−1. All analyses were performed separately for each simulation repeat. The averages and standard deviations over independent repeats are reported. […]

Pipeline specifications

Application Protein structure analysis
Organisms Saccharomyces cerevisiae, Homo sapiens
Chemicals Adenosine Diphosphate