SET7/9 Catalytic Mutants Reveal the Role of Active Site Water Molecules in Lysine Multiple Methylation

Paul A Del Rizzo,Jean-François Couture,Lynnette M.A Dirk,Bethany S Strunk,Marijo S Roiko,Joseph S Brunzelle,Robert L Houtz,Raymond C Trievel

doi:10.1074/jbc.m110.114587

Abstract

SET domain lysine methyltransferases (KMTs) methylate specific lysine residues in histone and non-histone substrates. These enzymes also display product specificity by catalyzing distinct degrees of methylation of the lysine ε-amino group. To elucidate the molecular mechanism underlying this specificity, we have characterized the Y245A and Y305F mutants of the human KMT SET7/9 (also known as KMT7) that alter its product specificity from a monomethyltransferase to a di- and a trimethyltransferase, respectively. Crystal structures of these mutants in complex with peptides bearing unmodified, mono-, di-, and trimethylated lysines illustrate the roles of active site water molecules in aligning the lysine ε-amino group for methyl transfer with S-adenosylmethionine. Displacement or dissociation of these solvent molecules enlarges the diameter of the active site, accommodating the increasing size of the methylated ε-amino group during successive methyl transfer reactions. Together, these results furnish new insights into the roles of active site water molecules in modulating lysine multiple methylation by SET domain KMTs and provide the first molecular snapshots of the mono-, di-, and trimethyl transfer reactions catalyzed by these enzymes.

Highlights

SET domain enzymes represent a family of S-adenosylmethionine (AdoMet)3-dependent methyltransferases that catalyze the site-specific methylation of protein lysyl residues in a host of proteins, including histones, transcription factors, chromatin-modifying enzymes, ribosomal subunits, and other substrates [1,2,3]
Peptides were allowed to bind at room temperature for 30 min, We examined whether the Y305F mutant dimethylated and the resin was collected by centrifugation (ϳ9000 ϫ g). the TAF10-K189 peptide via a processive or a distributive
How does the active site constrain the motion of the lysine ⑀-amino group to align it for methyl transfer with AdoMet, while providing adequate volume to accommodate the mono, di, and trimethylated lysine side chain generated during multiple methyl transfer reactions? The structures of the Y305F and Y245A mutants resolve this paradox, illustrating that alterations in the positions or occupancies of water molecules within their active sites generate the space required to

Summary

EXPERIMENTAL PROCEDURES

Expression, and Purification of the SET7/9 Mutants— The Y245A and Y305F mutants were introduced into the pHIS2 SET7/9 expression vector encoding residues 110 –366 [27] via QuikChange site-directed mutagenesis (Stratagene) and were verified by dideoxy DNA sequencing. In the course of characterizing WT SET7/9, we observed that the enzyme copurified with AdoMet or another contaminant that resulted in technical difficulties in the isothermal titration calorimetry (ITC) experiments and co-crystallization trials with the TAF10 peptides. To overcome this problem, a denaturation and refolding step was inserted in the purification scheme. Fluorescent Methyltransferase Assay—A coupled fluorescent methyltransferase assay was used to measure the kinetic parameters of WT SET7/9 and the Y245A and Y305F mutants as reported previously, with the exception that 50 –150 nM enzyme, 100 ␮M AdoMet, and varying concentrations of TAF10 peptide substrate were used [27, 36]. WT SET7/9 and the Y245A and Y305F mutants displayed ligand:protein binding stoichiometries (N values) between 0.8 and 1.0, demonstrating

RESULTS

Kma kcata

Amount of product formeda nmol nmol

DISCUSSION