Computational protocol: A Mechanism for Actin Filament Severing by Malaria Parasite Actin Depolymerizing Factor 1 via a Low Affinity Binding Interface*

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Protocol publication

[…] Recombinant ADF/cofilins (50 μm) were incubated with G-actin (24 μm) in the presence of sulfo-SDA or EDC in G-buffer for 0.5 or 2 h, respectively. For F-actin-ADF/cofilin cross-linking, G-actin was first induced to polymerize by the additional 1/10th volume of ×10 F-buffer (500 mm KCl, 20 mm MgCl2) for 1 h at room temperature. EDC and ADF/cofilins were then added to F-actin and incubated at room temperature for 2 h. Cross-linked F-actin·ADF/cofilin complexes were pelleted at 100,000 × g in a TLA 100 rotor for 1 h before sample analysis. Sulfo-SDA-treated samples were subsequently photoactivated by UV illumination at 360 nm for 20 min. Cross-linked actin·ADF/cofilin complexes were separated by SDS-PAGE and complex bands were excised, followed by manual in-gel reduction, alkylation, and tryptic digestion. Extracted peptides were injected and separated by nano-flow reversed-phase liquid chromatography on a nano-LC system (1200 series, Agilent) using a nanoAcquity C18 150 × 0.15-mm inner diameter column (Waters) with a linear 60-min gradient set at a flow rate of 1.2 μl/min at 45 °C from 100% solvent A (0.1% formic acid in Milli-Q water) to 100% solvent B (0.1% formic acid, 60% acetonitrile (Mallinckrodt Baker, NJ), 40% Milli-Q water). For SDA cross-linked samples, the nano-HPLC was coupled on-line to an LTQ-Orbitrap XL mass spectrometer equipped with a nanoelectrospray ion source (Thermo Fisher Scientific) for automated MS/MS. The Orbitrap was run in a data-dependent acquisition mode with the Orbitrap resolution set at 60,000 and the top five multiply charged species selected for fragmentation in the linear ion trap by collision-induced dissociation (single charged species were ignored). The ion threshold was set to 15,000 counts for MS/MS. The activation time was set to 30 ms. For EDC cross-linked samples, digested samples were injected and separated by nano-flow reversed-phase liquid chromatography on a nano LC system (Waters nanoAcquity) using a nanoAcquity C18 150 × 0.075-mm inner diameter column (Waters) with a linear 60-min gradient set at a flow rate of 0.4 μl/min from 95% solvent A (0.1% formic acid in Milli-Q water) to 100% solvent B (0.1% formic acid, 80% acetonitrile (Mallinckrodt Baker, NJ), 20% Milli-Q water). The nano-UPLC was coupled on-line to a Q-Exactive mass spectrometer equipped with a nano-electrospray ion source (Thermo Fisher Scientific) set to acquire full scan (70,000 resolution) and the top 10 multiply charged species selected for fragmentation using the high energy collision disassociation (single charged species were ignored). Fragment ions where analyzed with resolution set at 17,500 with the ion threshold set to 1e5 intensity. The activation time was set to 30 ms and the normalized collision energy set to 24. Raw files consisting of full-scan MS, low-resolution MS/MS (OrbiTrap XL), and high-resolution MS/MS spectra (Q-Exactive) were converted to the MGF data format with Proteome Discoverer 1.4 and searched using Xcomb generated databases with the Mascot or pLink algorithms, respectively (). Databases for every possible cross-linked pair were generated by xComb version 1.3 (). The amino acid sequences for the expressed recombinant ADF proteins were derived from its genetic sequence and the Uniprot entry for rabbit actin were uploaded in Uniprot FASTA format. Trypsin (two missed cleavages) was chosen for enzyme digestion. Both intra- and inter-protein cross-links were created. The minimum peptide length for each peptide of the pair was four amino acids with at least one trypsin missed cleavage selected with amine cross-linking. The cross-link database was uploaded in Mascot version 2.3. Mascot parameters for each search included no enzyme cleavages and a fixed modification in the form of carboxymethyl at Cys residues. Spectra were searched with a mass tolerance of 20 ppm in MS mode and 0.5 Da in MS/MS mode for the Orbitrap XL. The MS/MS fragmentation of cross-linked peptides identified by the Mascot search was manually analyzed to assign ion peaks using mMass () and annotated with assistance from the recently published Expert System GUI as a guide (). For high resolution MS/MS Q-Exactive data, we searched for cross-linked peptides using the pLink algorithm (). The database consisted of the Uniprot annotated entries for all recombinant proteins used and pLink configuration set default mass accuracy settings. Cross-linker settings were also edited to include two EDC entries (−18.0152 Da) for Lys linked to Glu and Asp residues. XL-MS determined cross-linked peptide interfaces and the crystal structures of rabbit actin (PDB code 1J6Z), PfADF1 (PDB code 3Q2B), and HsCOF1 (PDB code 1Q8X) were used to reconstruct the monomeric actin-ADF/cofilin structural models using the built in rotation and translation functions within PyMol (Delano Scientific LLC). The F-actin-PfADF1 and F-actin-HsCOF1 structural models (PDB codes 3G37 (ref) 3Q2B and 1Q8X (ref), respectively) were built using the same method. […]

Pipeline specifications

Software tools Proteome Discoverer, pLink, mMass, PyMOL
Databases xComb
Application MS-based untargeted proteomics
Organisms Plasmodium falciparum, Homo sapiens
Diseases Hypoprothrombinemias, Malaria
Chemicals Amino Acids