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.

In certain crystal structures of complexes for which the native crystals have been generated using PEG as the precipitant, PEG oligomers can be seen within the gorge. For example, in the galanthamine/TcAChE complex (1dx6), a . The galanthamine is shown in space-filling format in green, and above it a PEG tetramer in magenta.

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., .

In the DECA-PEG/TcAChE structure (5e2i) a , between the proximal quaternary group of the DECA and the indole ring of Trp84. DECA molecule is colored red, and the PEG moiety blue. Due to the presence of the PEG molecule oligomer at the bottom of the gorge in the DECA-PEG/TcAChE structure, the DECA molecule is positioned further up the gorge than in the DECA-AS/TcAChE structure. . The DECA in the DECA-PEG/TcAChE structure is shown as red ball-and-sticks, and the PEG oligomers as blue ball-and-sticks. The DECA in the DECA-AS/TcAChE structure is shown as cyan ball-and-sticks. .

(5dlp). The MB molecule is colored purple. In MB-PEG/TcAChE complex (5e4t) . Consequently, the MB molecule is positioned further up the gorge than in the MB-AS/TcAChE complex. . The MB in the MB-PEG/TcAChE structure is shown as purple ball-and-sticks, and the PEG/PGE oligomers as blue ball-and-sticks. The MB in the MB-AS/TcAChE structure is shown as pink ball-and-sticks. .

. The PEG2 trimer at the bottom of the gorge is in the same position as the PEG dimer seen in the DECA-PEG/TcAChE structure, and both overlap with the proximal segment of the thioPEG heptamer (PE7) in its complex with TcAChE. A ball-and-sticks representation of PEG-SH-350 (the thioPEG heptamer) in its complex with TcAChE (PDB entry 1jjb) is shown, with carbon atoms in cyan, and the sulfur as a darkorange sphere. Several aromatic residues lining the active-site gorge are also shown in ball-and-sticks format, with carbons in yellow. Overlayed are ball-and-sticks models of the PEG oligomers adjacent to Trp84 in MB-PEG/TcAChE (carbons in green) and the PGE in the DECA-PEG/TcAChE (carbons in blue).

Docking of MB into the X-ray structure obtained using crystals grown from PEG200, viz., MB-PEG/TcAChE, yields a . The experimental MB in each structure is depicted as ball-and-sticks with the carbon atoms colored purple, while the docked structures are shown as ball-and-sticks with the carbon atoms colored green. PEG molecules are shown in blue, and two key residues, viz., Trp84 and Trp279, are shown with carbon atoms in yellow, oxygen atoms in red, and nitrogens in blue. . However, , or , closer to the position in the crystal structure of the complex in which the crystals had been grown using AS as the precipitant, MB-AS/TcAChE. . Docking of MB into the crystal structure of the MB/TcAChE complex (5dlp) obtained using crystals grown from AS from which the MB had been removed, . For comparison, the docking of MB to MB-PEG/TcAChE, from which the two PEGs and waters were removed is compared to the experimentally observed MB-AS/TcAChE structure. .

. The indole of Trp84 is displayed in space-filling mode, with carbon atoms colored green. MB and DECA are depicted as ball-and-sticks models, with their carbon atoms in cyan and magenta, respectively. Whereas the proximal quaternary group of MB interacts with the five-membered ring of the indole, the DECA extends further, such that its proximal quaternary group interacts with the indole’s six-membered ring.