Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Chromatin Assembly and Disassembly , Transcription Factors/chemistry , Transcription Factors/metabolism , Xenopus laevis , Adenosine Triphosphatases/genetics , Animals , Crystallography, X-Ray , Models, Molecular , Protein Structure, Quaternary , Protein Structure, Tertiary , Structural Homology, Protein , Transcription Factors/genetics , Xenopus laevis/geneticsABSTRACT
DIM-5 is a SUV39-type histone H3 Lys9 methyltransferase that is essential for DNA methylation in N. crassa. We report the structure of a ternary complex including DIM-5, S-adenosyl-L-homocysteine, and a substrate H3 peptide. The histone tail inserts as a parallel strand between two DIM-5 strands, completing a hybrid sheet. Three post-SET cysteines coordinate a zinc atom together with Cys242 from the SET signature motif (NHXCXPN) near the active site. Consequently, a narrow channel is formed to accommodate the target Lys9 side chain. The sulfur atom of S-adenosyl-L-homocysteine, where the transferable methyl group is to be attached in S-adenosyl-L-methionine, lies at the opposite end of the channel, approximately 4 A away from the target Lys9 nitrogen. Structural comparison of the active sites of DIM-5, an H3 Lys9 trimethyltransferase, and SET7/9, an H3 Lys4 monomethyltransferase, allowed us to design substitutions in both enzymes that profoundly alter their product specificities without affecting their catalytic activities.
Subject(s)
Histone-Lysine N-Methyltransferase , Histones/chemistry , Methyltransferases/chemistry , Neurospora crassa/enzymology , S-Adenosylhomocysteine/chemistry , Catalytic Domain/physiology , Cysteine/chemistry , Histone Methyltransferases , Lysine/chemistry , Macromolecular Substances , Models, Molecular , Molecular Structure , Peptides/chemistry , Protein Methyltransferases , Protein Structure, Tertiary/physiology , Sulfur/chemistry , Zinc/chemistryABSTRACT
AdoMet-dependent methylation of histones is part of the "histone code" that can profoundly influence gene expression. We describe the crystal structure of Neurospora DIM-5, a histone H3 lysine 9 methyltranferase (HKMT), determined at 1.98 A resolution, as well as results of biochemical characterization and site-directed mutagenesis of key residues. This SET domain protein bears no structural similarity to previously characterized AdoMet-dependent methyltransferases but includes notable features such as a triangular Zn3Cys9 zinc cluster in the pre-SET domain and a AdoMet binding site in the SET domain essential for methyl transfer. The structure suggests a mechanism for the methylation reaction and provides the structural basis for functional characterization of the HKMT family and the SET domain.
