Crystallization, optimization, and crystal structure determination of ligands in complex with galectin-8N

Abstract: This thesis focuses on the protein “galectin-8N” which is a lectin protein that takes part in several physiological functions such as inflammation, autophagy, cell migration, and more. The project has three objectives: to observe and increase the reproducibility of crystallization of two variants (wildtype and mutant) of the human protein galectin-8N and to analyze the binding site when three different novel ligands are each inserted. Lastly, attempts of crystallizing mouse galectin-8N was also investigated which there is no prior crystallization data of. To observe required conditions for crystallizing and to increase the reproducibility of the two variants of human galectin-8N, two commercial plates of “MorpheusTM” were used and four manual plates imitating the conditions of Morpheus. After crystals were observed in the droplets of the manual plates, one of the plates was used to soak the ligand MH-3-80 and the other two to cocrystallize with ligands MH-3-80, MH-1FH and NV9.1. All the produced crystals were brought to the MAX IV where data was collected from and through iterative refinement processes with phenix, five models of human galectin-8N were created. From the models, all interactions between inserted ligands and the binding site were observed via PyMOL. Lastly, crystallization attempts of mouse galectin-8N were experimented on in total of eight commercial screens, two buffer screens, and one Morpheus screen combined with “Seed Beads”. The results were that both variants of human galectin-8N crystallizes in the Morpheus commercial plates and well in manual screens imitating Morpheus’ conditions. Crystals were found to prefer conditions of: buffer 1, in precipitant mixture 4, and with additives such as ethylene glycols, monosaccharides and alcohols. Ligands MH-3-80 and MH-1FH were inserted successfully into both variants of the human proteins, while NV9.1 did not. The interactions that occurred between inserted ligands and the residues at the binding site were: Arg52, His72, Arg76, Asn86, and Trp93. Other interactions were also observed however those existed exclusively depending on the inserted ligands’ structure and their moieties. Lastly, the crystallization of mouse galectin-8N was unfortunately difficult and rather unsuccessful, however the commercial screen: JCSG+TM gave the most promising results and is highly recommended for future observations.