Journal:Protein Science:2

The Impact of Crystallization Conditions on Structure-Based Drug Design: a Case Study on the Methylene Blue/Acetylcholinesterase Complex

Orly Dym, Wanling Song, Clifford Felder, Esther Roth, Valery Shnyrov, Yacov, Ashani, Yechun Xu, Robbie P. Joosten, Lev Weiner, Joel L. Sussman, and Israel Silman ^[1]

Molecular Tour
Crystal structure of TcAChE (residues 4-305, purple, and residues 306-535, red). The located between the two sub-domains. There are several of the conserved aromatic residues: , in the peripheral anionic site (PAS) at the top of the gorge; , mid-way down the gorge, and , the principal element of the catalytic ‘anionic’ sub-site (CAS), near the bottom. CAS involves the . The carbon atoms of these conserved residues colored yellow.

Structure-based drug design utilizes experimental 3D apo-protein or complex structures that are usually retrieved from the PDB. Over 57% of the crystallographic PDB entries were obtained with polyethyleneglycols (PEGs) as precipitant and/or as cryoprotectant, but fewer than 6% of

these report the presence of individual PEGs. We here report a case in which presence of PEG in a crystal structure markedly affected the bound ligand’s position. Specifically, we compared the positions of methylene blue and of decamethonium in the acetylcholinesterase complexes obtained using isomorphous crystals precipitated with PEG200 or ammonium sulfate. The ligands’ positions within the active-site gorge in complexes obtained using PEG200 as precipitant are influenced by the presence of ethyleneglycol oligomers. In both cases, an ethyleneglycol dimer is bound to W84 at the gorge’s bottom, preventing their proximal quaternary groups’ interaction with its indole. Consequently, both ligands are ~2.5 Å further up the gorge than in complexes obtained using crystals precipitated with ammonium sulfate, in which these quaternary groups make a direct π-cation interaction with the indole. These findings have implications for structure-based drug design, since data for ligand-protein complexes with PEG as precipitant may not reflect the ligand’s position in its absence, and could result in selection of incorrect lead compounds for drug discovery. Docking methylene blue into the protein structure of the complex obtained with PEG200, but omitting the ethyleneglycol dimer yields results in poor agreement with the crystal structure; excellent agreement is obtained if the ethyleneglycol dimer is included. Many proteins display structural elements in which precipitants like PEG might lodge. It will be important to investigate presence of precipitants in published crystal structures, and whether it has resulted in misinterpretation of electron density maps, thus adversely affecting drug design.

The DECA molecule in the DECA-AS/TcAChE structure (5e4j) is aligned along the axis of the active-site gorge, spanning the CAS and the PAS. The DECA makes non-bonded interactions with six of the conserved aromatic residues that line the gorge surface, viz., .