Dissecting the Kinetic Mechanism of Human Lysine Methyltransferase 2D and Its Interactions with the WRAD2 Complex.

Autor:	Edwardes LV; Discovery Biology, Discovery Sciences, BioPharmaceuticals, R&D, AstraZeneca, Cambridge CB4 0WG, U.K., Caswell SJ; Discovery Biology, Discovery Sciences, BioPharmaceuticals, R&D, AstraZeneca, Cambridge CB4 0WG, U.K., Giurrandino M; Discovery Biology, Discovery Sciences, BioPharmaceuticals, R&D, AstraZeneca, Cambridge CB4 0WG, U.K., Zhai X; Mechanistic and Structural Biology, Discovery Sciences, BioPharmaceuticals, R&D, AstraZeneca, Boston, Massachusetts 02210, United States., Gohlke A; Mechanistic and Structural Biology, Discovery Sciences, BioPharmaceuticals, R&D, AstraZeneca, Cambridge CB4 0WG, U.K., Kostomiris DH; Mechanistic and Structural Biology, Discovery Sciences, BioPharmaceuticals, R&D, AstraZeneca, Boston, Massachusetts 02210, United States., Pollard HK; Discovery Biology, Discovery Sciences, BioPharmaceuticals, R&D, AstraZeneca, Cambridge CB4 0WG, U.K., Pflug A; Mechanistic and Structural Biology, Discovery Sciences, BioPharmaceuticals, R&D, AstraZeneca, Cambridge CB4 0WG, U.K., Hamm GR; Imaging and Data Analytics, Clinical Pharmacology and Safety Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge CB4 0WG, U.K., Jervis KV; Discovery Biology, Discovery Sciences, BioPharmaceuticals, R&D, AstraZeneca, Cambridge CB4 0WG, U.K., Clarkson PN; Discovery Biology, Discovery Sciences, BioPharmaceuticals, R&D, AstraZeneca, Cambridge CB4 0WG, U.K., Syson K; Discovery Biology, Discovery Sciences, BioPharmaceuticals, R&D, AstraZeneca, Cambridge CB4 0WG, U.K.
Jazyk:	angličtina
Zdroj:	Biochemistry [Biochemistry] 2022 Sep 20; Vol. 61 (18), pp. 1974-1987. Date of Electronic Publication: 2022 Sep 07.
DOI:	10.1021/acs.biochem.2c00385
Abstrakt:	Human lysine methyltransferase 2D (hKMT2D) is an epigenetic writer catalyzing the methylation of histone 3 lysine 4. hKMT2D by itself has little catalytic activity and reaches full activation as part of the WRAD2 complex, additionally comprising binding partners WDR5, RbBP5, Ash2L, and DPY30. Here, a detailed mechanistic study of the hKMT2D SET domain and its WRAD2 interactions is described. We characterized the WRAD2 subcomplexes containing full-length components and the hKMT2D SET domain. By performing steady-state analysis as a function of WRAD2 concentration, we identified the inner stoichiometry and determined the binding affinities for complex formation. Ash2L and RbBP5 were identified as the binding partners critical for the full catalytic activity of the SET domain. Contrary to a previous report, product and dead-end inhibitor studies identified hKMT2D as a rapid equilibrium random Bi-Bi mechanism with EAP and EBQ dead-end complexes. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS) analysis showed that hKMT2D uses a distributive mechanism and gives further insights into how the WRAD2 components affect mono-, di-, and trimethylation. We also conclude that the Win motif of hKMT2D is not essential in complex formation, unlike other hKMT2 proteins.
Databáze:	MEDLINE
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