Computational protocol: Agonistic Human Antibodies Binding to Lecithin-Cholesterol Acyltransferase Modulate High Density Lipoprotein Metabolism*

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Protocol publication

[…] Full-length human LCAT protein (aa 1–416) harboring mutations of L4F and N5D () and a tobacco etch virus-cleavable C-terminal His tag was expressed using the BacMam expression system (Thermo Fisher Scientific) in HEK293S cells. Protein was purified by affinity, ion exchange, and size exclusion chromatography. Tobacco etch virus protease was used to remove the His tag during the purification process. The 27C3 antibody with a caspase-3 cleavage site engineered on the heavy chain between the Fab and Fc domains was expressed in HEK293–6E cells. The 27C3 Fab was isolated by purification following cleavage with caspase-3. We found that the LCAT-27C3 complex did not crystallize, so we added a previously discovered tool Fab (Fab1) to assist in crystallization (). Fab1 was expressed and purified from E. coli. Ternary complex was made and purified by size exclusion chromatography. The purified ternary complex was washed in 10 mm Tris (pH 7.5), 25 mm NaCl and concentrated to 8 mg/ml for crystallization. Diffraction quality crystals were grown with the LCAT-27C3-Fab1 complex from 0.1 m Hepes (pH 7.0), 5% PEG 20000. Crystals were taken to the Berkeley Advanced Light Source beamline 5.0.2, and a 2.45 Å data set was collected. Images were processed with iMOSFLM () and Aimless () from the CCP4 Program Suite (). The LCAT-27C3-Fab1 crystals grow in the P212121 space group with a = 57.9, b = 127.6, and c = 256.1 Å with ∼60% solvent and one complex in the asymmetric unit. The crystal structures of the LCAT-Fab1 complex () and 27C3 Fab (2.05 Å structure, data not shown) were used as starting models for molecular replacement in Phaser (). The initial model was built with multiple rounds of model building in Quanta (Accelrys) and refinement with CNX (). Final model building and refinement were performed using Coot () and PHENIX (), respectively. Validation with MolProbity () shows an overall score of 2.15 with 93.7% of the amino acids in Ramachandran favored regions. The final model has an R of 18.9% and an Rfree of 24.2%. Structure figures were made using PyMOL (PyMOL Molecular Graphics System, Schrodinger, LLC). […]

Pipeline specifications

Software tools iMosflm, CCP4, Coot, PHENIX, MolProbity, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Macaca fascicularis, Homo sapiens, Dipturus trachyderma
Diseases Cardiovascular Diseases, Dyslipidemias
Chemicals Cholesterol