Water-assisted Dual Mode Cofactor Recognition by HhaI DNA Methyltransferase

Abstract

Methyltransferase-catalyzed modification of substrate DNA, RNA, or protein serves as a key signal in the regulation and maintenance of diverse biological processes in a range of organisms from prokaryotes to eukaryotes (1, 2). Subsequent to its isolation from Hemophilus haemolyticus nearly a quarter century ago, HhaI DNA methyltransferase (M.HhaI) 1 has been subjected to incisive investigations yielding detailed insights into the reaction chemistry (3-7). M.HhaI is an S-adenosyl-Lmethionine (AdoMet) dependent DNA methyltransferase that catalyzes the covalent attachment of a methyl group at the C-5 position of the aromatic ring of the first cytosine in the specific sequence 5Ј-GCGC-3Ј. Its ability to perform the methyl transfer within the B-DNA helix was rationalized by a localized "DNA backbone rotation" by nearly 180°(base flipping) without significantly bending or kinking of the rest of the duplex (8 -11). Base flipping serves to deliver the base into a concave catalytic site in the enzyme (8). The Gln 287 side chain fills up the gap generated concomitantly in the DNA bound with M.HhaI, leading to a stable and specific interruption of the aromatic -stacked base pairs, a feature recently utilized to evaluate long range electron migration through DNA (12). M.HhaI methylates all of the cytosines in poly(dG-dC) DNA (3), with hemimethylated DNA being the preferred substrate (13). Both the reaction product S-adenosyl-L-homocysteine (AdoHcy) (14) and, by implication, the cofactor AdoMet (7), appear to share the ability to drive M.HhaI to trap the target cytosine in the catalytic site to form a productive complex. These results portray AdoMet and AdoHcy as important modulators of the multiple binding modes of the DNA-bound enzyme.Despite a wealth of information on the structural, dynamic, and kinetic aspects of the M.HhaI mechanism, little attention has been paid to the energetics of AdoMet and AdoHcy recognition reactions. No molecule other than ATP serves as a cofactor in more diverse reactions than does AdoMet (15). It was, therefore, of interest to understand the thermodynamic basis of recognition of the ubiquitous cofactor AdoMet by methyltransferases, a feature shared among structurally related enzymes that catalyze reactions with diverse substrate specificities (16 -18). For example, a snapshot of a step that follows methyl transfer has been elucidated by crystallographic analyses of the DNA intermediate covalently bound to M.HhaI (8) and M.HaeIII (19). Despite its absence in the original crystallization mix of the ternary complex, it was AdoHcy that was found, fortuitously, to reside within the cofactor binding pocket (8) shared by AdoMet (16). This illustrates that the transfer of the methyl group from AdoMet to the target cytosine results in the formation not only of the modified (i.e. methylated) DNA but also the transformation of AdoMet into AdoHcy. On the other * This work was supported by grants from the Department of Biotechnology, Government of India (to D. N. R. and A. S.) and the Department of Scien...

Probing a rate-limiting step by mutational perturbation of AdoMet binding in the HhaI methyltransferase

“…Binding equilibria at several concentration points of labeled [ methyl - 3 H]AdoMet for each mutant were determined and K D values derived by fitting data to a single-site binding mechanism. The derived K D values for the WT (4.4 μM) and W41I mutant (14 μM) are in good agreement with those determined previously by fluorescent spectroscopy (6,7,13) or isothermal calorimetry (13,14). Overall, $K_{\text{D}}^{\text{AdoMet}}$ for the mutant MTases were higher than those of the WT protein by 1–2 orders of magnitude (four orders of magnitude for W41P) and increased in the order: WT < W41I < W41F, W41R, W41V < W41L, W41A < W41G ≪ W41P (Figure 3A).…”

“…Calorimetric analyses showed that the binding of cofactor to M.HhaI is exothermic (ΔΔ G = −6.7 and −7.8 kcal/mol for AdoMet and AdoHcy, respectively) and is driven by a favorable enthalpic change (14). Solution binding studies with truncated AdoHcy analogs showed that the thermodynamic contribution of the amino acid side chain is in the order of 2.3–2.6 kcal/mol (12).…”

“…Studies with synthetic AdoHcy analogs suggested that the molecular anchor for cofactor binding is its adenosyl moiety (12). Recently, conserved residues Phe18 (conserved motif I), Trp41 (motif II), Asp60 (motif III) and Leu100 (motif V) involved in cofactor binding have been subjected to site-directed mutagenesis and biochemical studies (13,14). However, only conservative replacements were analyzed in some detail, and the functional significance of these residues could not be fully evaluated.…”

Differences in Hydration Coupled to Specific and Nonspecific Competitive Binding and to Specific DNA Binding of the Restriction Endonuclease BamHI

“…We use an approach termed the osmotic stress technique (2,3) that measures changes in hydration coupled with changes in the functional state by measuring the effect of water activity on reaction equilibria and kinetics. The osmotic stress technique has been used previously to measure the changes in hydration accompanying the DNA binding of several regulatory proteins: Escherichia coli gal (4), lac (5), tyr (6), and Cro (7) repressors, E. coli CAP protein (8), Hin recombinase (9), Ultrathorax and Deformed homeodomains (10), the restriction endonucleases EcoRI (11)(12)(13)(14), BamHI (15,16), and EcoRV (17), HhaI methyltransferase (18), Sso7d protein (19), the TATA-binding protein (20,21), and the E2C protein from papillomavirus (22).…”

Sequestered Water and Binding Energy are Coupled in Complexes of λ Cro Repressor with Non-consensus Binding Sequences

“…The osmotic stress approach has been widely used to measure changes in water associated with the binding of free protein and DNA to form complexes. [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] We have been primarily interested in differences in sequestered water among specific, non-cognate, and non-specific complexes and have focused on competition binding reactions and dissociation rates. 13,[24][25][26][27][28][29][30] A linear dependence E-mail address of the corresponding author: raud@mail.nih.gov of relative binding free energies or dissociation rate constant activation energies on osmotic pressure indicates that the difference in the number of water molecules between the initial and final states is constant for that solute.…”