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Lens epithelium-derived growth factor p75 (LEDGF/p75) is an epigenetic reader involved in several human pathologies including human immunodeficiency virus (HIV) infection and cancers such as mixed-lineage leukemia-rearranged (MLL-r) leukemia. The N-terminal PWWP domain of LEDGF/p75 is essential for tethering the MLL protein to the chromatin. The PWWP domain recognizes di- and trimethylation marks on histone H3 Lys36. This process results in the transcription of MLL target genes. Once translocations occur in the MLL gene, oncogenic MLL fusion proteins could be formed and tethered to chromatin by LEDGF/p75. Eventually, this leads to aberrant gene transcription and leukemic conditions arise. Notably, LEDGF/p75 is essential for the onset of MLL-r leukemia, however it is dispensable for hematopoiesis, making this protein a promising drug target. Our aim is to block the methyllysine binding pocket of LEDGF PWWP domain through a rationally designed small-molecule inhibitor. To this end, X-ray crystallography-based fragment screening (FBS-X) with the PWWP domain has been initiated. While the LEDGF PWWP domain could never be made to produce crystals suitable for FBS-X, the closely related PWWP domain of hepatoma-derived growth factor 2 (HDGF2) could be used. Nevertheless, these crystals were losing diffraction quality within several weeks, which was hypothesized to be linked to a possible oxidation of the exposed Cys64 residue. To circumvent these difficulties, we have recombinantly produced the Cys64Ser mutant of HDGF2 PWWP domain (HDGF2 PWWP-C64S). Crystallization conditions for HDGF2 PWWP-C64S were identified and successfully optimized for FBS-X. A total of eight crystal forms were obtained and used for structure determination. The best crystals were stable over time and diffracted X-rays to a resolution of 1.1 Å which exceeds the resolution of the wild-type domain crystals. The C64S mutation did not impact the binding pose of a previously confirmed binding fragment. These results demonstrated that the mutant HDGF2 PWWP-C64S is suitable for fragment soaking. To rationalize the observed beneficial effect of the C64S mutation on crystallization, we have systematically compared the crystal lattice contacts (interfaces) in all crystal forms obtained (eight for HDGF2 PWWP-C64S, three for wild-type HDGF2 PWWP and one for LEDGF PWWP). Five recurring interfaces were observed, of which a particular interface including Ser64 was present in every HDGF2 PWWP-C64S crystal form. Typically it was also the largest interface. In contrast, this interface was less frequent in the wild-type crystal forms. It was suggested that oxidation of Cys64 in the wild-type could interfere with the stability of this important interface, resulting in crystal lattice degradation. At the same time, it cannot be excluded that oxidation of Cys64 is causing reduced crystallization propensity as well, yet this requires extra research.

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