Computational protocol: Biochemical and Mass Spectrometric Characterization of Human N-Acylethanolamine-Hydrolyzing Acid Amidase Inhibition

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Protocol publication

[…] The sequence for human NAAA was taken from the SWISS-PROT protein sequence database (amino acids 126–359 primary accession number Q02083). The homology model of hNAAA was constructed using the crystal structure of conjugated bile acid hydrolase (CBAH) from Clostridium Perfringens (PDB ID: 2BJF) as a template in Prime (1.6 ed., Schrödinger, LLC, New York, NY). An initial BLAST alignment between the two sequences was adjusted by taking secondary structure into account using SSpro and PSIPRED . This alignment was further refined manually to superimpose Asn204 and Asn287 of hNAAA with Asn82 and Asn175 of CBAH respectively as previously suggested . The resultant alignment (13% identity, 21% homology, 34% gaps) was used for construction of the initial hNAAA model. Loops 2–6 and 8–14 were refined using an ab initio loop prediction algorithm. The loop refinement step deletes the loop and reconstructs it from a backbone dihedral library; the loop is then exhaustively sampled to identify the lowest energy conformation. All other loops featured mainly homologous residues and contained no gaps or insertions. The protein underwent a truncated-Newton energy minimization, using the OPLS_2005 all-atom force field and a Generalized Born continuum solvation model.AM6701 and N-Cbz-serine β-lactone were prepared for docking using the LipPrep (2.2 ed., Schrödinger, LLC, New York, NY) protocol and the OPLS_2005 force field. The ligands were docked to hNAAA using the extra precision (XP) procedure in Glide (5.6 ed., Schrödinger, LLC, New York, NY). The top pose for each ligand was then used to create the product for reaction. A covalent bond was imposed between the carbonyl carbon of the ligand and the sulfur atom of Cys126, for AM6701 the leaving group was removed and for N-Cbz-serine β-lactone the ring was opened. Atom types were reassigned and the entire system underwent minimization. […]

Pipeline specifications

Software tools SSpro, PSIPRED
Databases UniProt
Application Protein structure analysis
Organisms Homo sapiens
Diseases Multiple Sclerosis
Chemicals Cysteine