Probing multiple enzymatic methylation events in real time with NMR spectroscopy

Abstract

Post-translational modification (PTM) of proteins is of critical importance to the regulation of many cellular processes in eukaryotic organisms. One of the most well-studied protein PTMs is methylation, wherein an enzyme catalyzes the transfer of a methyl group from a cofactor to a lysine or arginine side chain. Lysine methylation is especially abundant in the histone tails and is an important marker for denoting active or repressed genes. Given their relevance to transcriptional regulation, the study of methyltransferase function through in vitro experiments is an important stepping stone toward understanding the complex mechanisms of regulated gene expression. To date, most methyltransferase characterization strategies rely on the use of radioactive cofactors, detection of a methyl transfer byproduct, or discontinuous-type assays. Although such methods are suitable for some applications, information about multiple methylation events and kinetic intermediates is often lost. Herein, we describe the use of two-dimensional NMR to monitor mono-, di-, and trimethylation in a single reaction tube. To do so, we incorporated 13 C into the donor methyl group of the enzyme cofactor S-adenosyl methionine. In this way, we may study enzymatic methylation by monitoring the appearance of distinct resonances corresponding to mono-, di-, or trimethyl lysine without the need to isotopically enrich the substrate. To demonstrate the capabilities of this method, we evaluated the activity of three lysine methyltransferases, Set7, MWRAD 2 (MLL1 complex), and PRDM9, toward the histone H3 tail. We monitored mono-or multimethylation of histone H3 tail at lysine 4 through sequential short two-dimensional heteronuclear single quantum coherence experiments and fit the resulting progress curves to first-order kinetic models. In summary, NMR detection of PTMs in one-pot, real-time reaction using facile cofactor isotopic enrichment shows promise as a method toward understanding the intricate mechanisms of methyltransferases and other enzymes.

An acetylation-mediated chromatin switch governs H3K4 methylation read-write capability

“…HisDPY30 was expressed, purified, and cleaved to remove the 6HIS-tag as described (Patel et al, 2009; Usher et al, 2021). A two-fold molar excess of DPY30 was added to the MWRA sub-complex and incubated on ice for 1 hour.…”

“…Fractions containing 6HIS-tagged MWRA were identified by SDS-PAGE, pooled, and supplemented with 6HIS-tagged TEV protease (purified as described: Nautiyal and Kuroda, 2018) at a 1:100 enzyme to substrate molar ratio to cleave the 6HIS-tag on WDR5. This mixture was dialyzed against three changes of WB (each 2L for at least four hours at 4°C) and 6HIS-TEV removed from cleaved MWRA via IMAC (Usher et al, 2021). MWRA flow-through protein solution was concentrated to ~15 mL using a 30 kDa MWCO centrifugal filter ( EMD Millipore ), ensuring not to concentrate to where solution became yellow/cloudy and viscous.…”

“…Methods for the expression, purification, and assembly of MLL1 core complex (MLL1C) were adapted from published protocols (Usher et al, 2021). A polycistronic recombinant expression construct containing the MLL1 SET domain (Uniprot Q03164; residues 3745-3969), WDR5 (Uniprot P61964; residues 2-334), RbBP5 (Uniprot Q15291; residues 1-538) and Ash2L (Uniprot Q9UBL3-3; residues 1-534) (MWRA construct) in pST44 vector was a kind gift from Dr. Song Tan (Tan et al, 2005).…”

“…Fractions containing stoichiometric MWRA sub-complex were identified by SDS-PAGE, pooled, and concentrated to ~15 mL. HisDPY30 was expressed, purified, and cleaved to remove the 6HIS-tag as described (Patel et al, 2009;Usher et al, 2021). A two-fold molar excess of DPY30 was added to the MWRA sub-complex and incubated on ice for 1 hour.…”

Biophysical insights into glucose-dependent transcriptional regulation by PDX1

“…However, as evidenced by contradictory enzyme assignments and ambiguous consequences on PDX1 functions, we must explore in vitro approaches to complement in vivo studies of PDX1 phosphorylation. NMR is a tractable strategy to study possible structural consequences of PDX1 PTMs; we and others have leveraged NMR strategies toward kinetic and structural characterization of PTMs in disordered protein systems ( 190 , 191 , 192 , 193 ).Through such approaches, we may generate new hypotheses for PTM-regulated PDX1 function and, further, parse the findings from within very complex cellular systems.…”